EMC-dependent membrane insertion of IER3IP1 sustains efficient ER-to-Golgi trafficking

EMC-dependent membrane insertion of IER3IP1 sustains efficient ER-to-Golgi trafficking | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article EMC-dependent membrane insertion of IER3IP1 sustains efficient ER-to-Golgi trafficking Sang-Wook Kang, Yejin Shin, Minseok Shin, Kang-Sug Jo, Youngsup Song, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8560191/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The endoplasmic reticulum (ER) membrane complex (EMC) acts as an insertase for a distinct subset of membrane proteins, yet how defects in EMC-dependent membrane insertion propagate to downstream secretory pathway function remains poorly understood. Here, we identify Immediate Early Response 3 Interacting Protein 1 (IER3IP1) as an EMC-dependent ER membrane protein whose stable biogenesis is required to sustain efficient ER-to-Golgi trafficking of newly synthesized secretory cargos. Loss of EMC activity destabilizes IER3IP1, resulting in delayed ER export of folding- and trafficking-sensitive proteins, including clusterin and the prion protein, without overt disruption of global ER structure or protein synthesis. We further show that membrane insertion alone is insufficient for IER3IP1 function: a transmembrane domain–dependent intramembrane interaction mediated by the N-terminal segment is required to support cargo export competence. Together, these findings illustrate how defects in membrane protein biogenesis can indirectly constrain secretory pathway output through loss of specific EMC-dependent membrane proteins, establishing a mechanistic link between membrane insertion efficiency, protein stability, and ER export. Biological sciences/Biochemistry/Proteins/Membrane proteins Biological sciences/Molecular biology/Protein folding/Endoplasmic reticulum Biological sciences/Cell biology/Organelles/Endoplasmic reticulum Biological sciences/Cell biology/Protein transport/Protein translocation ER membrane complex (EMC) IER3IP1 Membrane protein insertion Early secretory pathway Secretory protein export Clusterin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The early secretory pathway ensures efficient export of proteins synthesized in the endoplasmic reticulum (ER) toward the Golgi apparatus and downstream compartments 1 . Proper execution of this pathway requires not only translocation of secretory proteins into the ER lumen but also the coordinated biogenesis and function of ER membrane proteins that support cargo maturation and export 2 , 3 . Perturbations in these processes can lead to selective defects in ER-to-Golgi trafficking and disproportionate stress on folding-sensitive or rapidly exported cargos, thereby compromising cellular proteostasis 4 , 5 . Membrane protein biogenesis in the ER is inherently complex, as newly synthesized proteins display diverse topologies and transmembrane domain (TMD) hydrophobicities 3 . To accommodate this diversity, eukaryotic cells rely on multiple, partially overlapping insertion pathways rather than a single universal mechanism 6 . While the Sec61 translocon mediates cotranslational insertion of most secretory and type I membrane proteins, substrates with weakly hydrophobic or topologically constrained TMDs require alternative machineries 6 . Among these, the ER membrane complex (EMC) has emerged as a conserved insertase that supports membrane integration of a discrete subset of ER membrane proteins that are inefficiently handled by Sec61 alone 7 , 8 . Extensive biochemical and genetic studies have established the importance of the EMC in the biogenesis and stability of its client proteins 9 , 10 , 11 . Loss of EMC activity destabilizes a specific cohort of membrane proteins, leading to misinsertion and degradation 7 , 12 , and mutations in EMC subunits are associated with neurodevelopmental and metabolic disorders 8 . Despite these advances, it remains unclear how defects in EMC-dependent insertion translate into downstream cellular phenotypes. In particular, whether EMC dysfunction affects ER-to-Golgi trafficking directly or indirectly through loss of functional client proteins has not been resolved. Several observations suggest that EMC-dependent effects on secretory trafficking may be mediated indirectly, through client proteins that themselves participate in cargo export or quality control. Indeed, impaired anterograde transport of selected cargos has been reported in EMC-deficient contexts, raising the possibility that individual EMC clients act as functional intermediates linking membrane protein biogenesis to trafficking efficiency 13 , 14 . However, the identities of such client proteins and the mechanisms by which they influence ER export remain poorly defined. Recent advances in proximity labeling–based proteomic approaches have enabled systematic interrogation of membrane protein biogenesis within the ER 15, 16 . By allowing spatially restricted, live-cell labeling of ER-localized proteins, these strategies facilitate identification of membrane proteins whose insertion or stability depends on specific machineries and enable discrimination between primary biogenesis defects and secondary proteostatic consequences 17 , 18 . One such protein is Immediate Early Response 3 Interacting Protein 1 (IER3IP1) 19 , a small multipass ER membrane protein whose loss-of-function mutations are linked to neurodevelopmental and β-cell survival disorders 20 , 21 , 22 . Previous studies have implicated IER3IP1 in ER-to-Golgi trafficking and selective cargo secretion in multiple experimental systems 23 , 24 , yet its mode of membrane insertion and the relationship between its biogenesis and trafficking function remain unclear. Here, we investigate how EMC-dependent membrane protein biogenesis influences the early secretory pathway by focusing on IER3IP1, a poorly characterized ER membrane protein previously implicated in ER-to-Golgi trafficking. Using a combination of EMC perturbation, topology-sensitive reporters, and ER-targeted proximity labeling, we examine the requirements for IER3IP1 membrane insertion, stability, and functional competence. By placing IER3IP1 within the context of EMC-dependent protein biogenesis, this work aims to clarify how defects in ER membrane protein insertion can selectively impact secretory pathway output. Results ER-TurboID identifies EMC-dependent ER membrane proteins To identify ER-localized proteins whose abundance or membrane association depends on the ER membrane complex (EMC), we employed proximity-dependent biotinylation using TurboID, a promiscuous biotin ligase that labels neighboring proteins in living cells 15 . To confine labeling to the ER lumen, we engineered a V5-tagged TurboID construct containing an N-terminal BiP signal sequence for ER targeting and a C-terminal KDEL motif for luminal retention (hereafter “ER-TurboID”). This construct was stably integrated into Flp-In T-REx 293 cells to generate an isogenic, doxycycline-inducible cell line (Fig. 1 A). Upon addition of biotin, ER-TurboID rapidly labeled ER-resident proteins, as detected by streptavidin–HRP (Fig. 1 B), and biotinylated proteins were efficiently recovered on streptavidin resin within 10 min (Fig. S1 A). Labeling was restricted to the ER lumen, as evidenced by selective biotinylation of luminal chaperones such as BiP and calnexin, whose extended luminal domains are accessible to TurboID (Fig. 1 C). Indirect immunofluorescence confirmed co-localization of TurboID and biotinylated proteins within the ER network (Fig. S1 B). Together, these results establish the spatial specificity and temporal responsiveness of ER-TurboID and validate its suitability for interrogating ER-localized proteomes under defined genetic perturbations. Because EMC5 is a core subunit whose loss destabilizes the entire EMC complex 7 , we compared ER-proximal proteomes between wild-type (WT) cells and EMC5-deficient (ΔEMC) cells. An EMC-null ER-TurboID cell line was generated by CRISPR/Cas9-mediated genome editing, and disruption of EMC integrity was confirmed by immunoblotting for multiple EMC subunits (Fig. 1 D). Functional impairment of EMC activity was further validated using tail-anchored reporters bearing an opsin-based glycosylation tag (OPT), which discriminates between properly inserted and uninserted species; loss of EMC resulted in a marked reduction of the inserted form (Fig. S1 C). ER-TurboID–labeled proteins from WT and ΔEMC cells were affinity-purified and quantified by mass spectrometry for comparative analysis (Fig. S1 D). Among the biotinylated proteins identified (Table S1 ), ER membrane proteins were of particular interest because the EMC contributes to the insertion of a subset of transmembrane domains (TMDs) 25 . As expected, numerous abundant ER membrane protein biogenesis factors—including the Sec61 translocon, TRAP, Sec63, TRAM1, the SRP receptor, oligosaccharyltransferase subunits, and additional ER membrane complexes—were recovered at comparable levels in WT and ΔEMC cells (Table S2 ), indicating that global ER integrity and luminal labeling efficiency were preserved. Applying a defined statistical threshold (log₂FC ≥ 0.5; p < 0.20) (Table S3 ) revealed reduced recovery of all membrane-integrated EMC subunits and several established EMC client proteins, including squalene synthase and cytochrome b5 (Fig. 1 E; Fig. S1 E), consistent with impaired insertion or stability upon EMC loss. Beyond these known substrates, several previously unrecognized EMC-dependent proteins were identified. Among them, Immediate Early Response 3 Interacting Protein 1 (IER3IP1) exhibited the most pronounced reduction in ER-proximal recovery upon EMC depletion (Fig. 1 E; Table S3 ), nominating it as a strong candidate EMC-dependent ER membrane protein. EMC is required for efficient insertion and stability of IER3IP1 The EMC dependence of IER3IP1 was validated by immunoblotting and metabolic pulse-labeling, both of which revealed a marked reduction in newly synthesized IER3IP1 in EMC-deficient cells (Fig. 2 A). In contrast, depletion of IER3IP1 did not alter the abundance of EMC subunits or the membrane insertion of the established EMC client Sec61β (Fig. S2 A), indicating that IER3IP1 is not required for EMC assembly or general EMC function. To assess the membrane topology of IER3IP1, we employed an optimized opsin-based glycosylation reporter (OPT) containing two N-glycan acceptor sites that report on luminal exposure only when adjacent TMDs are correctly inserted (Fig. 2 B). Pulse-labeling combined with endoglycosidase digestion established a reproducible modification pattern in WT cells: both acceptor sites were glycosylated when OPT was fused to the N-terminus, whereas only one site was modified when OPT was fused to the C-terminus (Fig. 2 C). This topology-dependent glycosylation pattern was faithfully recapitulated in a mammalian cell-free translation system supplemented with dog pancreas rough microsomes (Fig. S2 B), providing independent confirmation of the inferred membrane orientation. By contrast, depletion of EMC completely abolished OPT glycosylation at both the N- and C-terminal fusions (Fig. 2 D; Fig. S2 C), demonstrating that proper insertion of both TMDs of IER3IP1 is strictly dependent on EMC activity. Consistent with the fate of uninserted membrane proteins 26 , IER3IP1 underwent proteasome-dependent degradation when EMC function was compromised (Fig. S2 D), accounting for its reduced steady-state abundance in ΔEMC cells (Fig. 2 D). Increasing the hydrophobicity of the N-terminal TMD through single-point substitutions (S7L or Q10L) restored insertion and stability of IER3IP1 in EMC-deficient cells (Fig. 2 E), indicating that the marginal hydrophobicity of this segment underlies its EMC dependence 9 . EMC-dependent IER3IP1 promotes efficient ER-to-Golgi trafficking of clusterin Given prior reports linking IER3IP1 to selective secretory trafficking 13 , 14 , 23 , we examined whether EMC-dependent biogenesis of IER3IP1 influences ER-to-Golgi transport of clusterin (CLU), a folding-sensitive secretory glycoprotein. To visualize CLU dynamics, we employed the Retention Using Selective Hooks (RUSH) system, which synchronizes cargo release from the ER upon biotin addition 27 . In WT cells, CLU–GFP efficiently redistributed from the ER to a perinuclear compartment consistent with the GM130-positive Golgi apparatus within 1 h of biotin addition (Fig. 3 A; Fig. S3 A). By contrast, CLU–GFP remained largely confined to the ER during the same interval in EMC- or IER3IP1-depleted cells (Fig. 3 B). Notably, and in contrast to findings reported by another group 23 , this trafficking defect was not attributable to global Golgi dysfunction, as brefeldin A–induced Golgi disassembly and reassembly proceeded normally in IER3IP1-deficient cells (Fig. S3 B). To quantify the trafficking delay more precisely, we developed a biochemical pulse–chase variant of the RUSH assay using CLU–SBP–HA (Fig. 4 A), in which newly synthesized CLU can be resolved into an ER-resident core-glycosylated precursor (CLUα/β) and a Golgi-processed mature species (CLUβ). (Fig. S4 A). However, the Golgi-processed CLUβ accumulated poorly at steady state as a result of its rapid secretion within 1 h of biotin addition (Fig. S4 B, C). We therefore monitored the intracellular abundance and turnover of newly synthesized CLUα/β as a quantitative proxy for ER-to-Golgi trafficking efficiency. In WT cells, CLUα/β declined progressively during the biotin chase in a brefeldin A–sensitive manner (Fig. 4 B; Fig. S4 B), confirming its dependence on ER-to-Golgi transport. In contrast, turnover of CLUα/β was delayed in IER3IP1- or EMC-deficient cells (Fig. 4 C), indicating impaired ER export. Maturation of the GPI-anchored prion protein (PrP) was similarly impaired in IER3IP1-deficient cells (Fig. S4 D), suggesting that loss of IER3IP1 affects ER-to-Golgi trafficking of multiple secretory cargos. Re-expression of IER3IP1 restored normal trafficking kinetics not only in ΔIER3IP1 cells but also in ΔEMC cells (Fig. 4 D, E; Fig. S4 E), with concordant rescue effects observed by fluorescence microscopy (Fig. S4 F). Together, these results indicate that efficient ER-to-Golgi transport of selected cargos requires EMC-dependent generation of a functional IER3IP1 pool. The N-terminal TMD of IER3IP1 mediates heteromeric assembly within the ER membrane Given its small size, lack of enzymatic domains, and EMC-dependent biogenesis, we reasoned that IER3IP1 is likely to function through direct physical interactions with other ER-resident membrane proteins rather than acting as an autonomous regulator. Accordingly, we investigated whether IER3IP1 engages in specific physical interactions with other ER-resident membrane proteins. To this end, we employed thiol-specific crosslinking using bismaleimidohexane (BMH), an irreversible homobifunctional reagent that covalently links cysteine residues in close proximity within the membrane (Fig. 5 A). In pulse-labeled cells expressing wild-type IER3IP1, BMH treatment produced a distinct higher–molecular-weight species of approximately 18 kDa, migrating above monomeric IER3IP1 (~ 10 kDa) under reducing SDS–PAGE conditions. This crosslinked product (XLP) was reproducibly detected by both autoradiography and immunoblotting (Fig. 5 B; Fig. S5 A). Notably, substitution of the sole endogenous cysteine within the N-terminal TMD of IER3IP1 (C15A) abolished XLP formation, indicating that this species reflects a cysteine-dependent covalent interaction mediated by the N-terminal TMD. To assess whether the C-terminal TMD of IER3IP1 contributes to intermolecular interactions, we introduced a valine-to-cysteine substitution at position 65 (V65C) on the C15A background to enable BMH-dependent crosslinking within this TMD (Fig. 5 A). Unlike the higher–molecular-weight N-terminal XLP, crosslinking via the C-terminal TMD produced a smaller species consistent with IER3IP1 homodimers (Fig. 5 C). This conclusion was supported by mixed-dimer formation between FLAG- and V5-tagged IER3IP1 variants detected by immunoprecipitation and immunoblotting (Fig. 5 D). Together, these findings indicate that the two TMDs of IER3IP1 mediate distinct interaction modes, with the N-terminal TMD favoring heteromeric interactions and the C-terminal TMD promoting homomerization. Both heteromeric and homomeric crosslinked species were reduced but remained detectable in EMC-deficient cells (Fig. S5 B), indicating that these interactions do not strictly depend on EMC once IER3IP1 reaches the membrane. Because the heteromeric XLP arises from a native cysteine and correlates with trafficking phenotypes, subsequent analyses focused on this N-terminal TMD–dependent interaction. A disease-associated mutation in the N-terminal TMD impairs efficient CLU export To assess the functional relevance of the N-terminal TMD–mediated interaction, we generated a panel of point mutations within the N-terminal transmembrane helix designed to alter its hydrophobicity (Fig. 6 A). Crosslinking analysis showed that all tested substitutions markedly reduced N-terminal XLP formation relative to wild-type IER3IP1 (Fig. 6 B), underscoring the stringent conformational constraints of this intramembrane interaction. Among these variants, we focused on a homozygous alanine-to-valine substitution at position 18 (A18V), a disease-associated mutation linked to microcephaly in humans and mice 20 , 21 . Notably, the A18V variant showed the most pronounced reduction in XLP formation while maintaining normal membrane insertion, as assessed by glycosylation-based topology reporters (Fig. 6 B; Fig. S6 A). Having identified A18V as a separation-of-function variant that selectively disrupts the N-terminal TMD interaction without affecting membrane insertion, we next examined its impact on ER-to-Golgi trafficking. Re-expression of the A18V variant in IER3IP1-deficient cells failed to restore efficient ER-to-Golgi trafficking of newly synthesized CLU, as measured by synchronized RUSH release assays (Fig. 6 C; Fig. S6 C). A similar defect was observed in EMC-deficient cells expressing the A18V variant (Fig. 6 D; Fig. S6 D, S6E), and fluorescence microscopy confirmed persistent ER retention of CLU after cargo release (Fig. 6 E; Fig. S4 F). Together, these results establish that the N-terminal TMD–mediated heteromeric interaction of IER3IP1 is required for efficient CLU export. Collectively, these findings establish that heteromeric assembly of IER3IP1 via its N-terminal TMD enhances the efficiency of early secretory trafficking of CLU, and that disruption of this interaction by engineered or disease-associated mutations compromises cargo export. (Fig. 7 ). Discussion The results presented here establish a framework for understanding how selective defects in ER membrane protein biogenesis can indirectly shape early secretory pathway output. Rather than assigning the ER membrane protein complex (EMC) a direct role in the core machinery of ER export, our findings support a model in which EMC-dependent membrane insertion sustains the abundance and functional competence of a subset of ER-resident membrane proteins that, in turn, promote efficient cargo trafficking. Within this framework, IER3IP1 emerges as a representative EMC-dependent factor whose impaired biogenesis has the potential to give rise to cargo-selective trafficking defects without globally disrupting ER-to-Golgi transport, thereby preserving overall secretory pathway integrity. A central observation of this study is that loss of either EMC components or IER3IP1 selectively impairs ER-to-Golgi transport of newly synthesized cargos such as clusterin (CLU). Importantly, re-expression of IER3IP1 restored trafficking efficiency even under EMC-compromised conditions, indicating that the observed transport defects primarily reflect the loss of functional IER3IP1 rather than a generalized failure of secretory pathway organization. These findings provide functional evidence that EMC activity influences secretory output indirectly, through its effects on specific downstream client proteins, rather than through direct involvement in the vesicle trafficking machinery 7 , 8 . This distinction is conceptually important, as it resolves a key ambiguity in the interpretation of EMC-associated trafficking phenotypes reported in previous studies 11 , 13 , 14 . While EMC dysfunction has been linked to defects in secretion, these effects have often been difficult to reconcile with the well-established role of the EMC as a membrane insertase rather than a canonical trafficking factor. Our data support a parsimonious model in which EMC-dependent insertion is required to maintain a functional pool of regulatory ER membrane proteins that themselves modulate cargo export efficiency. In this framework, defects in secretion arise not from direct perturbation of vesicle formation or trafficking architecture, but from the secondary loss of specific EMC clients whose functions are rate-limiting for the export of selected cargos. In line with prevailing models of ER export, ER exit sites and COPII vesicle formation are generally permissive rather than intrinsically cargo selective. Cargo specificity is primarily imposed at the level of cargo capture and packaging—through Sec24 isoform usage, cargo receptors, or the folding and maturation state of individual cargos—rather than through selective assembly or remodeling of ER exit sites themselves 28 , 29 , 30 . Accordingly, our data do not support a role for IER3IP1 in ER exit site organization or COPII vesicle formation. Rather, they indicate that IER3IP1 modulates the export competence of a subset of cargos upstream of, or parallel to, COPII engagement, thereby influencing ER-to-Golgi trafficking efficiency without directly perturbing the core vesicle biogenesis machinery, as also suggested by independent studies 23 , 24 . The identification of IER3IP1 as such an EMC-dependent trafficking factor was enabled by the application of an ER-targeted TurboID proximity-labeling strategy, which captures transient, low-abundance, or unstable ER membrane proteins in living cells 15 . By operating within the intact ER lumen, this approach overcomes limitations inherent to detergent-based biochemical fractionation, which often leads to the loss of weakly associated or rapidly degraded membrane proteins. The spatial and temporal resolution afforded by ER-TurboID allowed us to distinguish primary consequences of defective membrane insertion from secondary perturbations in secretory proteostasis, facilitating the detection of IER3IP1 as a previously unrecognized EMC client. More broadly, these results highlight the utility of proximity-labeling approaches for uncovering functionally important membrane proteins that may be underrepresented in conventional proteomic datasets due to intrinsic instability. The biophysical properties of IER3IP1 offer a compelling explanation for its reliance on EMC-mediated insertion. IER3IP1 is an unusually compact membrane protein composed of two closely spaced transmembrane domains separated by a minimal cytosolic loop, providing limited extramembrane surface for independent stabilization. Notably, the N-terminal transmembrane domain exhibits marginal hydrophobicity near the threshold for spontaneous membrane insertion 31 . This feature aligns with emerging models in which the EMC preferentially inserts substrates with weakly hydrophobic, short, or topologically constrained transmembrane segments 8 . In the absence of EMC activity, such substrates are prone to misinsertion and rapid degradation 10 . The pronounced instability of IER3IP1 observed in EMC-deficient cells is therefore consistent with its predicted biophysical vulnerability and illustrates how the EMC safeguards the biogenesis of a specific class of membrane proteins. Beyond its dependence on EMC for membrane insertion, our data further indicate that specific intramembrane features of IER3IP1 are critical for its trafficking function. We identify a transmembrane domain–dependent heteromeric interaction mediated by the N-terminal segment, the disruption of which—either by engineered mutations or by a disease-associated variant—impairs efficient ER export of CLU without preventing membrane insertion per se. This separation-of-function phenotype indicates that correct transmembrane domain insertion is necessary but not sufficient for IER3IP1 activity, and that specific intramembrane interactions are required to support its role in secretory trafficking. Functionally, these findings argue against a model in which IER3IP1 acts as a structural organizer of the early secretory pathway or as a direct regulator of vesicle biogenesis. Instead, they are consistent with a mechanism in which IER3IP1 modulates cargo export efficiency indirectly, likely by influencing the folding, maturation, retention, or export competence of selected cargos within the ER. Such a mechanism provides a coherent explanation for why loss of IER3IP1 slows, but does not abolish, ER-to-Golgi transport and why the resulting trafficking defects are cargo selective rather than global. This interpretation is consistent with prior studies linking IER3IP1 to early secretory trafficking. Loss-of-function mutations in IER3IP1 have been shown to cause selective secretion defects across diverse cellular contexts, including neuronal and pancreatic β-cell systems, without inducing a generalized collapse of the secretory pathway 22 , 23 , 32 . In these settings, IER3IP1 deficiency has been associated with impaired secretion of specific cargos and with cellular dysfunction in highly secretory or stress-sensitive cell types. Our observation that IER3IP1 depletion delays ER-to-Golgi transport of both CLU and the GPI-anchored prion protein further supports the idea that IER3IP1 acts on a subset of cargos that share common biophysical or folding constraints, rather than functioning as a universal trafficking factor. Several important questions remain unanswered. A key priority for future work will be the identification of the molecular partners that interact with the IER3IP1 N-terminus and clarification of how these interactions support efficient ER-to-Golgi trafficking. Determining whether such partners are themselves EMC-dependent clients or components of cargo maturation pathways will be essential for defining the molecular logic of IER3IP1 function. An equally important issue concerns the scope of cargos whose ER export depends on IER3IP1. While our data identify folding- and trafficking-sensitive secretory proteins as particularly affected, it remains to be determined which shared biochemical or biogenetic features render certain cargos vulnerable to loss of IER3IP1 activity, and whether this requirement extends beyond the cargos examined here. It will also be important to clarify the cellular context in which IER3IP1 exerts its effects on cargo export, including how its activity is coordinated with broader ER proteostasis networks. More broadly, our findings raise the possibility that additional EMC-dependent membrane proteins modulate secretory pathway output upstream of vesicle formation, not through direct regulation of ER exit sites themselves, but through effects on cargo maturation, stability, or export competence. In this context, the EMC–IER3IP1 axis illustrates how membrane insertion energetics, intramembrane interactions, and protein stability can converge to influence ER-to-Golgi trafficking efficiency. Together, these findings suggest that perturbations in membrane protein biogenesis can give rise to cargo-specific secretion phenotypes without compromising overall secretory pathway integrity. By linking EMC-dependent insertion to the functional availability of a trafficking-modulatory ER membrane protein, this work provides a conceptual framework for understanding how defects in membrane protein biogenesis influence secretory throughput and ER homeostasis, and highlights how early steps in membrane protein integration can exert downstream effects by shaping the composition and function of the ER membrane proteome. Methods Antibodies and Reagents The following antibodies were used in this study: anti-Sec61β, anti-TRAPα, and anti-Flag antibodies were previously described 33 , 34 . Commercial antibodies were obtained as follows: anti-EMC2 (TTC35, 25443-1-AP), anti-EMC4 (TMEM85, 2708-1-AP), anti-EMC7 (C15orf24, 27550-1-AP), and anti-EMC8 (COX4NB, 19889-1-AP) were purchased from Proteintech (Rosemont, IL, USA); anti-EMC10 (C19orf63, ab180148) and anti-IER3IP1 (ab181247) were from abcam (Cambridge, UK), anti-GM130 (A5344) was obtained from ABclonal (Woburn, MA, USA); anti-V5 (R96025) was acquired from Invitrogen (Carlsbad, CA, USA); anti-EMC5 (MMGT1, A305-833A) was purchased from Bethyl Laboratories (Montgomery, TX, USA); high-affinity anti-HA (11867431001) was obtained from Merck KGaA (Darmstadt, Germany); anti-Calnexin (2679), anti-BiP (3177), and anti-Hsp90 (4877) were from Cell Signaling Technology (Danvers, MA, USA); HRP-conjugated streptavidin (N100) was from Thermo Fisher Scientific (Waltham, MA, USA); anti-CD230 (PrP, clone 3F4, 800307) was obtained from BioLegend (San Diego, CA, USA). The following magnetic (Mag) beads were used: anti-HA (88837) and streptavidin (HY-K0208) from Thermo Fisher Scientific; protein A/G (HY-K0202) from MedChemExpress (Monmouth Junction, NJ, USA); V5-Trap (V5tma) from Proteintech; and anti-Flag M2 (M8823) from Sigma-Aldrich Korea (Seoul, South Korea). Endoglycosidase H (Endo H), PNGase F, SP6 RNA polymerase, and all restriction enzymes for molecular cloning were purchased from New England Biolabs (Ipswich, MA, USA). Ribonuclease inhibitor (RNasin) and rabbit reticulocyte lysate (RRL) were obtained from Promega (Madison, WI, USA). Brefeldin A (HY-16592) was from MedChemExpress (Monmouth Junction, NJ, USA). Flag peptide (F3290) and biotin (B4501) were obtained from Sigma-Aldrich Korea (Seoul, South Korea). BMH (bis-maleimidohexane; 22330) was acquired from Thermo Fisher Scientific (Waltham, MA, USA). Cell culture analyses Flp-In™ T-REx™ 293 cells (R78007; Invitrogen) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere containing 5% CO₂. Plasmid transfections were performed using Lipofectamine™ 2000 reagent (11668019; Thermo Fisher Scientific) according to the manufacturer’s instructions. Isogenic stable cell lines expressing TurboID or IER3IP1-V5/FLAG were generated by co-transfection with a target protein expression vector and the pOG44 Flp recombinase vector. Protein expression was driven by a CMV promoter and induced with doxycycline (10 ng/mL) for 15 hours. Knockout cell lines were generated using the CRISPR/Cas9 system. Cells were co-transfected with pSpCas9(BB)-2A-Puro (PX459) V2.0 (a gift from Feng Zhang; Addgene plasmid #62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988) encoding the target sgRNAs. Single-cell clones were isolated by serial dilution in puromycin-containing medium. For rescue experiments, plasmids encoding IER3IP1-V5/FLAG with silent mutations at the sgRNA target sites were used to prevent cleavage by the CRISPR/Cas9 machinery. For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by incubation with the appropriate primary antibodies. Biotinylated proteins, GM130, and TurboID localization were detected by immunostaining. Clusterin localization was visualized via GFP fluorescence. Fluorescence images were acquired using a Zeiss LSM880 microscope equipped with Airyscan (Carl Zeiss, Oberkochen, Germany) and processed using ZEN 3.4 software (Blue edition). Molecular biology Coding sequences of IER3IP1 (GenBank accession no. AF371963), EMC5 (BC033588), clusterin (DQ894623), Sec61β (CR456883), cytochrome b5 (Cb5; CR456990), squalene synthase (SQS; CR457033), and prion protein (PRNP; M13899) were PCR-amplified from human cDNAs prepared from Flp-In T-REx 293 cells and subcloned into the pcDNA5-FRT/TO expression vector (Invitrogen). To generate the ER-TurboID construct, the coding sequence of V5-TurboID was PCR-amplified from V5-TurboID-NES_pcDNA3 (a gift from Alice Ting; Addgene plasmid #107169; http://n2t.net/addgene:107169 ; RRID:Addgene_107169), subcloned into the pcDNA5-FRT/TO vector, and further modified by appending the coding sequence of the N-terminal BiP signal peptide and a C-terminal KDEL retention motif to ensure ER localization. Single-guide RNA (sgRNA) sequences were designed using CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ), and annealed, phosphorylated oligonucleotides were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector. Constructs encoding OPT, Flag, and V5 tags were generated by annealing and phosphorylating synthetic oligonucleotides, followed by insertion at either the N- or C-terminus of the target coding sequences. Mutant constructs were produced using conventional site-directed mutagenesis. RUSH constructs were generated by subcloning the clusterin coding sequence downstream of the IRES in Str-KDEL_ST-SBP-EGFP (a gift from Franck Perez; Addgene plasmid #65264; http://n2t.net/addgene:65264 ; RRID:Addgene_65264). For biochemical RUSH assays, the EGFP coding sequence was replaced with an HA tag. All constructs were verified by Sanger sequencing (Cosmogenetech, Seoul, South Korea). Comparative proximity proteomics For proximity labeling, ER-TurboID–expressing cells were induced with doxycycline (10 ng/mL) for 15 h and subsequently incubated with biotin (50 µM) for 30 min. To remove cytosolic proteins, cells were treated with semi-permeabilization buffer (0.015% digitonin, 110 mM potassium acetate, 20 mM HEPES, and 2 mM magnesium acetate) at 4°C for 10 min. Cells were then lysed in buffer D (50 mM Hepes, pH 7.5, 150 mM NaCl, 5mM MgAc, 1mM DTT, 1xprotease inhibitor) containing 2% digitonin, and biotinylated proteins were enriched using streptavidin magnetic beads. Beads were sequentially washed with the following buffers: twice with buffer D containing 0.2% digitonin (2 min each), 1 M KCl (2 min), 0.1 M Na₂CO₃ (10 s), 2 M urea in 10 mM Tris-HCl (pH 8.0, 10 s), and twice again with buffer D containing 0.2% digitonin (2 min each). Washed beads were resuspended in 2× SDS–PAGE sample buffer containing 2 mM biotin and used for immunoblotting. For on-bead trypsin digestion, bead pellets were resuspended in 5% SDS with 50 mM ammonium bicarbonate, reduced with 20 mM dithiothreitol (10 min, 95°C), and alkylated with 40 mM iodoacetamide (30 min, room temperature, dark). Samples were acidified with 12% phosphoric acid and mixed with S-Trap binding buffer (90% methanol, 100 mM TEAB, pH 7.55). Lysates were loaded onto S-Trap spin columns (ProtiFi) and washed three times with binding buffer. On-column digestion was performed overnight at 37°C using a Trypsin/Lys-C mix (protein:enzyme = 100:1) in 50 mM TEAB 35 . Peptides were sequentially eluted with 50 mM TEAB, 0.2% formic acid, and 0.2% formic acid/50% acetonitrile, dried, and stored at − 80°C. Dried peptides were reconstituted in 0.1% formic acid and analyzed on an Ultimate 3000 RSLC system coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) using previously described LC gradients and DDA settings 36 , 37 . Spectra were searched with SequestHT (Proteome Discoverer 2.4) against the UniProt Homo sapiens database. Search parameters included ± 10 ppm precursor tolerance, 0.02 Da fragment tolerance, cysteine carbamidomethylation (fixed), and N-terminal acetylation and methionine oxidation (variable), allowing two missed cleavages. Peptide spectral matches were filtered to a 1% FDR using Percolator. Label-free quantification was performed using intensities of unique and razor peptides, excluding oxidized methionine–containing peptides. Miscellaneous biochemistry For immunoblotting, cells cultured in 6-well plates were washed with PBS and lysed in buffer K (1% SDS and 100 mM Tris-HCl, pH 7.5). Lysates were denatured by boiling for > 5 min, and proteins were resolved by SDS–PAGE (10% or 12% Tris–Tricine gels) and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk in PBS-T (1× PBS, 0.1% Tween-20) for 30 min, incubated with primary antibodies for 1 h, and then with HRP-conjugated secondary antibodies for 1 h. Detection was performed using SuperSignal™ West Pico PLUS (Thermo Fisher Scientific) and imaged with a C-DiGit Blot Scanner (LI-COR Biosciences). Pulse–chase experiments were performed to monitor IER3IP1 synthesis and cross-linking or to assess ER-to-Golgi trafficking of clusterin (CLU). Cells were seeded in 6-well plates, transfected with the indicated constructs, and induced with doxycycline (10 ng/mL) for 15 h. After starvation for 30 min in methionine/cysteine-free, serum-free medium, cells were pulse-labeled with [³⁵S]-methionine/cysteine (PerkinElmer, Waltham, MA) for 30 min. The chase was carried out in complete medium, and cells were lysed at the indicated time points. For RUSH assays 27 , biotin (40 µM) was added at the onset of the chase to trigger synchronized cargo release. For chemical cross-linking, cells were washed with reaction buffer (1× PBS, 10 mM EDTA) and incubated with 0.2 mM BMH (bis-maleimidohexane) for 1 h at room temperature. After quenching and washing, cells were lysed in buffer K and diluted 10-fold in immunoprecipitation (IP) buffer (1% Triton X-100, 50 mM HEPES, 150 mM NaCl). Lysates were incubated with FLAG-or HA-magnetic beads for 90 min. Beads were washed five times with IP buffer and eluted in 30 µL of 2× SDS–PAGE sample buffer. A 10 µL aliquot of each sample was resolved by SDS–PAGE and analyzed by autoradiography. In vitro synthesis of IER3IP1 was performed as described previously 38 . DNA templates containing the SP6 promoter were amplified by PCR and used for transcription reactions (10 µL total volume) carried out at 37°C for 1 h. Each reaction contained 5–10 ng/µL PCR product, 0.8 U/µL RNasin, 0.4 U/µL SP6 RNA polymerase, 40 mM HEPES, 6 mM MgCl₂, 20 mM spermidine, 10 mM reduced glutathione, 10 mM each NTP, and 0.5 mM m⁷G(5′)ppp(5′)G RNA cap analog. Translation (10 µL total) was carried out in rabbit reticulocyte lysate (RRL; Promega, L4960) supplemented with an amino-acid mix lacking methionine, 0.5 µCi/µL [³⁵S]-methionine, 0.5 µL RNA transcript, and 1 µL canine rough microsomes (gift of Dr. Ramanujan S. Hegde, MRC, UK). Reactions were incubated at 30°C for 30 min, and translation products (0.5 µL) were combined with 10 µL 1× SDS–PAGE sample buffer and analyzed by autoradiography. Glycosidase sensitivity and co-immunoprecipitation assays were conducted as previously described 39 . For protein analysis, cells were lysed in buffer K, denatured by boiling, and resolved by SDS–PAGE using 10% or 12% Tris–Tricine gels. Equal loading was verified by immunoblotting for Sec61β or TRAPα. Detailed experimental parameters are provided in the corresponding figure legends. Quantitation and statistical analysis Autoradiographs of newly synthesized [³⁵S]-labeled clusterin were converted to grayscale images and analyzed using ImageJ software (NIH). Quantitative values represent the mean ± SD obtained from at least three independent biological replicates. Statistical significance was evaluated with two-tailed Student’s t -tests, and significance thresholds were defined as follows: p < 0.05 (* ), p < 0.01 (**), and p < 0.005 (*** ). AI use statement Generative AI tools (ChatGPT, OpenAI; GPT-5.2) were used solely for linguistic refinement of selected sections of the manuscript. No AI tools were used to design experiments, generate or process data, prepare figures, or interpret results related to EMC-dependent membrane protein biogenesis, ER exit site remodeling, or IER3IP1-mediated cargo trafficking. All experimental design, data acquisition, analysis, and scientific interpretation were carried out entirely by the authors. Declarations Acknowledgements We thank the members of the ER Lab for insightful discussions, and the Mass Spectrometry and Confocal Microscopy Core Facilities at the Convergence Medicine Research Center (CREDIT), Asan Medical Center, for their technical support and instrumentation. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-RS-2023-00271127 and NRF-RS-2024-00450920) and in part by the Asan Institute for Life Sciences (2023IL0005, 2024IL0011, and 2025IL0015). Author contributions S.-W.K. conceived and designed experiments, analyzed data, and wrote manuscript. Y.S. performed experiments, analyzed data, and contributed to writing the manuscript. M.S. and K.-S.J. performed some experiments. K.K. and Y.S. provided resources and assisted with data analysis. 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Supplementary Files Fig.S6.jpg Supplementary Figure 6 Fig.S5.jpg Supplementary Figure 5 Fig.S4.jpg Supplementary Figure 4 TableS1.xlsx Supplementary Table 1 TableS2.xlsx Supplementary Table 2 TableS3.xlsx Supplementary Table 3 Fig.S1.jpg Supplementary Figure 1 SIText.docx Supplementary Figure & Table Legend Fig.S2.jpg Supplementary Figure 2 Fig.S3.jpg Supplementary Figure 3 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8560191","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":588436331,"identity":"7d68b3f2-93e0-48a1-b280-208d9acafeae","order_by":0,"name":"Sang-Wook Kang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFACNhBhA+fKMPAQpyUNzuUhVsthErTIz0hL/Fzw67y8bvsB5g8fKg4DtZx9gFeLwY20w9Iz+24bbjuTwCY54wxQC2+7AX4t0ukN0rw9txm33WBgY+ZtA2rhZyPgsNnpzb95e87ZA7Uwf/5LjBaG22nHpHl+HEgEamGQZgRp4W3Dr8Pg/rM0a96G5ORtZxLbJHvOpPOw8Rwj4LCeY8a3ef7Y2W47fvjwhx8V1nL8PGn4tYABI9gpjA1gDiGfQMEf4pSNglEwCkbBCAUAmndClwiMTxcAAAAASUVORK5CYII=","orcid":"","institution":"University of Ulsan","correspondingAuthor":true,"prefix":"","firstName":"Sang-Wook","middleName":"","lastName":"Kang","suffix":""},{"id":588436332,"identity":"a3800a16-798c-4c6b-b6fb-f0c83dc6c88c","order_by":1,"name":"Yejin Shin","email":"","orcid":"","institution":"University of Ulsan","correspondingAuthor":false,"prefix":"","firstName":"Yejin","middleName":"","lastName":"Shin","suffix":""},{"id":588436333,"identity":"f56d4cfa-36d3-4785-b693-71f1bd6b54ef","order_by":2,"name":"Minseok Shin","email":"","orcid":"","institution":"University of Ulsan","correspondingAuthor":false,"prefix":"","firstName":"Minseok","middleName":"","lastName":"Shin","suffix":""},{"id":588436334,"identity":"52292250-986f-47a8-93b7-e11fc890c1c3","order_by":3,"name":"Kang-Sug Jo","email":"","orcid":"","institution":"University of Ulsan","correspondingAuthor":false,"prefix":"","firstName":"Kang-Sug","middleName":"","lastName":"Jo","suffix":""},{"id":588436335,"identity":"561c3978-1658-465f-ba3c-dcc46db3ba5f","order_by":4,"name":"Youngsup Song","email":"","orcid":"","institution":"University of Ulsan","correspondingAuthor":false,"prefix":"","firstName":"Youngsup","middleName":"","lastName":"Song","suffix":""},{"id":588436336,"identity":"464eb939-fee6-4306-b423-6c1f9cd5e3e5","order_by":5,"name":"Kyunggon Kim","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kyunggon","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2026-01-09 11:01:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8560191/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8560191/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102442097,"identity":"4fb51663-ec26-49bd-8284-338e6e96c855","added_by":"auto","created_at":"2026-02-11 17:03:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":300802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of EMC-dependent membrane proteins using ER-targeted TurboID proximity labeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e A doxycycline-inducible, ER-resident TurboID construct (BiP–V5–TurboID–KDEL) was stably integrated into Flp-In T-REx 293 cells. Upon biotin addition, TurboID catalyzed proximity-dependent biotinylation of ER-luminal and membrane-associated proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Cells were induced with doxycycline and labeled with biotin for 0, 10, or 30 min. Whole-cell lysates were analyzed by SDS–PAGE and immunoblotting. Biotinylated species were detected with streptavidin–HRP (SA–HRP), and TurboID expression was verified with anti-V5 antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Biotinylated proteins were enriched using streptavidin-conjugated beads. Input and bound fractions were immunoblotted for ER markers (BiP, CANX, Sec61β) to verify selective labeling of luminally exposed domains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Immunoblotting of wild-type (WT) and EMC5-deficient (ΔE) cells confirmed loss of the EMC complex (EMC4, 5, 7, 8, 10), while unrelated ER proteins (CANX, TRAPα) were unchanged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eStreptavidin-enriched proteins were subjected to LC–MS/MS analysis. The resulting volcano plot displays log₂ fold change (ΔE/WT) versus –log₁₀ p-value, highlighting EMC-dependent ER membrane proteins (red), including IER3IP1. Blue symbols indicate EMC subunits that were selectively diminished in ΔE cells. A representative subset of enriched proteins, together with their abundance ratio (log₂ fold change), sequence coverage, number of unique peptides, and amino acid length, is provided.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/56d47760484c1590a5c42bbb.jpg"},{"id":102746321,"identity":"cefc6ab5-281d-4c38-ae20-352d82e770b4","added_by":"auto","created_at":"2026-02-16 08:56:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":302760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEMC dependence of IER3IP1 membrane integration and topology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u0026nbsp;WT and EMC-deficient (ΔE) cell lysates were immunoblotted for EMC5, IER3IP1, and TRAPα. For metabolic labeling, cells expressing IER3IP1–V5 were pulse-labeled with [³⁵S]-methionine, followed by immunoprecipitation of IER3IP1–V5 to assess the effect of EMC loss on its de novo synthesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;Schematic of the opsin-tag topology assay. The N-terminal opsin tag (\u003cstrong\u003eMNGTEGPNFYVPFSNKTVD\u003c/strong\u003e) contains two N-glycosylation sites (red), enabling detection of luminal exposure. IER3IP1 variants fused to the opsin tag are shown with predicted orientations; red circles indicate potential glycans and yellow circles denote V5 epitopes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u0026nbsp;WT cells expressing the constructs in (B) were pulse-labeled and immunoprecipitated with anti-V5, followed by PNGase F or Endo H digestion. Unglycosylated species (open dot) and the corresponding mobility shifts for singly (filled dot), doubly (double dots), and triply (triple dots) glycosylated forms are indicated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u0026nbsp;WT and ΔE cells expressing OPT-tagged variants were analyzed as in (C). Altered glycosylation in ΔE cells indicated defective EMC-dependent topogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e\u0026nbsp;Apparent free energies of membrane insertion (Δ\u003cem\u003eG\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e) for TMD1 and hydrophobicity-enhanced mutants (S7L, Q10L) were calculated using the ΔG Prediction Server. OPT-tagged variants expressed in WT and ΔE cells were analyzed as in (D) to test whether increased hydrophobicity shifts IER3IP1 insertion from an EMC-dependent to an EMC-independent mode.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/7ff3e9ee67e0e13b8dca6887.jpg"},{"id":102745662,"identity":"009f4706-fd30-48e3-bb4f-2d492bca7a9b","added_by":"auto","created_at":"2026-02-16 08:53:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":241705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization of CLU trafficking using the RUSH system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The CLU–RUSH construct encodes an ER-localized hook (streptavidin–KDEL) and a fluorogenic CLU reporter (CLU–SBP–GFP). In the absence of biotin, CLU–SBP–GFP is captured by streptavidin–KDEL and retained in the ER, whereas biotin addition triggers its synchronized release and ER-to-Golgi transport. Representative images show ER retention (–biotin) and Golgi redistribution (+biotin, 1 h). Arrowheads indicate Golgi-localized CLU. Scale bar, 10 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e WT, ΔEMC, and ΔIER3IP1 cells expressing CLU-RUSH were imaged at 1 h after ± biotin. CLU–SBP–GFP accumulated at the Golgi in WT cells but remained ER-localized in ΔEMC and ΔIER3IP1 cells, indicating dependence on both EMC and IER3IP1 for ER-to-Golgi trafficking. Arrowheads denote Golgi-localized CLU. Scale bar, 10 µm.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/007b8df70b0b787069f63d4f.jpg"},{"id":102442106,"identity":"5b9d06f4-86c5-4130-897e-9d35faa5b80e","added_by":"auto","created_at":"2026-02-11 17:03:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1113996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical RUSH assay to monitor IER3IP1-dependent ER-to-Golgi trafficking of CLU\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic of the RUSH–pulse labeling assay used to biochemically monitor CLU maturation. CLU-RUSH cells expressing CLU–SBP–HA under an IRES promoter were pulse-labeled and chased with ± biotin. Anti-HA immunoprecipitates were analyzed by SDS–PAGE to distinguish ER-resident CLUα/β–SBP–HA from Golgi-processed CLUβ–SBP–HA fragment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Cells were pulse-labeled for 30 min and chased for 0.5–4 h. Autoradiographs were quantified using ImageJ to determine relative levels of CLUα/β and CLUβ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e WT, ΔIER3IP1, and ΔEMC cells were analyzed in parallel, and CLUα/β levels were normalized to the 0 h chase to compare trafficking kinetics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D, E)\u003c/strong\u003e Transient expression of V5-tagged IER3IP1 in ΔIER3IP1 (D) or ΔEMC (E) cells restored CLUα/β processing. Quantification shows mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). Statistical significance was determined by two-tailed Student’s t-test (*p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/252010dee3ac95b0c34844bf.jpg"},{"id":102745626,"identity":"7cd8477e-bceb-436b-bd8a-1bdb961d3f12","added_by":"auto","created_at":"2026-02-16 08:52:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":268326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCysteine-based crosslinking of IER3IP1 through its transmembrane domains (TMDs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Single (C15A or V65C) and double (C15A and V65C) cysteine mutants of IER3IP1–FLAG were generated to probe TMD-dependent interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e WT or C15A IER3IP1–FLAG was pulse-labeled and treated with the thiol crosslinker BMH (0–200 µM, 1 h, RT). Anti-FLAG immunoprecipitates were analyzed by SDS–PAGE and autoradiography. Higher-molecular-weight bands (*) indicate BMH-dependent intermolecular crosslinking.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Single- and double-cysteine mutants were analyzed as in (B). Distinct crosslinked species (N-XL, C-XL) reflected site-specific crosslinking between IER3IP1 and adjacent molecules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e FLAG- or V5-tagged IER3IP1 variants were coexpressed and crosslinked with BMH. Anti-FLAG immunoprecipitates were immunoblotted with anti-FLAG, and anti-V5 antibodies. Co-precipitation of IER3IP1–V5 with IER3IP1–FLAG confirmed C-terminal TMD (V65C)-mediated homomeric association.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/533f8483449234178fe4851a.jpg"},{"id":102745695,"identity":"26fba277-07c4-4848-acff-18843af590e1","added_by":"auto","created_at":"2026-02-16 08:53:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1123843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of the A18V mutation on IER3IP1 heteromeric complex formation and CLU trafficking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u0026nbsp;Point mutations (red) were introduced into N-terminal TMD of IER3IP1, and Δ\u003cem\u003eG\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e values were predicted using the Δ\u003cem\u003eG\u003c/em\u003e Prediction Server. BMH-reactive cysteine residues are indicated in blue. A18V* corresponds to a disease-associated allele.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;WT and mutants, prepared as in (A), were pulse-labeled and crosslinked with BMH. Anti-FLAG immunoprecipitates were analyzed by SDS–PAGE and autoradiography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C, D)\u003c/strong\u003e\u0026nbsp;ΔIER3IP1 (C) and ΔEMC (D) cells coexpressing CLU–SBP–HA and WT or A18V IER3IP1 were pulse-labeled for 30 min and chased for 2 h. Anti-HA immunoprecipitates were analyzed by SDS–PAGE to quantify ER-resident CLUα/β. Data represent mean ± SD (\u003cem\u003en\u003c/em\u003e\u0026nbsp;= 3). Statistical significance was determined by two-tailed Student’s t-test (*p \u0026lt; 0.05; **p \u0026lt; 0.005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e\u0026nbsp;ΔIER3IP1 and ΔEMC cells coexpressing CLU–SBP–GFP and IER3IP1(A18V) were incubated with ± biotin for 1 h and imaged by confocal microscopy. Scale bar, 10 µm.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/8dc55bde21433c720abf10e8.jpg"},{"id":102442101,"identity":"c712eb05-266b-4ffd-ab9f-5983fc7b79df","added_by":"auto","created_at":"2026-02-11 17:03:01","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":160948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorking model for EMC-dependent IER3IP1 function in ER export\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEMC-dependent membrane insertion stabilizes IER3IP1, a compact ER membrane protein with marginally hydrophobic transmembrane domains. Properly inserted IER3IP1 engages in intramembrane interactions required for its trafficking activity. Loss of EMC function or disruption of these interactions impairs the export of selected secretory cargos, exemplified by clusterin (CLU), resulting in delayed ER-to-Golgi transport. IER3IP1 is proposed to modulate cargo export competence upstream of, or parallel to, COPII engagement, without directly regulating ER exit site organization or vesicle biogenesis.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/d83dc6f17e8d78a4f48c63ca.jpg"},{"id":102750554,"identity":"a7e874ca-f3a0-44ea-baac-13a5c8583e3e","added_by":"auto","created_at":"2026-02-16 09:20:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5355667,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/505b5b00-ce2f-4b49-9cae-29133397e2cb.pdf"},{"id":102442099,"identity":"144f99da-33a9-4e98-a5f2-11d744b2bcf0","added_by":"auto","created_at":"2026-02-11 17:03:01","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":857385,"visible":true,"origin":"","legend":"Supplementary Figure 6","description":"","filename":"Fig.S6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/0f47fe6662e38f81f448a629.jpg"},{"id":102442093,"identity":"f1416b3a-d0b1-45f5-bd1e-425f8b6da156","added_by":"auto","created_at":"2026-02-11 17:03:01","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":112118,"visible":true,"origin":"","legend":"Supplementary Figure 5","description":"","filename":"Fig.S5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/4de5fe94702360aa8eab0c72.jpg"},{"id":102746074,"identity":"67cfa418-bf63-4c08-bd6c-a364f65cca60","added_by":"auto","created_at":"2026-02-16 08:55:34","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1093170,"visible":true,"origin":"","legend":"Supplementary Figure 4","description":"","filename":"Fig.S4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/24e05454b2c3a992577ab2c2.jpg"},{"id":102745806,"identity":"b87aa1ed-1e94-43f4-b9a0-a0c0e528da0d","added_by":"auto","created_at":"2026-02-16 08:54:06","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":297865,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/0c290c56710a7b83560547eb.xlsx"},{"id":102746075,"identity":"bd180fef-68f9-4bef-a086-89edee6758f0","added_by":"auto","created_at":"2026-02-16 08:55:34","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":90974,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/5bd7320aa0c7ac13cd071d5a.xlsx"},{"id":102746036,"identity":"925687ae-f835-4d98-b142-a5b19f4f6e6c","added_by":"auto","created_at":"2026-02-16 08:55:20","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13289,"visible":true,"origin":"","legend":"Supplementary Table 3","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/04cf109402b866b2efd5bc0e.xlsx"},{"id":102442108,"identity":"80a290aa-f046-4f21-bd36-bb659733390a","added_by":"auto","created_at":"2026-02-11 17:03:02","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":317099,"visible":true,"origin":"","legend":"Supplementary Figure 1","description":"","filename":"Fig.S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/8b7b610838cc29df471e61e7.jpg"},{"id":102442110,"identity":"8ef73b06-26db-4722-9f4c-0a4c4cfcd716","added_by":"auto","created_at":"2026-02-11 17:03:02","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":33458,"visible":true,"origin":"","legend":"Supplementary Figure \u0026 Table Legend","description":"","filename":"SIText.docx","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/29a8bb284a84c63a22b006ff.docx"},{"id":102745939,"identity":"51bcca15-d416-41c1-8aad-9a713c4b9907","added_by":"auto","created_at":"2026-02-16 08:54:49","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":217257,"visible":true,"origin":"","legend":"Supplementary Figure 2","description":"","filename":"Fig.S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/ae2b7972780149193fe816af.jpg"},{"id":102442109,"identity":"aeaa8443-e103-4a1e-89ac-54080375cb26","added_by":"auto","created_at":"2026-02-11 17:03:02","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":173074,"visible":true,"origin":"","legend":"Supplementary Figure 3","description":"","filename":"Fig.S3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560191/v1/ea90f44ec87e6ec20cb8d1ea.jpg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"EMC-dependent membrane insertion of IER3IP1 sustains efficient ER-to-Golgi trafficking","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe early secretory pathway ensures efficient export of proteins synthesized in the endoplasmic reticulum (ER) toward the Golgi apparatus and downstream compartments \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Proper execution of this pathway requires not only translocation of secretory proteins into the ER lumen but also the coordinated biogenesis and function of ER membrane proteins that support cargo maturation and export \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Perturbations in these processes can lead to selective defects in ER-to-Golgi trafficking and disproportionate stress on folding-sensitive or rapidly exported cargos, thereby compromising cellular proteostasis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMembrane protein biogenesis in the ER is inherently complex, as newly synthesized proteins display diverse topologies and transmembrane domain (TMD) hydrophobicities \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To accommodate this diversity, eukaryotic cells rely on multiple, partially overlapping insertion pathways rather than a single universal mechanism \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While the Sec61 translocon mediates cotranslational insertion of most secretory and type I membrane proteins, substrates with weakly hydrophobic or topologically constrained TMDs require alternative machineries \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Among these, the ER membrane complex (EMC) has emerged as a conserved insertase that supports membrane integration of a discrete subset of ER membrane proteins that are inefficiently handled by Sec61 alone \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExtensive biochemical and genetic studies have established the importance of the EMC in the biogenesis and stability of its client proteins \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Loss of EMC activity destabilizes a specific cohort of membrane proteins, leading to misinsertion and degradation \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and mutations in EMC subunits are associated with neurodevelopmental and metabolic disorders \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Despite these advances, it remains unclear how defects in EMC-dependent insertion translate into downstream cellular phenotypes. In particular, whether EMC dysfunction affects ER-to-Golgi trafficking directly or indirectly through loss of functional client proteins has not been resolved.\u003c/p\u003e \u003cp\u003eSeveral observations suggest that EMC-dependent effects on secretory trafficking may be mediated indirectly, through client proteins that themselves participate in cargo export or quality control. Indeed, impaired anterograde transport of selected cargos has been reported in EMC-deficient contexts, raising the possibility that individual EMC clients act as functional intermediates linking membrane protein biogenesis to trafficking efficiency \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, the identities of such client proteins and the mechanisms by which they influence ER export remain poorly defined.\u003c/p\u003e \u003cp\u003eRecent advances in proximity labeling\u0026ndash;based proteomic approaches have enabled systematic interrogation of membrane protein biogenesis within the ER \u003csup\u003e15, 16\u003c/sup\u003e. By allowing spatially restricted, live-cell labeling of ER-localized proteins, these strategies facilitate identification of membrane proteins whose insertion or stability depends on specific machineries and enable discrimination between primary biogenesis defects and secondary proteostatic consequences \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne such protein is Immediate Early Response 3 Interacting Protein 1 (IER3IP1) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, a small multipass ER membrane protein whose loss-of-function mutations are linked to neurodevelopmental and β-cell survival disorders \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Previous studies have implicated IER3IP1 in ER-to-Golgi trafficking and selective cargo secretion in multiple experimental systems \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, yet its mode of membrane insertion and the relationship between its biogenesis and trafficking function remain unclear.\u003c/p\u003e \u003cp\u003eHere, we investigate how EMC-dependent membrane protein biogenesis influences the early secretory pathway by focusing on IER3IP1, a poorly characterized ER membrane protein previously implicated in ER-to-Golgi trafficking. Using a combination of EMC perturbation, topology-sensitive reporters, and ER-targeted proximity labeling, we examine the requirements for IER3IP1 membrane insertion, stability, and functional competence. By placing IER3IP1 within the context of EMC-dependent protein biogenesis, this work aims to clarify how defects in ER membrane protein insertion can selectively impact secretory pathway output.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eER-TurboID identifies EMC-dependent ER membrane proteins\u003c/h2\u003e \u003cp\u003eTo identify ER-localized proteins whose abundance or membrane association depends on the ER membrane complex (EMC), we employed proximity-dependent biotinylation using TurboID, a promiscuous biotin ligase that labels neighboring proteins in living cells \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To confine labeling to the ER lumen, we engineered a V5-tagged TurboID construct containing an N-terminal BiP signal sequence for ER targeting and a C-terminal KDEL motif for luminal retention (hereafter \u0026ldquo;ER-TurboID\u0026rdquo;). This construct was stably integrated into Flp-In T-REx 293 cells to generate an isogenic, doxycycline-inducible cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon addition of biotin, ER-TurboID rapidly labeled ER-resident proteins, as detected by streptavidin\u0026ndash;HRP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and biotinylated proteins were efficiently recovered on streptavidin resin within 10 min (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Labeling was restricted to the ER lumen, as evidenced by selective biotinylation of luminal chaperones such as BiP and calnexin, whose extended luminal domains are accessible to TurboID (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Indirect immunofluorescence confirmed co-localization of TurboID and biotinylated proteins within the ER network (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Together, these results establish the spatial specificity and temporal responsiveness of ER-TurboID and validate its suitability for interrogating ER-localized proteomes under defined genetic perturbations.\u003c/p\u003e \u003cp\u003eBecause EMC5 is a core subunit whose loss destabilizes the entire EMC complex \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, we compared ER-proximal proteomes between wild-type (WT) cells and EMC5-deficient (ΔEMC) cells. An EMC-null ER-TurboID cell line was generated by CRISPR/Cas9-mediated genome editing, and disruption of EMC integrity was confirmed by immunoblotting for multiple EMC subunits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Functional impairment of EMC activity was further validated using tail-anchored reporters bearing an opsin-based glycosylation tag (OPT), which discriminates between properly inserted and uninserted species; loss of EMC resulted in a marked reduction of the inserted form (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eER-TurboID\u0026ndash;labeled proteins from WT and ΔEMC cells were affinity-purified and quantified by mass spectrometry for comparative analysis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Among the biotinylated proteins identified (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), ER membrane proteins were of particular interest because the EMC contributes to the insertion of a subset of transmembrane domains (TMDs) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. As expected, numerous abundant ER membrane protein biogenesis factors\u0026mdash;including the Sec61 translocon, TRAP, Sec63, TRAM1, the SRP receptor, oligosaccharyltransferase subunits, and additional ER membrane complexes\u0026mdash;were recovered at comparable levels in WT and ΔEMC cells (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), indicating that global ER integrity and luminal labeling efficiency were preserved.\u003c/p\u003e \u003cp\u003eApplying a defined statistical threshold (log₂FC\u0026thinsp;\u0026ge;\u0026thinsp;0.5; p\u0026thinsp;\u0026lt;\u0026thinsp;0.20) (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) revealed reduced recovery of all membrane-integrated EMC subunits and several established EMC client proteins, including squalene synthase and cytochrome b5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE), consistent with impaired insertion or stability upon EMC loss. Beyond these known substrates, several previously unrecognized EMC-dependent proteins were identified. Among them, Immediate Early Response 3 Interacting Protein 1 (IER3IP1) exhibited the most pronounced reduction in ER-proximal recovery upon EMC depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE; Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), nominating it as a strong candidate EMC-dependent ER membrane protein.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEMC is required for efficient insertion and stability of IER3IP1\u003c/h3\u003e\n\u003cp\u003eThe EMC dependence of IER3IP1 was validated by immunoblotting and metabolic pulse-labeling, both of which revealed a marked reduction in newly synthesized IER3IP1 in EMC-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, depletion of IER3IP1 did not alter the abundance of EMC subunits or the membrane insertion of the established EMC client Sec61β (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), indicating that IER3IP1 is not required for EMC assembly or general EMC function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the membrane topology of IER3IP1, we employed an optimized opsin-based glycosylation reporter (OPT) containing two N-glycan acceptor sites that report on luminal exposure only when adjacent TMDs are correctly inserted (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Pulse-labeling combined with endoglycosidase digestion established a reproducible modification pattern in WT cells: both acceptor sites were glycosylated when OPT was fused to the N-terminus, whereas only one site was modified when OPT was fused to the C-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This topology-dependent glycosylation pattern was faithfully recapitulated in a mammalian cell-free translation system supplemented with dog pancreas rough microsomes (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB), providing independent confirmation of the inferred membrane orientation. By contrast, depletion of EMC completely abolished OPT glycosylation at both the N- and C-terminal fusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC), demonstrating that proper insertion of both TMDs of IER3IP1 is strictly dependent on EMC activity.\u003c/p\u003e \u003cp\u003eConsistent with the fate of uninserted membrane proteins \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, IER3IP1 underwent proteasome-dependent degradation when EMC function was compromised (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD), accounting for its reduced steady-state abundance in ΔEMC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Increasing the hydrophobicity of the N-terminal TMD through single-point substitutions (S7L or Q10L) restored insertion and stability of IER3IP1 in EMC-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), indicating that the marginal hydrophobicity of this segment underlies its EMC dependence \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eEMC-dependent IER3IP1 promotes efficient ER-to-Golgi trafficking of clusterin\u003c/h3\u003e\n\u003cp\u003eGiven prior reports linking IER3IP1 to selective secretory trafficking \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we examined whether EMC-dependent biogenesis of IER3IP1 influences ER-to-Golgi transport of clusterin (CLU), a folding-sensitive secretory glycoprotein. To visualize CLU dynamics, we employed the Retention Using Selective Hooks (RUSH) system, which synchronizes cargo release from the ER upon biotin addition \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In WT cells, CLU\u0026ndash;GFP efficiently redistributed from the ER to a perinuclear compartment consistent with the GM130-positive Golgi apparatus within 1 h of biotin addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). By contrast, CLU\u0026ndash;GFP remained largely confined to the ER during the same interval in EMC- or IER3IP1-depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Notably, and in contrast to findings reported by another group \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, this trafficking defect was not attributable to global Golgi dysfunction, as brefeldin A\u0026ndash;induced Golgi disassembly and reassembly proceeded normally in IER3IP1-deficient cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo quantify the trafficking delay more precisely, we developed a biochemical pulse\u0026ndash;chase variant of the RUSH assay using CLU\u0026ndash;SBP\u0026ndash;HA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), in which newly synthesized CLU can be resolved into an ER-resident core-glycosylated precursor (CLUα/β) and a Golgi-processed mature species (CLUβ). (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). However, the Golgi-processed CLUβ accumulated poorly at steady state as a result of its rapid secretion within 1 h of biotin addition (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB, C). We therefore monitored the intracellular abundance and turnover of newly synthesized CLUα/β as a quantitative proxy for ER-to-Golgi trafficking efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn WT cells, CLUα/β declined progressively during the biotin chase in a brefeldin A\u0026ndash;sensitive manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB), confirming its dependence on ER-to-Golgi transport. In contrast, turnover of CLUα/β was delayed in IER3IP1- or EMC-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating impaired ER export. Maturation of the GPI-anchored prion protein (PrP) was similarly impaired in IER3IP1-deficient cells (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eD), suggesting that loss of IER3IP1 affects ER-to-Golgi trafficking of multiple secretory cargos. Re-expression of IER3IP1 restored normal trafficking kinetics not only in ΔIER3IP1 cells but also in ΔEMC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E; Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eE), with concordant rescue effects observed by fluorescence microscopy (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF). Together, these results indicate that efficient ER-to-Golgi transport of selected cargos requires EMC-dependent generation of a functional IER3IP1 pool.\u003c/p\u003e\n\u003ch3\u003eThe N-terminal TMD of IER3IP1 mediates heteromeric assembly within the ER membrane\u003c/h3\u003e\n\u003cp\u003eGiven its small size, lack of enzymatic domains, and EMC-dependent biogenesis, we reasoned that IER3IP1 is likely to function through direct physical interactions with other ER-resident membrane proteins rather than acting as an autonomous regulator. Accordingly, we investigated whether IER3IP1 engages in specific physical interactions with other ER-resident membrane proteins.\u003c/p\u003e \u003cp\u003eTo this end, we employed thiol-specific crosslinking using bismaleimidohexane (BMH), an irreversible homobifunctional reagent that covalently links cysteine residues in close proximity within the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In pulse-labeled cells expressing wild-type IER3IP1, BMH treatment produced a distinct higher\u0026ndash;molecular-weight species of approximately 18 kDa, migrating above monomeric IER3IP1 (~\u0026thinsp;10 kDa) under reducing SDS\u0026ndash;PAGE conditions. This crosslinked product (XLP) was reproducibly detected by both autoradiography and immunoblotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA). Notably, substitution of the sole endogenous cysteine within the N-terminal TMD of IER3IP1 (C15A) abolished XLP formation, indicating that this species reflects a cysteine-dependent covalent interaction mediated by the N-terminal TMD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess whether the C-terminal TMD of IER3IP1 contributes to intermolecular interactions, we introduced a valine-to-cysteine substitution at position 65 (V65C) on the C15A background to enable BMH-dependent crosslinking within this TMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Unlike the higher\u0026ndash;molecular-weight N-terminal XLP, crosslinking via the C-terminal TMD produced a smaller species consistent with IER3IP1 homodimers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This conclusion was supported by mixed-dimer formation between FLAG- and V5-tagged IER3IP1 variants detected by immunoprecipitation and immunoblotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Together, these findings indicate that the two TMDs of IER3IP1 mediate distinct interaction modes, with the N-terminal TMD favoring heteromeric interactions and the C-terminal TMD promoting homomerization.\u003c/p\u003e \u003cp\u003eBoth heteromeric and homomeric crosslinked species were reduced but remained detectable in EMC-deficient cells (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eB), indicating that these interactions do not strictly depend on EMC once IER3IP1 reaches the membrane. Because the heteromeric XLP arises from a native cysteine and correlates with trafficking phenotypes, subsequent analyses focused on this N-terminal TMD\u0026ndash;dependent interaction.\u003c/p\u003e\n\u003ch3\u003eA disease-associated mutation in the N-terminal TMD impairs efficient CLU export\u003c/h3\u003e\n\u003cp\u003eTo assess the functional relevance of the N-terminal TMD\u0026ndash;mediated interaction, we generated a panel of point mutations within the N-terminal transmembrane helix designed to alter its hydrophobicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Crosslinking analysis showed that all tested substitutions markedly reduced N-terminal XLP formation relative to wild-type IER3IP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), underscoring the stringent conformational constraints of this intramembrane interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong these variants, we focused on a homozygous alanine-to-valine substitution at position 18 (A18V), a disease-associated mutation linked to microcephaly in humans and mice \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Notably, the A18V variant showed the most pronounced reduction in XLP formation while maintaining normal membrane insertion, as assessed by glycosylation-based topology reporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB; Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eHaving identified A18V as a separation-of-function variant that selectively disrupts the N-terminal TMD interaction without affecting membrane insertion, we next examined its impact on ER-to-Golgi trafficking. Re-expression of the A18V variant in IER3IP1-deficient cells failed to restore efficient ER-to-Golgi trafficking of newly synthesized CLU, as measured by synchronized RUSH release assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eC). A similar defect was observed in EMC-deficient cells expressing the A18V variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD; Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eD, S6E), and fluorescence microscopy confirmed persistent ER retention of CLU after cargo release (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE; Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF). Together, these results establish that the N-terminal TMD\u0026ndash;mediated heteromeric interaction of IER3IP1 is required for efficient CLU export.\u003c/p\u003e \u003cp\u003eCollectively, these findings establish that heteromeric assembly of IER3IP1 via its N-terminal TMD enhances the efficiency of early secretory trafficking of CLU, and that disruption of this interaction by engineered or disease-associated mutations compromises cargo export. (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results presented here establish a framework for understanding how selective defects in ER membrane protein biogenesis can indirectly shape early secretory pathway output. Rather than assigning the ER membrane protein complex (EMC) a direct role in the core machinery of ER export, our findings support a model in which EMC-dependent membrane insertion sustains the abundance and functional competence of a subset of ER-resident membrane proteins that, in turn, promote efficient cargo trafficking. Within this framework, IER3IP1 emerges as a representative EMC-dependent factor whose impaired biogenesis has the potential to give rise to cargo-selective trafficking defects without globally disrupting ER-to-Golgi transport, thereby preserving overall secretory pathway integrity.\u003c/p\u003e \u003cp\u003eA central observation of this study is that loss of either EMC components or IER3IP1 selectively impairs ER-to-Golgi transport of newly synthesized cargos such as clusterin (CLU). Importantly, re-expression of IER3IP1 restored trafficking efficiency even under EMC-compromised conditions, indicating that the observed transport defects primarily reflect the loss of functional IER3IP1 rather than a generalized failure of secretory pathway organization. These findings provide functional evidence that EMC activity influences secretory output indirectly, through its effects on specific downstream client proteins, rather than through direct involvement in the vesicle trafficking machinery \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis distinction is conceptually important, as it resolves a key ambiguity in the interpretation of EMC-associated trafficking phenotypes reported in previous studies \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. While EMC dysfunction has been linked to defects in secretion, these effects have often been difficult to reconcile with the well-established role of the EMC as a membrane insertase rather than a canonical trafficking factor. Our data support a parsimonious model in which EMC-dependent insertion is required to maintain a functional pool of regulatory ER membrane proteins that themselves modulate cargo export efficiency. In this framework, defects in secretion arise not from direct perturbation of vesicle formation or trafficking architecture, but from the secondary loss of specific EMC clients whose functions are rate-limiting for the export of selected cargos.\u003c/p\u003e \u003cp\u003eIn line with prevailing models of ER export, ER exit sites and COPII vesicle formation are generally permissive rather than intrinsically cargo selective. Cargo specificity is primarily imposed at the level of cargo capture and packaging\u0026mdash;through Sec24 isoform usage, cargo receptors, or the folding and maturation state of individual cargos\u0026mdash;rather than through selective assembly or remodeling of ER exit sites themselves \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Accordingly, our data do not support a role for IER3IP1 in ER exit site organization or COPII vesicle formation. Rather, they indicate that IER3IP1 modulates the export competence of a subset of cargos upstream of, or parallel to, COPII engagement, thereby influencing ER-to-Golgi trafficking efficiency without directly perturbing the core vesicle biogenesis machinery, as also suggested by independent studies \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe identification of IER3IP1 as such an EMC-dependent trafficking factor was enabled by the application of an ER-targeted TurboID proximity-labeling strategy, which captures transient, low-abundance, or unstable ER membrane proteins in living cells \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. By operating within the intact ER lumen, this approach overcomes limitations inherent to detergent-based biochemical fractionation, which often leads to the loss of weakly associated or rapidly degraded membrane proteins. The spatial and temporal resolution afforded by ER-TurboID allowed us to distinguish primary consequences of defective membrane insertion from secondary perturbations in secretory proteostasis, facilitating the detection of IER3IP1 as a previously unrecognized EMC client. More broadly, these results highlight the utility of proximity-labeling approaches for uncovering functionally important membrane proteins that may be underrepresented in conventional proteomic datasets due to intrinsic instability.\u003c/p\u003e \u003cp\u003eThe biophysical properties of IER3IP1 offer a compelling explanation for its reliance on EMC-mediated insertion. IER3IP1 is an unusually compact membrane protein composed of two closely spaced transmembrane domains separated by a minimal cytosolic loop, providing limited extramembrane surface for independent stabilization. Notably, the N-terminal transmembrane domain exhibits marginal hydrophobicity near the threshold for spontaneous membrane insertion \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This feature aligns with emerging models in which the EMC preferentially inserts substrates with weakly hydrophobic, short, or topologically constrained transmembrane segments \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In the absence of EMC activity, such substrates are prone to misinsertion and rapid degradation \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The pronounced instability of IER3IP1 observed in EMC-deficient cells is therefore consistent with its predicted biophysical vulnerability and illustrates how the EMC safeguards the biogenesis of a specific class of membrane proteins.\u003c/p\u003e \u003cp\u003eBeyond its dependence on EMC for membrane insertion, our data further indicate that specific intramembrane features of IER3IP1 are critical for its trafficking function. We identify a transmembrane domain\u0026ndash;dependent heteromeric interaction mediated by the N-terminal segment, the disruption of which\u0026mdash;either by engineered mutations or by a disease-associated variant\u0026mdash;impairs efficient ER export of CLU without preventing membrane insertion per se. This separation-of-function phenotype indicates that correct transmembrane domain insertion is necessary but not sufficient for IER3IP1 activity, and that specific intramembrane interactions are required to support its role in secretory trafficking.\u003c/p\u003e \u003cp\u003eFunctionally, these findings argue against a model in which IER3IP1 acts as a structural organizer of the early secretory pathway or as a direct regulator of vesicle biogenesis. Instead, they are consistent with a mechanism in which IER3IP1 modulates cargo export efficiency indirectly, likely by influencing the folding, maturation, retention, or export competence of selected cargos within the ER. Such a mechanism provides a coherent explanation for why loss of IER3IP1 slows, but does not abolish, ER-to-Golgi transport and why the resulting trafficking defects are cargo selective rather than global.\u003c/p\u003e \u003cp\u003eThis interpretation is consistent with prior studies linking IER3IP1 to early secretory trafficking. Loss-of-function mutations in IER3IP1 have been shown to cause selective secretion defects across diverse cellular contexts, including neuronal and pancreatic β-cell systems, without inducing a generalized collapse of the secretory pathway \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In these settings, IER3IP1 deficiency has been associated with impaired secretion of specific cargos and with cellular dysfunction in highly secretory or stress-sensitive cell types. Our observation that IER3IP1 depletion delays ER-to-Golgi transport of both CLU and the GPI-anchored prion protein further supports the idea that IER3IP1 acts on a subset of cargos that share common biophysical or folding constraints, rather than functioning as a universal trafficking factor.\u003c/p\u003e \u003cp\u003eSeveral important questions remain unanswered. A key priority for future work will be the identification of the molecular partners that interact with the IER3IP1 N-terminus and clarification of how these interactions support efficient ER-to-Golgi trafficking. Determining whether such partners are themselves EMC-dependent clients or components of cargo maturation pathways will be essential for defining the molecular logic of IER3IP1 function. An equally important issue concerns the scope of cargos whose ER export depends on IER3IP1. While our data identify folding- and trafficking-sensitive secretory proteins as particularly affected, it remains to be determined which shared biochemical or biogenetic features render certain cargos vulnerable to loss of IER3IP1 activity, and whether this requirement extends beyond the cargos examined here. It will also be important to clarify the cellular context in which IER3IP1 exerts its effects on cargo export, including how its activity is coordinated with broader ER proteostasis networks.\u003c/p\u003e \u003cp\u003eMore broadly, our findings raise the possibility that additional EMC-dependent membrane proteins modulate secretory pathway output upstream of vesicle formation, not through direct regulation of ER exit sites themselves, but through effects on cargo maturation, stability, or export competence. In this context, the EMC\u0026ndash;IER3IP1 axis illustrates how membrane insertion energetics, intramembrane interactions, and protein stability can converge to influence ER-to-Golgi trafficking efficiency.\u003c/p\u003e \u003cp\u003eTogether, these findings suggest that perturbations in membrane protein biogenesis can give rise to cargo-specific secretion phenotypes without compromising overall secretory pathway integrity. By linking EMC-dependent insertion to the functional availability of a trafficking-modulatory ER membrane protein, this work provides a conceptual framework for understanding how defects in membrane protein biogenesis influence secretory throughput and ER homeostasis, and highlights how early steps in membrane protein integration can exert downstream effects by shaping the composition and function of the ER membrane proteome.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and Reagents\u003c/h2\u003e \u003cp\u003eThe following antibodies were used in this study: anti-Sec61β, anti-TRAPα, and anti-Flag antibodies were previously described \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Commercial antibodies were obtained as follows: anti-EMC2 (TTC35, 25443-1-AP), anti-EMC4 (TMEM85, 2708-1-AP), anti-EMC7 (C15orf24, 27550-1-AP), and anti-EMC8 (COX4NB, 19889-1-AP) were purchased from Proteintech (Rosemont, IL, USA); anti-EMC10 (C19orf63, ab180148) and anti-IER3IP1 (ab181247) were from abcam (Cambridge, UK), anti-GM130 (A5344) was obtained from ABclonal (Woburn, MA, USA); anti-V5 (R96025) was acquired from Invitrogen (Carlsbad, CA, USA); anti-EMC5 (MMGT1, A305-833A) was purchased from Bethyl Laboratories (Montgomery, TX, USA); high-affinity anti-HA (11867431001) was obtained from Merck KGaA (Darmstadt, Germany); anti-Calnexin (2679), anti-BiP (3177), and anti-Hsp90 (4877) were from Cell Signaling Technology (Danvers, MA, USA); HRP-conjugated streptavidin (N100) was from Thermo Fisher Scientific (Waltham, MA, USA); anti-CD230 (PrP, clone 3F4, 800307) was obtained from BioLegend (San Diego, CA, USA). The following magnetic (Mag) beads were used: anti-HA (88837) and streptavidin (HY-K0208) from Thermo Fisher Scientific; protein A/G (HY-K0202) from MedChemExpress (Monmouth Junction, NJ, USA); V5-Trap (V5tma) from Proteintech; and anti-Flag M2 (M8823) from Sigma-Aldrich Korea (Seoul, South Korea).\u003c/p\u003e \u003cp\u003eEndoglycosidase H (Endo H), PNGase F, SP6 RNA polymerase, and all restriction enzymes for molecular cloning were purchased from New England Biolabs (Ipswich, MA, USA). Ribonuclease inhibitor (RNasin) and rabbit reticulocyte lysate (RRL) were obtained from Promega (Madison, WI, USA). Brefeldin A (HY-16592) was from MedChemExpress (Monmouth Junction, NJ, USA). Flag peptide (F3290) and biotin (B4501) were obtained from Sigma-Aldrich Korea (Seoul, South Korea). BMH (bis-maleimidohexane; 22330) was acquired from Thermo Fisher Scientific (Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell culture analyses\u003c/h2\u003e \u003cp\u003eFlp-In\u0026trade; T-REx\u0026trade; 293 cells (R78007; Invitrogen) were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. Plasmid transfections were performed using Lipofectamine\u0026trade; 2000 reagent (11668019; Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. Isogenic stable cell lines expressing TurboID or IER3IP1-V5/FLAG were generated by co-transfection with a target protein expression vector and the pOG44 Flp recombinase vector. Protein expression was driven by a CMV promoter and induced with doxycycline (10 ng/mL) for 15 hours. Knockout cell lines were generated using the CRISPR/Cas9 system. Cells were co-transfected with pSpCas9(BB)-2A-Puro (PX459) V2.0 (a gift from Feng Zhang; Addgene plasmid #62988; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:62988\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:62988\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:Addgene_62988) encoding the target sgRNAs. Single-cell clones were isolated by serial dilution in puromycin-containing medium. For rescue experiments, plasmids encoding IER3IP1-V5/FLAG with silent mutations at the sgRNA target sites were used to prevent cleavage by the CRISPR/Cas9 machinery.\u003c/p\u003e \u003cp\u003eFor immunofluorescence analysis, cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by incubation with the appropriate primary antibodies. Biotinylated proteins, GM130, and TurboID localization were detected by immunostaining. Clusterin localization was visualized via GFP fluorescence. Fluorescence images were acquired using a Zeiss LSM880 microscope equipped with Airyscan (Carl Zeiss, Oberkochen, Germany) and processed using ZEN 3.4 software (Blue edition).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMolecular biology\u003c/h2\u003e \u003cp\u003eCoding sequences of IER3IP1 (GenBank accession no. AF371963), EMC5 (BC033588), clusterin (DQ894623), Sec61β (CR456883), cytochrome b5 (Cb5; CR456990), squalene synthase (SQS; CR457033), and prion protein (PRNP; M13899) were PCR-amplified from human cDNAs prepared from Flp-In T-REx 293 cells and subcloned into the pcDNA5-FRT/TO expression vector (Invitrogen). To generate the ER-TurboID construct, the coding sequence of V5-TurboID was PCR-amplified from V5-TurboID-NES_pcDNA3 (a gift from Alice Ting; Addgene plasmid #107169; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:107169\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:107169\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:Addgene_107169), subcloned into the pcDNA5-FRT/TO vector, and further modified by appending the coding sequence of the N-terminal BiP signal peptide and a C-terminal KDEL retention motif to ensure ER localization. Single-guide RNA (sgRNA) sequences were designed using CRISPick (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portals.broadinstitute.org/gppx/crispick/public\u003c/span\u003e\u003cspan address=\"https://portals.broadinstitute.org/gppx/crispick/public\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and annealed, phosphorylated oligonucleotides were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector. Constructs encoding OPT, Flag, and V5 tags were generated by annealing and phosphorylating synthetic oligonucleotides, followed by insertion at either the N- or C-terminus of the target coding sequences. Mutant constructs were produced using conventional site-directed mutagenesis. RUSH constructs were generated by subcloning the clusterin coding sequence downstream of the IRES in Str-KDEL_ST-SBP-EGFP (a gift from Franck Perez; Addgene plasmid #65264; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:65264\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:65264\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:Addgene_65264). For biochemical RUSH assays, the EGFP coding sequence was replaced with an HA tag. All constructs were verified by Sanger sequencing (Cosmogenetech, Seoul, South Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComparative proximity proteomics\u003c/h2\u003e \u003cp\u003eFor proximity labeling, ER-TurboID\u0026ndash;expressing cells were induced with doxycycline (10 ng/mL) for 15 h and subsequently incubated with biotin (50 \u0026micro;M) for 30 min. To remove cytosolic proteins, cells were treated with semi-permeabilization buffer (0.015% digitonin, 110 mM potassium acetate, 20 mM HEPES, and 2 mM magnesium acetate) at 4\u0026deg;C for 10 min. Cells were then lysed in buffer D (50 mM Hepes, pH 7.5, 150 mM NaCl, 5mM MgAc, 1mM DTT, 1xprotease inhibitor) containing 2% digitonin, and biotinylated proteins were enriched using streptavidin magnetic beads. Beads were sequentially washed with the following buffers: twice with buffer D containing 0.2% digitonin (2 min each), 1 M KCl (2 min), 0.1 M Na₂CO₃ (10 s), 2 M urea in 10 mM Tris-HCl (pH 8.0, 10 s), and twice again with buffer D containing 0.2% digitonin (2 min each). Washed beads were resuspended in 2\u0026times; SDS\u0026ndash;PAGE sample buffer containing 2 mM biotin and used for immunoblotting.\u003c/p\u003e \u003cp\u003eFor on-bead trypsin digestion, bead pellets were resuspended in 5% SDS with 50 mM ammonium bicarbonate, reduced with 20 mM dithiothreitol (10 min, 95\u0026deg;C), and alkylated with 40 mM iodoacetamide (30 min, room temperature, dark). Samples were acidified with 12% phosphoric acid and mixed with S-Trap binding buffer (90% methanol, 100 mM TEAB, pH 7.55). Lysates were loaded onto S-Trap spin columns (ProtiFi) and washed three times with binding buffer. On-column digestion was performed overnight at 37\u0026deg;C using a Trypsin/Lys-C mix (protein:enzyme\u0026thinsp;=\u0026thinsp;100:1) in 50 mM TEAB \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Peptides were sequentially eluted with 50 mM TEAB, 0.2% formic acid, and 0.2% formic acid/50% acetonitrile, dried, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003eDried peptides were reconstituted in 0.1% formic acid and analyzed on an Ultimate 3000 RSLC system coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) using previously described LC gradients and DDA settings \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Spectra were searched with SequestHT (Proteome Discoverer 2.4) against the UniProt Homo sapiens database. Search parameters included\u0026thinsp;\u0026plusmn;\u0026thinsp;10 ppm precursor tolerance, 0.02 Da fragment tolerance, cysteine carbamidomethylation (fixed), and N-terminal acetylation and methionine oxidation (variable), allowing two missed cleavages. Peptide spectral matches were filtered to a 1% FDR using Percolator. Label-free quantification was performed using intensities of unique and razor peptides, excluding oxidized methionine\u0026ndash;containing peptides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMiscellaneous biochemistry\u003c/h2\u003e \u003cp\u003eFor immunoblotting, cells cultured in 6-well plates were washed with PBS and lysed in buffer K (1% SDS and 100 mM Tris-HCl, pH 7.5). Lysates were denatured by boiling for \u0026gt;\u0026thinsp;5 min, and proteins were resolved by SDS\u0026ndash;PAGE (10% or 12% Tris\u0026ndash;Tricine gels) and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk in PBS-T (1\u0026times; PBS, 0.1% Tween-20) for 30 min, incubated with primary antibodies for 1 h, and then with HRP-conjugated secondary antibodies for 1 h. Detection was performed using SuperSignal\u0026trade; West Pico PLUS (Thermo Fisher Scientific) and imaged with a C-DiGit Blot Scanner (LI-COR Biosciences).\u003c/p\u003e \u003cp\u003ePulse\u0026ndash;chase experiments were performed to monitor IER3IP1 synthesis and cross-linking or to assess ER-to-Golgi trafficking of clusterin (CLU). Cells were seeded in 6-well plates, transfected with the indicated constructs, and induced with doxycycline (10 ng/mL) for 15 h. After starvation for 30 min in methionine/cysteine-free, serum-free medium, cells were pulse-labeled with [\u0026sup3;⁵S]-methionine/cysteine (PerkinElmer, Waltham, MA) for 30 min. The chase was carried out in complete medium, and cells were lysed at the indicated time points.\u003c/p\u003e \u003cp\u003eFor RUSH assays \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, biotin (40 \u0026micro;M) was added at the onset of the chase to trigger synchronized cargo release. For chemical cross-linking, cells were washed with reaction buffer (1\u0026times; PBS, 10 mM EDTA) and incubated with 0.2 mM BMH (bis-maleimidohexane) for 1 h at room temperature. After quenching and washing, cells were lysed in buffer K and diluted 10-fold in immunoprecipitation (IP) buffer (1% Triton X-100, 50 mM HEPES, 150 mM NaCl). Lysates were incubated with FLAG-or HA-magnetic beads for 90 min. Beads were washed five times with IP buffer and eluted in 30 \u0026micro;L of 2\u0026times; SDS\u0026ndash;PAGE sample buffer. A 10 \u0026micro;L aliquot of each sample was resolved by SDS\u0026ndash;PAGE and analyzed by autoradiography.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e synthesis of IER3IP1 was performed as described previously \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. DNA templates containing the SP6 promoter were amplified by PCR and used for transcription reactions (10 \u0026micro;L total volume) carried out at 37\u0026deg;C for 1 h. Each reaction contained 5\u0026ndash;10 ng/\u0026micro;L PCR product, 0.8 U/\u0026micro;L RNasin, 0.4 U/\u0026micro;L SP6 RNA polymerase, 40 mM HEPES, 6 mM MgCl₂, 20 mM spermidine, 10 mM reduced glutathione, 10 mM each NTP, and 0.5 mM m⁷G(5\u0026prime;)ppp(5\u0026prime;)G RNA cap analog. Translation (10 \u0026micro;L total) was carried out in rabbit reticulocyte lysate (RRL; Promega, L4960) supplemented with an amino-acid mix lacking methionine, 0.5 \u0026micro;Ci/\u0026micro;L [\u0026sup3;⁵S]-methionine, 0.5 \u0026micro;L RNA transcript, and 1 \u0026micro;L canine rough microsomes (gift of Dr. Ramanujan S. Hegde, MRC, UK). Reactions were incubated at 30\u0026deg;C for 30 min, and translation products (0.5 \u0026micro;L) were combined with 10 \u0026micro;L 1\u0026times; SDS\u0026ndash;PAGE sample buffer and analyzed by autoradiography.\u003c/p\u003e \u003cp\u003eGlycosidase sensitivity and co-immunoprecipitation assays were conducted as previously described \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. For protein analysis, cells were lysed in buffer K, denatured by boiling, and resolved by SDS\u0026ndash;PAGE using 10% or 12% Tris\u0026ndash;Tricine gels. Equal loading was verified by immunoblotting for Sec61β or TRAPα. Detailed experimental parameters are provided in the corresponding figure legends.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQuantitation and statistical analysis\u003c/h2\u003e \u003cp\u003eAutoradiographs of newly synthesized [\u0026sup3;⁵S]-labeled clusterin were converted to grayscale images and analyzed using ImageJ software (NIH). Quantitative values represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD obtained from at least three independent biological replicates. Statistical significance was evaluated with two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests, and significance thresholds were defined as follows: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*\u003cem\u003e), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 (***\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAI use statement\u003c/h2\u003e \u003cp\u003eGenerative AI tools (ChatGPT, OpenAI; GPT-5.2) were used solely for linguistic refinement of selected sections of the manuscript. No AI tools were used to design experiments, generate or process data, prepare figures, or interpret results related to EMC-dependent membrane protein biogenesis, ER exit site remodeling, or IER3IP1-mediated cargo trafficking. All experimental design, data acquisition, analysis, and scientific interpretation were carried out entirely by the authors.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the members of the ER Lab for insightful discussions, and the Mass Spectrometry and Confocal Microscopy Core Facilities at the Convergence Medicine Research Center (CREDIT), Asan Medical Center, for their technical support and instrumentation. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-RS-2023-00271127 and NRF-RS-2024-00450920) and in part by the Asan Institute for Life Sciences (2023IL0005, 2024IL0011, and 2025IL0015). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.-W.K. conceived and designed experiments, analyzed data, and wrote manuscript. Y.S. performed experiments, analyzed data, and contributed to writing the manuscript. M.S. and K.-S.J. performed some experiments. K.K. and Y.S. provided resources and assisted with data analysis.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no competing interests exist.\u003c/p\u003e\n\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarlowe, C.K., Miller, E.A.: Secretory protein biogenesis and traffic in the early secretory pathway. Genetics. \u003cb\u003e193\u003c/b\u003e, 383\u0026ndash;410 (2013)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaffer, K.L., Sharma, A., Snapp, E.L., Hegde, R.S.: Regulation of protein compartmentalization expands the diversity of protein function. Dev. Cell. \u003cb\u003e9\u003c/b\u003e, 545\u0026ndash;554 (2005)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHegde, R.S., Keenan, R.J.: The mechanisms of integral membrane protein biogenesis. Nat. Rev. Mol. Cell. 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Redox Biol. \u003cb\u003e56\u003c/b\u003e, 102456 (2022)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ER membrane complex (EMC), IER3IP1, Membrane protein insertion, Early secretory pathway, Secretory protein export, Clusterin","lastPublishedDoi":"10.21203/rs.3.rs-8560191/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8560191/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe endoplasmic reticulum (ER) membrane complex (EMC) acts as an insertase for a distinct subset of membrane proteins, yet how defects in EMC-dependent membrane insertion propagate to downstream secretory pathway function remains poorly understood. Here, we identify Immediate Early Response 3 Interacting Protein 1 (IER3IP1) as an EMC-dependent ER membrane protein whose stable biogenesis is required to sustain efficient ER-to-Golgi trafficking of newly synthesized secretory cargos. Loss of EMC activity destabilizes IER3IP1, resulting in delayed ER export of folding- and trafficking-sensitive proteins, including clusterin and the prion protein, without overt disruption of global ER structure or protein synthesis. We further show that membrane insertion alone is insufficient for IER3IP1 function: a transmembrane domain\u0026ndash;dependent intramembrane interaction mediated by the N-terminal segment is required to support cargo export competence. Together, these findings illustrate how defects in membrane protein biogenesis can indirectly constrain secretory pathway output through loss of specific EMC-dependent membrane proteins, establishing a mechanistic link between membrane insertion efficiency, protein stability, and ER export.\u003c/p\u003e","manuscriptTitle":"EMC-dependent membrane insertion of IER3IP1 sustains efficient ER-to-Golgi trafficking","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 17:02:50","doi":"10.21203/rs.3.rs-8560191/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5532e927-265b-4e9b-90dc-aff9698c360a","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62603942,"name":"Biological sciences/Biochemistry/Proteins/Membrane proteins"},{"id":62603943,"name":"Biological sciences/Molecular biology/Protein folding/Endoplasmic reticulum"},{"id":62603944,"name":"Biological sciences/Cell biology/Organelles/Endoplasmic reticulum"},{"id":62603945,"name":"Biological sciences/Cell biology/Protein transport/Protein translocation"}],"tags":[],"updatedAt":"2026-03-18T11:35:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 17:02:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8560191","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8560191","identity":"rs-8560191","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}