Neuronal endolysosomal acidification relies on interactions between transmembrane protein 184B (TMEM184B) and the vesicular proton pump

Neuronal endolysosomal acidification relies on interactions between transmembrane protein 184B (TMEM184B) and the vesicular proton pump | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Neuronal endolysosomal acidification relies on interactions between transmembrane protein 184B (TMEM184B) and the vesicular proton pump View ORCID Profile Elizabeth B. Wright , Erik G. Larsen , Marco Padilla-Rodriguez , Paul R. Langlais , View ORCID Profile Martha R.C. Bhattacharya doi: https://doi.org/10.1101/2025.02.01.635992 Elizabeth B. Wright 1 Department of Neuroscience , 1040 E 4th Street, Tucson, Arizona 85721, USA and Graduate Interdisciplinary Program in Neuroscience, University of Arizona Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elizabeth B. Wright Erik G. Larsen 1 Department of Neuroscience , 1040 E 4th Street, Tucson, Arizona 85721, USA and Graduate Interdisciplinary Program in Neuroscience, University of Arizona Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marco Padilla-Rodriguez 2 University of Arizona Cancer Center , 1515 N Campbell Ave, Tucson, AZ 85724, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paul R. Langlais 3 Department of Medicine , 1501 N. Campbell Ave, Tucson, AZ 85724, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Martha R.C. Bhattacharya 1 Department of Neuroscience , 1040 E 4th Street, Tucson, Arizona 85721, USA and Graduate Interdisciplinary Program in Neuroscience, University of Arizona Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martha R.C. Bhattacharya For correspondence: marthab1{at}arizona.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Disruption of endolysosomal acidification is a hallmark of several neurodevelopmental and neurodegenerative disorders. Impaired acidification causes accumulation of toxic protein aggregates and disrupts neuronal homeostasis, yet the molecular mechanisms regulating endolysosomal pH in neurons remain poorly understood. A critical regulator of lumenal acidification is the vacuolar ATPase (V-ATPase), a proton pump whose activity depends on dynamic assembly of its V0 and V1 subdomains. In this study, we identify transmembrane protein 184B (TMEM184B) as a novel regulator of endolysosomal acidification in neurons. TMEM184B is an evolutionarily conserved 7-pass transmembrane protein required for synaptic structure and function, and sequence variation in TMEM184B causes neurodevelopmental disorders, but the mechanism for this effect is unknown. We performed proteomic analysis of TMEM184B-interacting proteins and identified enrichment of components involved in endosomal trafficking and function, including the V-ATPase. TMEM184B localizes to early and late endosomes, further supporting a role in the endosomal system. Loss of TMEM184B results in significant reductions in endolysosomal acidification within cultured mouse cortical neurons. This alteration in pH is associated with impaired assembly of the V-ATPase V0 and V1 subcomplexes in the TMEM184B mutant mouse brain, suggesting a mechanism by which TMEM184B promotes flux through the endosomal pathway. Overall, these findings identify a new contributor in maintaining endosomal function and provide a mechanistic basis for disrupted neuronal function in human TMEM184B-associated nervous system disorders. Significance Statement Endolysosomal acidification is essential for neuronal protein homeostasis, yet its regulation in neurons remains poorly understood. Here, we identify TMEM184B as a key regulator of this process, establishing its first known cellular role. We show that TMEM184B interacts with vacuolar ATPase (V-ATPase) components and promotes the assembly of its V0 and V1 subdomains, facilitating lumenal acidification. Loss of TMEM184B disrupts endolysosomal pH in neurons, potentially impairing proteostasis. These findings reveal a critical function for TMEM184B in neuronal maintenance and provide mechanistic insight into its link to neurological disorders. This work advances our understanding of endolysosomal regulation and suggests TMEM184B regulation could improve outcomes in diseases involving lysosomal dysfunction. Introduction Decreases in endolysosomal acidification contribute to cellular dysfunction in many neurological diseases. Cellular models of Lysosomal Storage Disorders (LSDs), Alzheimer’s Disease (AD), Parkinson’s Disease (PD), and Amyotrophic Lateral Sclerosis (ALS) all exhibit endolysosomal deacidification that causes the accumulation of toxic proteins (Arbo et al., 2020; Im et al., 2023; Hu et al., 2015 ; Lee et al., 2022 ; Lo & Zeng, 2023 ; Nixon & Rubinsztein, 2024 ; Root et al., 2021 ). In another disorder, Nieman Pick Type C Disease (NPCD), abnormally high pH in lysosomes causes failed trafficking of cholesterol cargos and neuron death ( Lloyd-Evans et al., 2008 ; Vivas et al., 2019 ). Enlarged endosomal compartments or accumulation of autophagic vesicles in neurons occurs frequently in neurodegenerative diseases, implying that cargo delivery or degradation is impaired ( Cataldo et al., 1996 , 2000 ; Root et al., 2021 ). How endolysosomal deacidification initially occurs, and the mechanistic link between deacidification and disease pathogenesis, is not fully understood. Endosomal trafficking relies on lumenal acidification mediated by the vesicular proton pump (V-ATPase). The V-ATPase consists of two subdomain complexes: the membrane-embedded V0 complex and the cytoplasmic V1 complex ( Cotter et al., 2015 ; Forgac, 2007 ; Toei et al., 2010 ). When assembled, the V1 complex hydrolyzes ATP, driving the translocation of protons through a cytoplasmic hemichannel within the V0a subunit into the lumen. This proton transport gradually reduces the lumenal pH, facilitating compartment progression through the endolysosomal pathway. The acidification of compartments is tightly regulated by the cell through dynamic assembly and disassembly of the V-ATPase in response to various factors, including amino acids, cholesterol, and intracellular signaling pathways ( Colacurcio & Nixon, 2016 ; Hurtado-Lorenzo et al., 2006 ; Kane, n.d.; Ratto et al., 2022 ; Zoncu et al., 2011 ). Additionally, resident transmembrane proteins can modulate V-ATPase activity by sensing the availability of charged molecules to counterbalance proton accumulation in the lumen ( Chadwick et al., 2021 ). For instance, the arginine transporter SLC38A9 is a well-characterized regulator of V-ATPase assembly, linking its activity to the activation of the mammalian target of rapamycin complex 1 (mTORC1) ( Wyant et al., 2017 ). Under low-nutrient conditions, the V-ATPase complex is disassembled, leading to reduced mTOR activity and the induction of autophagy. Transmembrane protein 184B (TMEM184B) is an evolutionarily conserved 7-pass transmembrane protein. TMEM184B is broadly expressed throughout the nervous system, with predominant expression in neurons (Bhattacharya et al., 2016; Larsen et al., 2022 ; Wright et al., 2023 ). Loss of TMEM184B leads to swollen presynaptic terminals, extra terminal branching, disrupted synaptic gene expression, and impaired synaptic transmission in multiple model systems, with concomitant disruptions in behavior (Bhattacharya et al., 2016; Cho et al., 2022 ). Electron microscopic analysis shows multilamellar inclusions within presynaptic terminals, suggesting a possible disruption in endolysosomal maturation, flux, or transport. Finally, TMEM184B human variants have been linked to a neurodevelopmental syndrome characterized by developmental delay, structural brain defects including corpus callosum hypoplasia and microcephaly, and seizures. In this study, TMEM184B patient-associated variants were linked to disruptions in cellular metabolism, as disruption of its function enhanced the nuclear localization of the stress-responsive transcription factor EB (TFEB) (Chapman et al., 2024). This finding suggests a possible role for TMEM184B in promoting cellular metabolism, which is interconnected with endolysosomal flux. Together, this evidence suggests that TMEM184B may contribute to neuronal structure and function by facilitating endolysosomal flux. How TMEM184B may accomplish this is unknown. In this study, we investigated the functional role of TMEM184B in the endolysosomal system. We found that TMEM184B associates with proteins known to regulate intracellular transport and endolysosomal trafficking, including the V-ATPase. Analysis of endolysosomal acidification in the absence of TMEM184B revealed an elevated endolysosomal pH compared to wild-type controls, underscoring a functional role in endolysosomal function. We find reduced assembly of V-ATPase subcomplexes V0 and V1, which likely causes the pH disruptions in mutant neurons. These findings identify a mechanistic basis by which TMEM184B regulates neuronal endolysosomal acidification to ensure synaptic structure and function. Our data also suggest an explanation for how TMEM184B functional alteration disrupts neural development. Methods Experimental design and statistical analysis For human cell TMEM184B localization, approximately 15-20 cells were imaged for each experimental group (TMEM184B-FLAG vs. GFP-FLAG) across 3 separate imaging sessions. For endolysosomal acidification assessment, a total of 13 mutant neurons and 10 wild-type primary cortical neurons were analyzed across 3 separate embryonic neuron dissections. Embryonic neurons were obtained from female mice at embryonic day 16. For V-ATPase assembly evaluation, 5 Tmem184b -mutant (3 male, 2 female) and 5 wild-type (3 male, 2 female) were euthanized for hippocampus dissection. All statistical analyses were performed in GraphPad Prism, and p-values less than 0.05 were considered statistically significant. Statistical significance was defined as follows: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). Data presented as the average ± SEM. For FIRE-pHly analysis, significance between wild-type and Tmem184b- mutant average puncta size was assessed using a Mann-Whitney test. All other analyses comparing Tmem184b -mutant and wild-type samples were evaluated by unpaired t-tests with Welch’s correction. Animals The use of mice for this study has been approved by the University of Arizona IACUC (protocol 17-216). TMEM184B -mutant mice used in this study contain a gene-trap insertion originally created by the Texas A&M Institute for Genomic Medicine allele Tmem184bGt (IST10294F4) on the C57BL/6 background. Mice with this insertion exhibit < 5% of wild-type TMEM184B mRNA expression by both qPCR and RNAseq (Bhattacharya et al., 2016; Larsen et al., 2022 ). We therefore call this line a “mutant” rather than a “knockout” throughout the manuscript. C57BL/6 mice (littermates when possible) were used as wild-type controls throughout this study. All mouse husbandry was in accordance with guidelines of the Institutional Animal Care and Use Committee (protocol 17-216) at the University of Arizona. Reagents Antibodies include: mouse monoclonal anti-V5-epitope antibody (Invitrogen, # R96025), myc-tag (9B11) mouse mAb (Cell Signalling Technology, # 2276S), rabbit polyclonal anti-ATP6V0A1 (Novus Biologicals, #NBP1-89342), rabbit polyclonal anti-ATP6V1A (Proteintech, # 17115-1-AP), rabbit polyclonal anti-ATP6V1H (abcam, # 187706), rat IgG Horseradish peroxidase-conjugated antibody (R&D systems, #HAF005), anti-rabbit IgG HRP-linked antibody (Cell Signalling Technology, #7074), monoclonal rat anti-LAMP1 (DSHB, # 1D4B), rabbit monoclonal anti-Cathepsin D (abcam, #75852), Cy3-goat anti-rabbit IgG (Jackson ImmunoResearch, # 111-165-144), Cy5-goat anti-rat IgG(Jackson ImmunoResearch, # 112-175-143), and purified anti-DYKDDDDK Tag antibody (BioLegend, #637301). pcDNA4 CMV c-Myc-tag –GFP ORF –2xflag-tag and FCIV CMV BirA ORF-V5-tag IRES-Venus were ordered from Addgene. pcDNA3.1 TMEM184B vectors were ordered from GenScript. FLAG-hTMEM184B-GFP was generated by Twist Biosciences. FLAG-GFP was made in house. mCherry-Rab7a-7 was a gift from Michael Davidson (Addgene plasmid # 55127; http://n2t.net/addgene:55127 ; RRID: Addgene_55127). mCherry-Rab5a-7 was a gift from Michael Davidson (Addgene plasmid # 55126; http://n2t.net/addgene:55126 ; RRID: Addgene_55126). DsRed-rab11 WT was a gift from Richard Pagano (Addgene plasmid # 12679; http://n2t.net/addgene:12679 ; RRID: Addgene_12679). pLJM1-FIRE-pHLy was a gift from Aimee Kao (Addgene plasmid # 170775; http://n2t.net/addgene:170775 ; RRID: Addgene_170775). Cell culture and lysis HEK293T cells were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco) containing 10% fetal bovine serum, 110mg/mL sodium pyruvate, and 10,000 U/mL Penicillin/Streptomycin. Cells were incubated at 5% CO 2 at 37°C. For immunoprecipitation coupled with tandem mass spectrometry (IP-MS) analysis and follow-up experiments, cells were removed from incubation and placed on ice for the entire lysis protocol. Media was aspirated from the plate and replaced twice with ice-cold PBS (Gibco) to gently wash cells. Cell scrapers (RPI) were used to pool cells for transfer to clean 1.5mL Eppendorf tubes. Cells were gently centrifuged at 800x g for 2 min. Cell pellets were resuspended with RIPA lysis buffer (150mM NaCl, 5mM EDTA, 50mM Tris, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing EDTA-free protease inhibitor (Biomake). Cells were incubated at 4°C for 1 hour on a rotator followed by centrifugation at 21.1x g for 5 min. Supernatants were collected and passed through a 29 ga. Syringe (Exel International, 26028) seven times on ice prior to protein content quantification using a Pierce BCA protein assay kit (Thermofisher). Cells were stored at −80 °C for future IP-MS experiments. Primary cortical neuron culture Cortices were collected from Tmem184b- mutant and wild-type mice at embryonic day 16. Neurons were dissociated with 0.25% Trypsin (Gibco) for 15 min at 37°C. Trypsin was replaced with 10% fetal bovine serum (FBS)-containing medium and incubated for 3 min at room temperature. Neurons were washed 3 times with ice-cold HBSS (Gibco) and triturated with fire polished glass Pasteur pipets (Fisher Scientific) of decreasing diameter 10 times each. Suspended neurons were filtered through 40µm cell strainers (Chemglass) and resuspended in Neurobasal Medium (Gibco) containing 0.5 mM L-glutamine, 10,000 U/mL Penicillin/Streptomycin (Gibco), and 1X B27 supplement. Approximately 20K neurons were plated in 8-well chambered #1.5 cover glass (Thermo Fisher) previously coated with 0.15ug/mL poly-D-lysine. Half of the media was replaced the next day prior to transfections. Neurons were incubated at 5% CO 2 at 37°C. Transfections Transfections in HEK293T cells were done using GeneJuice (EMD Millipore) and serum-free OptiMem GLUTAMAX (Gibco). For the entire IP-MS workflow, procedure was performed as previously described ( Parker et al., 2019 ): expression vectors of TMEM184B variants were tagged with either the c-Myc epitope tag or the V5 epitope tag sequence. Control vectors included bifunctional ligase/repressor (BirA) tagged with the Myc tag or V5 tag. Each vector was then transfected into in duplicate. To assess TMEM184B localization in the endosomal system, HEK293T cells were transfected with pHIV-FLAG-TMEM184B-myc or FLAG-GFP. To mark endosomes, cells were transfected with mCherry-Rab7 (late endosomes), mCherry-Rab5 (early endosomes), or dsRed-Rab11 (recycling endosomes)( Choudhury et al., 2002 ). Lysosome markers (CTSD and LAMP1) were evaluated using immunocytochemistry. Transfections in primary cortical neurons were done using FuGENE HD transfection reagent (Promega) and OptiMem. After transfection with pFUGW-FIRE-pHly for pH analysis, neurons were incubated for 6 hr before media was changed. Imaging was performed 2 days after transfections. Immunocytochemistry HEK293T cells were gently washed 3 times with 1X PBS-1% Tween 20 (PBST). Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min on ice. PFA was removed, cells were washed with 1X PBST for 5 min twice. Blocking solution (5% NGS in 1X PBST) was applied to cells and incubated for 30 min at room temperature on a rotator. Cells were gently washed 3 times with 1X PBST for 5 min to remove the remaining blocking solution. Primary antibodies against LAMP1 (1:500) and Cathepsin D (1:500) in 0.3% NGS and 1X PBST were applied to cells. Chambered slides were wrapped in parafilm and incubated overnight at 4°C. Primary antibodies were removed, and cells were gently washed 3 times with 1X PBST for 5 min. Secondary antibodies, goat, anti-rat Cy 5 (1:500) and anti-rabbit Cy3 (1:500) in 1X PBST, were added to cells. Chambered slides were covered in foil and incubated at room temperature for 1 hr. Cells were gently washed 3 times with 1X PBST for 5 min to remove any remaining secondary antibody and once with 1X PBS. Vectashield + DAPI (Fisher Scientific) was added to slides prior to applying #1.5 micro cover glass (Electron Microscopy Sciences) Hippocampus dissection Mice were humanely euthanized with carbon dioxide, and hippocampi were removed within 30 min of euthanasia. Isolated hippocampi were immediately placed into RIPA lysis buffer with protease inhibitor. Hippocampi were crushed using clean pestles and sonicated before centrifugation at 21k x g for 5 min. Protein concentrations from the final supernatant were quantified via BCA. Hippocampi were collected from 5 Tmem184b -mutant and 5 wild-type controls. All mice were approximately 6 months old. Immunoprecipitation Constructs containing mouse TMEM184B (NP_766196.1, 407 amino acids) or human TMEM184B (NP_036396.2, 407 amino acids) were used as appropriate. Mouse and human TMEM184B sequences are 96% identical (391/407) and 97% similar (395/407). 500 ug of Protein A/G Magnetic Beads (MedChemExpress, HY-K0202) were loaded into 1.5 mL Eppendorf tubes and washed 3 times with PBS prior to use. Beads were conjugated with primary antibody with incubation for 2 hr at room temperature on a rotator. Bead-antibody conjugates were washed 4 times with 1X PBS before addition of lysates and incubated overnight at 4°C on a rotator. Lysate supernatants were removed, and antibody-antigen were washed 4 times with PBS and transferred to new 1.5mL Eppendorf tubes to avoid non-specific binding to any remaining lysates. Proteins were eluded twice with 1X Laemmli sample buffer (Cold Spring Harbor) for a final volume of 40ul. Eluents were incubated for 5 min (95°C or 37 °C for TMEM184B samples) prior to loading into gel. For mass spectrometry analyses, 5µg of anti-V5-epitope antibody (Invitrogen) was used for bead conjugation. 1.5 mg whole cell lysates were added to conjugated beads prior to incubation overnight. For V-ATPase assembly analysis, 0.16 µg anti-ATP6V0A1 primary antibody was used to conjugate beads. 100 µg of hippocampus protein was added to conjugated beads. Gel electrophoresis, Coomassie, and Immunoblotting For V-ATPase assembly analysis, whole hippocampus lysates were mixed with 1X Laemmli Sample Buffer and incubated for 5 min at 95°C. Whole lysate controls and IP eluents were loaded into 4-20% Mini-PROTEAN TGX protein gels (Bio-Rad). Proteins were transferred onto 0.45m PVDF transfer membranes (Thermo Scientific) and blocked with 5% casein in 1X TBST (Sigma-Aldrich). Membranes were incubated overnight at 4°C in primary antibody against ATP6V1H or ATP6V1A in 0.3% casein and 1X TBST. Primary antibodies were removed, and membranes washed before incubation with horse-radish peroxidase-conjugated antibody. Blots were developed using ECL blotting substrate (Bio-Rad) and quantified using Image Lab software (Bio-Rad). For IP samples, V1A and V1H quantification was normalized to the total amount of V0A1 in the IP lane. Whole cell lysate controls were normalized to total amount of protein loaded into the sample lane. For IP-MS, eluents were run fresh on the day of MS analysis. IP-MS gels were stained with mass-spectrometry grade Coomassie Blue, imaged for annotation purposes, and processed and analyzed by the University of Arizona College of Medicine’s Quantitative Proteomics Laboratory. Mass Spectrometry Data Acquisition and Analysis To identify TMEM184B true protein-protein interaction candidates, a multiple epitope tags co-IP approach was used: two separate plasmids were created containing the same TMEM184B protein coding sequence but with either a c-Myc tag sequence or a V5 tag sequence at the end of TMEM184B’s C-terminus. In parallel, HEK293T 100mm plates were transfected with one vector in duplicate– thus, 8 plates in total were transfected: duplicates of TMEM184B-c-Myc, TMEM184B-V5, GFP-c-Myc, and BirA-V5. Mass spectrometry data was acquired as previously described ( Kruse et al., 2017 ; Parker et al., 2019 ). Once acquired, Scaffold software-derived (version 5, Proteome Software Inc., Portland, OR) total spectrum counts (TSCs) were compiled into a CSV and uploaded into the RStudio GUI (version 4.1.0) using the 64bit R software (version 4.0.4). Upon uploading Scaffold-mediated TSC CSV files into R, the data was wrangled to enable mathematical operations on TSCs. The only departure from the methods above was a lack of deployment of SAINT scoring and analysis of SAINT-scored hits ( Choi et al., 2010 ). To analyze the resulting MS data, we filtered to remove low abundance proteins (< 5 TSCs in both TMEM184B IP duplicates and < 10 TSCs in every TMEM184B sample) ( Teo et al., 2013 )). Following filtering, subsequent processing was performed on each tag dataset separately. Values of 0.1 were added to proteins with 0 TSCs to enable fold change calculation( Choi et al., 2010 ; Sardiu et al., 2008 ; Sowa et al., 2009 ). TSCs were then normalized by molecular weight (MW) and averaged between duplicates. These averages were used to calculate fold changes (FCs), one for each tag (e.g., avg TMEM184B-c-Myc MW-normalized TSC / avg GFP-c-Myc MW-normalized TSC), and a combined FC (e.g., avg of all TMEM184B MW-normalized TSC / avg of all control bait MW-normalized TSC). Candidates with ≥ 2 Avg FC in both tag system and a ≥ 2 combined FC were retained, and common contaminants were filtered out as previously described ( Mellacheruvu et al., 2013 ). R scripts, markdowns, and notebooks were developed to adapt multiple algorithms, to analyze subsequent candidates via gene ontology, and to visualize results. Fluorescent microscopy All imaging was taken on the Nikon Spinning-Disk SoRa super-resolution microscope in the University of Arizona Cancer Center (UACC) Microscopy Shared Resource. Images were acquired on a Nikon CSU-W1 SoRa Spinning-Disk Confocal microscope equipped with a Photometrics Kinetix sCMOS camera. Single slice images were acquired using a Nikon 60x Plan Apo 1.40NA objective lens. Prior to imaging live primary neurons, neurobasal media was removed from 8-well cover glass chambers and replaced with pre-warmed Live Cell Imaging Solution (Thermo Fisher). Neurons were allowed to acclimate to the microscope live cell imaging chamber (5% CO 2 and 37°C) for 30 minutes prior to imaging. The 488 and 561 nm lasers were used to excite mTFP1 and mCherry, respectively. Single slices were obtained and exported as ND2 files for later analysis in Nikon NIS Elements AR. Wild-type (10) and Tmem184b-mutant (13) neurons were imaged across 3 separate groups of 1-2 pooled embryos per genotype. For HEK293T localization experiments, the 405, 488, and 640 nm lasers were used to excite DAPI, GFP, and Cy5, respectively. The 561nm laser was used to excite mCherry, dsRed, or Cy3 according to the specific compartment. Single slice images were acquired for analysis in ImageJ (FIJI). For each compartment assessed, 15-20 images were taken for TMEM184B and GFP controls each. Colocalization analysis Raw CZI files were imported into ImageJ (FIJI) for processing, including background subtraction, prior to analysis. To assess the co-localization of TMEM184B with other cellular compartments, images from the red and green channels were binarized and segmented using the watershed function. Overlapping puncta between the binarized images were identified and analyzed using the “AND” function in the Image Calculator. Both the merged puncta and individual puncta from each channel were quantified using the “Analyze Particles” function to determine puncta area (µm²). The degree of TMEM184B localization to each compartment was calculated by dividing the area of merged puncta by the total area of TMEM184B+ puncta. For lysosome localization, images labeled with CTSD (Cathepsin D) and LAMP1 (Lysosomal-associated membrane protein 1) were first processed using the “AND” function to identify puncta associated with lysosomes. This resulting image was then intersected with TMEM184B+ puncta to assess co-localization. Additionally, separate calculations for LAMP1 and TMEM184B were performed to examine TMEM184B localization to late endosomes or deacidified lysosomes, distinguishing these from conventional lysosomal compartments. FIRE-pHly puncta analysis Nikon NIS Elements AR 5.42.03 software with the General Analysis 3 (GA3) module was used for image processing and analysis. To quantify red channel detections within each cell, a new image channel was created by applying a gaussian filter (sigma = 8) and rolling ball background subtraction (radius = 18µm) using the green channel. A signal threshold was applied to this “Cell Body” channel to properly segment the cell body and only quantify the red channel detections within each cell. The following image preprocessing tools were used for both the green and red channels: low pass filter = 4px and rolling ball background subtraction radius = 1.2µm. The Bright Spots Detection tool (diameter = 1.5µm) was used to detect the red channel within the Cell Body and the intensity threshold was adjusted per image to achieve accurate segmentation. The following data was quantified for each cell: cell body area (µm 2 ), total channel detections, and mean intensity of green and red fluorescence. Weights were determined as follows: dividing each cell’s number of puncta by all the puncta in the study and then multiplying each cell’s fraction by 10 to re-scale the value of the weight near a whole mouse. The green-to-red (G/R) fluorescence ratio for each identified puncta was calculated by dividing the mean green fluorescence intensity by the mean red fluorescent intensity. Results TMEM184B interacts with multiple proteins involved in endosomal trafficking and autophagy To clarify the role of TMEM184B in cellular processes that could contribute to neuronal maintenance (Bhattacharya et al., 2016; Cho et al., 2022 ), we first sought to identify its protein interactors within human cells. Because currently available antibodies show nonspecific binding in mutant tissue (M.R.C.B., personal communication) we could not cleanly immunoprecipitate endogenous TMEM184B from human or mouse tissue. We therefore performed immunoprecipitation (IP) of expressed, tagged TMEM184B coupled with tandem mass spectrometry (IP-MS/MS) analysis in HEK293T cells. We performed IP with two independent epitope tags (V5 and Myc) in duplicate for each tag to improve confidence in hits ( Fig. 1A-B ). After filtering out common contaminants ( Mellacheruvu et al., 2013 ) and intersecting potential candidate interactors from both datasets, we identified 136 unique proteins as candidate interactors of TMEM184B ( Fig. 1C and Extended Data Table 1-1). Download figure Open in new tab Fig 1: TMEM184B interacts with multiple proteins involved in endosomal trafficking and autophagy. A , Schematic depicting overall workflow of IP-MS analysis. B, Example western blot showing a successful pulldown of TMEM184B targeting its V5 tag. C, Average spectral counts of TMEM184B versus control (myc or V5) immunoprecipitation from HEK293T cells. Data is from 4 independent IP-MS experiments using two different affinity tags with 2 replicates each. Blue represents potential candidates significantly enriched as interactors with TMEM184B. All other proteins are denoted as “Background” in gray, and “TMEM184B” is marked in gold. Dashed box represents area in which all 136 enriched proteins are located. D-F, Cropped scatterplots (from blue dashed box in C) highlighting enriched proteins in functional categories. Light blue represents all protein candidates and dark blue represents proteins within designated functional category. Pink in D represents other vacuolar acidification GO term members. G-H, Top 10 significant results from gene ontology biological process (G) and cellular component (H) analyses, sorted by fold enrichment and adjusted p-value of 136 significant IP-MS hits. Interestingly, TMEM184B demonstrated significant interactions with two isoforms of the V-ATPase subunit V0a (a1 and a2) (FC = 3.38 and 3.18, respectively). To confirm a potential interaction between FLAG-TMEM184B-GFP and ATP6V0A1 (the V0a1 subunit), we performed co-IP assays (Extended Data Figure 1-2). We first used the FLAG-tagged proteins as bait and probed for V0a1. Results confirmed an interaction between V0a1 and TMEM184B We performed an inverse co-IP using V0a1 as bait and probed for FLAG-tagged proteins to increase confidence in this interaction and found similar results. Regulators of V-ATPase assembly were also found among the interacting protein candidates, including the Drosophila melanogaster X chromosomal gene-like proteins (DMXL1 and DMXL2), also known as Rabconnectin-3 (FC = 4.05 and 4.44, respectively) ( Eaton et al., 2024 ). Together, these data suggest that TMEM184B and the V-ATPase are associated in human cells. Among hits in the mass spectrometry analysis, many proteins were involved in cellular trafficking or autophagy, suggesting a possible influence of TMEM184B in these dynamic processes (Extended Data Table 1-1). Notably, proteins involved in regulating endosomal trafficking to the plasma membrane include ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor 2 (ARFGEF2, FC = 4.72), AAA-ATPase VPS4B (FC = 3.25), Coiled-Coil Domain Containing 22 (CCDC22, FC = 3.43), and the EH Domain Containing protein family 3 (EHD3, FC = 3.35) ( Bar et al., 2013 ; Singla et al., 2019 ; Tseng et al., 2021 ; Zhu et al., 2022 ). Collectively, these proteins contribute to endosomal trafficking, each fulfilling a distinct function in vesicle trafficking, protein recycling, and the maintenance of cellular homeostasis. Autophagy-related proteins Tuberous Sclerosis Complex (TSC2, FC = 2.55) and Activating molecule in BECN1-regulated autophagy protein 1 (AMBRA, FC = 2.433) also were present among TMEM184B interactors. These proteins regulate autophagy through distinct mechanisms: TSC2 modulates mTORC1 and AMPK signaling pathways, and AMBRA1 directly participates in autophagosome formation ( Albishi, 2023 ; Di Bartolomeo et al., 2010 ; Li et al., 2022 ; Nardo et al., 2014 ; Ng et al., 2011 ). To identify enriched functional categories of TMEM184B-interacting protein candidates, we performed Gene Ontology (GO) analyses for Biological Processes (BP) and Cellular Components (CC). Multiple categories indicated involvement in vacuolar acidification, encompassing the V-ATPase and known regulators (fold enrichment = 20.71) and intracellular transport (fold enrichment = 2.48) ( Fig. 1F ). For CC enrichment, the Regulator of the H+-ATPase of vacuolar and endosomal membranes (RAVE) complex (Jaskolka, Tarsio, et al., 2021; Jaskolka, Winkley, et al., 2021; Smardon et al., 2002 ) showed the highest fold enrichment (99.42, FDR = 0.008) ( Fig. 1G ). Other significantly enriched GO categories included transport-related mechanisms, such as vesicle transport, membrane fusion, and Golgi trafficking (Extended Data Table 1-3). These results suggest that TMEM184B localizes to multiple organellar membranes. TMEM184B localizes to early and late endosomes in human cells TMEM184B colocalizes with recycling endosomes in mouse sensory neurons and in fly motor neurons (Bhattacharya et al., 2016; Cho et al., 2022 ), but its distribution in human cells has not been established. Because many of the TMEM184B interactors we identified reside on endosomal membranes or participate in endosomal trafficking ( Fig. 1D-E ), we hypothesized that TMEM184B may at least partially localize to these compartments. We first quantified the fraction of TMEM184B + puncta that overlapped with the endosomal markers Rab5 (early endosomes), Rab7 (late endosomes), and Rab11 (recycling endosomes) relative to the total population of TMEM184B + puncta in each cell. Of all TMEM184B puncta identified, 45% showed early endosome colocalization, 34% showed late endosome colocalization, and 13% showed recycling endosome colocalization. ( Fig. 2A-C, E ). We further examined the prevalence of TMEM184B on each type of endosome. TMEM184B is present on ∼73% of early endosomes, ∼59% of late endosomes, and ∼45% of recycling endosomes. There is a small amount of TMEM184B at the plasma membrane, likely resulting from recycling endosome fusion. Together, these data indicate that TMEM184B is broadly distributed across endosomal populations, consistent with IP-MS results. Download figure Open in new tab Fig 2: TMEM184B localizes to early and late endosomes in human cells. All images show HEK293T cells expressing FLAG-TMEM184B-GFP. DAPI (blue) marks the nucleus. Images represent single slices of human cells. A-D, images depicting localization of FLAG-TMEM184B-GFP to early endosomes (mCherry-Rab5, A), late endosomes (mCherry-Rab7, B), recycling endosomes (dsRed-Rab11, C), and lysosomes (LAMP1+CTSD, D). White arrows highlight overlapping puncta. Scale bars = 2 μm. n = 45-60 cells per marker, imaged over 3 sessions each. E, Fraction of merged puncta over total TMEM184B puncta area. F, Fraction of merged puncta over the total red pixel area of endosomal markers. G, Fraction of merged puncta (TMEM184B + LAMP1 + CTSD) over total TMEM184B pixel area. H, Fraction of merged puncta over the total red pixel area (LAMP1 + CTSD) of lysosomal markers. Error bars represent SEM. To assess lysosomal localization, we used antibodies against LAMP1 and CTSD. Notably, LAMP1 is not exclusively localized to lysosomes but is also present in late endosomes ( Cheng et al., 2018 ; Yap et al., 2022 ; Yap & Winckler, 2022 ). Therefore, the addition of CTSD is required for accurate evaluation of lysosomes. We first quantified the overlap between TMEM184B + and LAMP1 + CTSD + puncta relative to the total number of TMEM184B + puncta in HEK293T cells. Notably, TMEM184B + puncta exhibited minimal colocalization with the lysosomal markers LAMP1 and CTSD ( Fig. 2G ). However, when considering the total lysosomal population, approximately 24% of lysosomes contained TMEM184B, indicating its presence on a subset of lysosomes ( Fig. 2H ). These findings suggest that TMEM184B is broadly distributed throughout the endosomal system, with a greater enrichment in early endosomal compartments. TMEM184B loss reduces endolysosomal acidification The interactions between TMEM184B and the V-ATPase ( Fig. 1D and 1-2) suggest that TMEM184B may regulate V-ATPase function. The endosomal system comprises a dynamic network of compartments that undergo progressive acidification during maturation ( Hu et al., 2015 ; Wang et al., 2017 ). We focused on TMEM184B’s role in endolysosomal acidification because of recent attention towards lysosomal deacidification in neurodegenerative disorders and aging ( Boland et al., 2008 ; Colacurcio & Nixon, 2016 ; J. H. Lee et al., 2022 ; Lie & Nixon, 2019 ; Nixon & Yang, 2012 ; Wolfe et al., 2013 ). To explore this, we utilized FIRE-pHly ( Chin et al., 2021 ), a pH-sensitive sensor tagged to LAMP1, to assess the acidity of these compartments in primary embryonic cortical neurons from Tmem184b -mutant and wild-type mice. Changes in compartment acidification were assessed by the ratio of green fluorescence (mTFP1) to red fluorescence (mCherry) in FIRE-pHly + puncta. FIRE-pHly + puncta are primarily localized near the soma and initial projection segments, as expected for lysosomes ( Fig 3A-B ). Notably, we observed that Tmem184b -mutant neurons occasionally displayed large puncta with prominent green fluorescence ( Fig. 3B ). We suspect these abnormal, enlarged compartments represent deacidified late endosomes or lysosomes, as wild-type neurons did not display similarly shaped puncta. In the absence of TMEM184B, FIRE-pHly + puncta exhibited a significantly increased average green-to-red (G/R) fluorescence ratio in each neuron (p = 0.0189), indicating deacidification of these compartments compared to wild-type controls ( Fig. 3C ). Tmem184b -mutant neurons exhibit deacidified puncta regardless of size (Extended Data Figure 3-1). Average puncta size showed no significant difference between Tmem184b- mutant neurons and wild-type controls ( Fig. 3D ). Furthermore, no significant change in the density of FIRE-pHly + puncta was observed ( Fig. 3E ). Together, this data shows that TMEM184B is necessary for proper acidification of the endolysosomal system. Download figure Open in new tab Fig 3: TMEM184B loss perturbs endolysosomal acidification. All images are single slices of the cell body and initial projections. A-B, Cell bodies and initial segments from wild-type (A) and Tmem184b -mutant (B) embryonic cortical neurons transfected with FIRE-pHly. Cropped images depict enlarged, abnormal green puncta indicated with white boxes. Scale bars = 5 µm. C, Comparison of weighted wild-type and mutant average G/R fluorescence intensity (p = 0.0189) using unpaired t-test with Welch’s correction. D, Weighted average puncta area (µm 2 ) of wild-type and mutant neurons (p = 0.751) using Mann Whitney test. E, Number of puncta normalized to the cell body area per 100 μm 2 for each neuron (p = 0.059). Using unpaired t-test with Welch’s correction. Error bars represent SEM. Loss of TMEM184B disrupts assembly of the vesicular proton pump V-ATPase activity is regulated by the assembly of its V0 and V1 subcomplexes. The formation of these complexes is influenced by lumenal nutrient levels and growth signaling pathways, adjusting to the cell’s metabolic needs. As we saw reduced endolysosomal acidification in the absence of TMEM184B, we wanted to determine if this reduction was due to reduced V-ATPase assembly through co-IP assays. Our results revealed significantly reduced interactions between the subunit V0a1 and both V1A and V1H subunits in Tmem184b -mutant mice ( Fig. 4A-B ) relative to wild-type controls. The interaction between V0a1 and V1H was reduced by 31.72% in the absence of TMEM184B, while the interaction between V0a1 and V1A was reduced by 64% ( Fig. 4C, E ). Quantification of total V1H and V1A levels in lysates indicated that the reduced interaction was not due to a decrease in protein expression ( Fig. 4D, F ). These findings indicate that the absence of TMEM184B disrupts V-ATPase assembly, which offers a plausible mechanistic explanation for the decreased endolysosomal acidification seen in Tmem184b -mutant neurons. Download figure Open in new tab Fig 4: Loss of TMEM184B disrupts assembly of the vesicular proton pump. A , Diagram of the vesicular proton pump adapted from previous work ( Sun-Wada & Wada, 2015 ). Red stars indicate specific subunits targeted in assembly assay. B, Blots of mouse hippocampal lysates show presence of V1H (top) and V1A (bottom) after V0a1 pulldown in wild-type and mutant mice. C and E, Quantification of the interaction between V0 and V1 subunits in Tmem184b- mutants and wild-type controls. Results of IP analysis normalized to the average total of V0A1 band intensity in wild-type IP samples. p = 0.0039 (C), p = 0.0028 (E). D and F, Quantification of total V1H (D) and V1A (F) normalized to the total amount of V0a1 input for IP. p = 0.32 (D), p = 0.33 (F). For all graphs, statistical evaluation used unpaired t-test with Welch’s correction. Error bars represent SEM. Discussion Neurons are highly polarized cells with complex morphologies, characterized by multiple elongated projections extending from the cell soma. As post-mitotic cells, neurons encounter unique challenges in preserving their structure and function over their extended lifespan. Endolysosomal trafficking plays an essential role in supporting neuronal growth and long-term maintenance by facilitating the proper localization of cellular proteins to membranes, regulating intracellular signaling pathways, and coordinating the clearance of endocytosed cargo ( Kuijpers et al., 2021 ; Roney et al., 2022 ). Dysregulated endolysosomal trafficking, particularly due to compartmental deacidification, contributes to cellular dysfunction associated with many neurological disorders. Despite significant progress, the mechanisms by which neurons maintain endolysosomal pH remain incompletely understood. Elucidating these mechanisms may reveal critical therapeutic targets for preventing the accumulation of toxic proteins and mitigating neurodegeneration. Our data indicate that TMEM184B interacts with candidate proteins associated with endosomal trafficking and macroautophagy. Among these were the V0a subunit (a1 and a2 isoforms) of the V-ATPase. Evidence has shown that a1 resides on lysosomes and endosomes while a2 is present on the Golgi and early endosomes ( Tuli & Kane, 2023 ). We also found an association with the RAVE complex and DMXL1 (Rabconnectin-3a), both well-established regulators of V-ATPase complex assembly. These findings prompted us to investigate the hypothesis that TMEM184B is not only localized within the endosomal system beyond that of recycling endosomes but also that TMEM184B plays a role in regulating endosomal acidification. TMEM184B has not been identified before in association studies that defined the proteins contributing to the V-ATPase subcomplexes or their regulatory proteins ( Merkulova et al., 2015 ). This could be because TMEM184B is not highly expressed (Bhattacharya et al., 2016; Larsen et al., 2022 ) and therefore may have fallen under the threshold for inclusion as a true positive interactor in directed immunoprecipitation or in high-throughput studies of proteome-wide interactions. In fact, the most highly cited association study acknowledges that their thresholds were set high and thus true interactors at low abundances may not be identified ( Merkulova et al., 2015 ). Because we wanted to ensure our IP-MS results were robust, we used strict filtering parameters to identify positive interacting candidates. Although our interaction data is derived from over-expression models of TMEM184B, we observed a robust and bi-directional interaction between TMEM184B and the V0a1 subunit ( Fig. 1 - 2 ). In human cells, TMEM184B is localized to early endosomes, late endosomes and recycling endosomes ( Fig. 2 ). During the transition from early to late endosomes, a stepwise process occurs in which Rab7 replaces Rab5 on the endosomal membrane ( Langemeyer et al., 2020 ; Mottola, 2014 ; Skjeldal et al., 2021 ). Therefore, both Rab GTPases can transiently coexist on the same compartment ( van der Beek et al., 2022 ). This may explain the high fractions of TMEM184B puncta overlapping with Rab5 or Rab7. In contrast, very few TMEM18B puncta are localized to lysosomes. These findings indicate that while TMEM184B localizes to both early and late endosomes, its primary function may be associated with the early stages of endocytosis rather than the terminal degradation phase. One possible explanation for this that TMEM184B is degraded in lysosomes or retained in late endosomes during late endosome-lysosome (LEL) fusion. If TMEM184B is sorted into intraluminal vesicles (ILVs) of late endosomes, it would be directed towards lysosomal degradation. Overexpression of TMEM184B may increase its degradation in lysosomes, consistent with the observation that approximately 20% of the total lysosomal population (LAMP1 + CTSD + ) colocalized with TMEM184B. TMEM184B exhibited partial localization at the plasma membrane, which may result from fusion with recycling endosomes rather than endogenous surface expression. The absence of TMEM184B leads to reduced acidification of late endosomes and lysosomes in neurons ( Fig. 3 ). As endosome maturation is tightly regulated by acidification, we hypothesized that these compartments’ size and morphology may also be perturbed. However, while we did occasionally observe enlarged and deacidified endolysosomes, we did not see a significant difference in the size of endolysosomes or the density of endolysosomes in the cell body. This suggests that the reduced endolysosomal acidification observed in mutant mice does not affect the morphology or distribution of degradative compartments within the cell body. Together, these results could suggest a perturbation in late endosome-lysosome fusion, leading to alkaline lysosomes residing in the cell body. An alternative explanation may be that impaired axonal retrograde late endosome transport reduces late endosome-lysosome fusion in the absence of TMEM184B. An interesting aspect to our findings is that TMEM184B appears to regulate lysosomal acidification without prominent lysosomal localization. It may achieve this by facilitating the budding and trafficking of the V0 subcomplex from the Golgi or endosomes, an essential step for V-ATPase assembly on endosomes and lysosomes. We identified interactors that participate in Golgi transport and vesicular tethering, suggesting that TMEM184B could participate early in the packaging or delivery of V-ATPase subunits from the Golgi throughout the endosomal system. Future studies should test these possibilities, which could illuminate how TMEM184B affects budding, fission, and fusion processes of membrane-bound compartments in neurons. In this study, we demonstrate a reduction in V-ATPase subdomain assembly in the absence of TMEM184B. IP-MS analysis showed enriched associations between TMEM184B and two isoforms of the V0a subunit, a1 and a2 ( Fig. 1 ). Given the observed reduction of endolysosomal acidification in TMEM184B-deficient cells ( Fig. 3 ), we hypothesized that V-ATPase assembly might also be compromised. Supporting this hypothesis, we observed an approximately two-fold decrease in the interaction between the V0a1 and V1A subunits compared to V1H ( Fig. 4 ), which was not attributable to reduced expression of either subunit. The V1H subunit is known to associate with the V0a1 subunit during complex assembly, and this substantial difference in interaction levels may reflect the spatial organization of these subunits. In the disassembled state, the V1H subunit undergoes a conformational change that brings it into closer contact with subunit F of the central stalk, effectively preventing ATP hydrolysis and rotational activity ( Collins & Forgac, 2020 ). Future biochemical and structure-function studies may enable us to understand how TMEM184B influences pump function. This study identifies a novel role for TMEM184B as a regulator of endolysosomal acidification. However, several aspects of its function remain unclear, including its precise biological role. The classification of TMEM184B within the transporter-opsin-G protein-coupled receptor (TOG) superfamily suggests a potential function as a transporter ( Yee et al., 2013 ). We suspect that the functional role of TMEM184B may affect both localization to the appropriate intracellular compartments and proton pump activity of the V-ATPase, perhaps through separate and possibly indirect mechanisms. While de-orphanizing putative transporters is technically challenging, identifying the molecular substrates that may be transported by TMEM184B is of significant interest. Some possibilities consistent with our results are that TMEM184B could participate as an endosomal nutrient sensor for the V-ATPase or that it could indirectly modulate V-ATPase activity via ion counterbalance within endosomes. Such investigations are expected to refine the working model of TMEM184B’s functional role and would build upon our results to better understand and treat TMEM184B-associated neurodevelopmental disorders and other nervous system disorders featuring endolysosomal dysfunction. Data and Code Availability All custom code developed for analysis will be freely available under an MIT license on Github at: https://github.com/eriklarsen4/ Conflicts of interest The authors declare no competing financial interests. Extended Data Table 1-1: Raw mass spectrometry data from IP-MS of TMEM184B (myc and V5-tagged) showing individual trial results, average values and fold changes of all proteins compared to controls. Extended Data Figure 1-2: IP-MS validation of V-ATPase interaction with TMEM184B. FLAG blots split to optimize visualization of TMEM184B input bands compared to GFP input. A, Representative blots showing V0a1 content following FLAG pulldown in FLAG-TMEM184B and FLAG-GFP expressing cells. (n = 3 per group). B , Quantification of V0a1 bands normalized to the total level of FLAG-tagged protein in corresponding lane. C, Representative blots of FLAG-tagged protein content following V0a1 subunit pulldown in FLAG-TMEM184B and FLAG-GFP expressing cells. (n = 3 per group). D , Quantification of FLAG signal normalized to the total amount of V0a1 in corresponding lane. Error bars represent SEM. Extended Data Table 1-3: Gene Ontology analysis results for the 136 proteins showing fold enrichment, adjusted p-values and false discovery fate (FDR) of biological process, cellular component and molecular function categories. Extended Data Figure 3-1: Puncta acidity is consistently higher in Tmem184b- neurons regardless of puncta size. Yellow dots represent wild-type neurons. Blue dots represent Tmem184b -mutant neurons. A , Comparing distribution of individual puncta area (µm 2 ) and corresponding G/R fluorescence intensity. 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Share Neuronal endolysosomal acidification relies on interactions between transmembrane protein 184B (TMEM184B) and the vesicular proton pump Elizabeth B. Wright , Erik G. Larsen , Marco Padilla-Rodriguez , Paul R. Langlais , Martha R.C. Bhattacharya bioRxiv 2025.02.01.635992; doi: https://doi.org/10.1101/2025.02.01.635992 Share This Article: Copy Citation Tools Neuronal endolysosomal acidification relies on interactions between transmembrane protein 184B (TMEM184B) and the vesicular proton pump Elizabeth B. Wright , Erik G. Larsen , Marco Padilla-Rodriguez , Paul R. Langlais , Martha R.C. 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