Negative feedback regulation of the hemi-arrestin MAPK scaffold Sms1 prevents untimely mating

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Negative feedback regulation of the hemi-arrestin MAPK scaffold Sms1 prevents untimely mating View ORCID Profile Boris Sieber , View ORCID Profile Laura Merlini , Wanlan Li , Maëlys Besomi , Laetitia Michon , Sushila Gordon-Lennox , View ORCID Profile Sophie G Martin doi: https://doi.org/10.1101/2025.09.24.678297 Boris Sieber 1 Department of Molecular and Cellular Biology, University of Geneva Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Boris Sieber For correspondence: Sophie.Martin{at}unige.ch Boris.Sieber{at}unige.ch Laura Merlini 1 Department of Molecular and Cellular Biology, University of Geneva Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Laura Merlini Wanlan Li 1 Department of Molecular and Cellular Biology, University of Geneva Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maëlys Besomi 1 Department of Molecular and Cellular Biology, University of Geneva Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laetitia Michon 1 Department of Molecular and Cellular Biology, University of Geneva Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sushila Gordon-Lennox 1 Department of Molecular and Cellular Biology, University of Geneva Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sophie G Martin 1 Department of Molecular and Cellular Biology, University of Geneva Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sophie G Martin For correspondence: Sophie.Martin{at}unige.ch Boris.Sieber{at}unige.ch Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Mitogen-activated protein kinases (MAPK) are ancestral kinases that form essential signalling cascades. However, scaffolds that recruit kinases to subcellular locations and promote signal transduction have only been described in few species. Notably, no scaffold was thought necessary for the MAPK cascade promoting sexual differentiation in fission yeast. Here, we identify the hemi-arrestin protein Sms1 as a structurally novel scaffold of this MAPK cascade. Interactions with PIP2 and the pheromone receptor–coupled Gα subunit target Sms1 to plasma membrane patches, where it assembles the active cascade by binding each MAP kinase. These interactions are essential for signal transduction and local signal interpretation for polarised growth. Phosphorylation, including by the MAPK itself, antagonises Sms1 membrane translocation, establishing a negative feedback that underlies polarity patch turnover and prevents untimely mating attempts. Thus, Sms1 is a MAPK scaffold with canonical functions despite its distinct structural fold, highlighting convergent evolution of MAPK scaffolds across eukaryotes. Introduction MAPK signalling, one of the best characterised transduction pathways across evolution, induces a wide range of responses ranging from cell proliferation to cell differentiation and sexual reproduction ( Avruch, 2007 ). The core of the pathway is formed by a three-tier kinase cascade wherein the MAP3K phosphorylates the MAP2K, which in turn phosphorylates the MAPK. These three kinases are widely conserved in all fungi, plants and mammals ( Kalapos et al., 2019 ; Widmann et al., 1999 ; Xu et al., 2017 ) and likely already existed in the last eukaryotic common ancestor ( Huang et al., 2025 ). As a single MAPK cascade converts distinct upstream stimuli into context-dependent outputs, a critical question is how kinase activity is spatiotemporally regulated, and how substrate specificity is determined. Key factors required for MAP kinases activation and substrate specificity are the MAPK scaffolds. MAPK scaffolds, which can be defined as proteins that interact with at least two MAP kinases, ensure signalling at specific, subcellular localisations by locally increasing kinases concentration and thus activity ( Park et al., 2003 ). The best characterised scaffolds of ERK-like MAPK include the budding yeast protein Ste5, and the metazoan proteins KSR1/2 and β-arrestins ( Fig. 1A ). Ste5 was first discovered and is essential for pheromone signalling ( Choi et al., 1994 ; Kranz et al., 1994 ; Marcus et al., 1994 ; Printen and Sprague, 1994 ). KSR1/2 is critical for Ras-induced tumorigenesis ( Frodyma et al., 2017 ). β-arrestins, beyond their canonical roles in binding phosphorylated G-protein coupled receptors (GPCR) to arrest signalling ( Gurevich, 2024 ), also scaffold MAPK signalling for cytosolic ERK activation ( DeFea et al., 2000 ; Luttrell et al., 2001 ; Tohgo et al., 2002 ). Interestingly, despite their divergent structures, these MAPK scaffolds display the same three critical features. First, they bind all three MAP kinases ( Choi et al., 1994 ; DeFea et al., 2000 ; Good et al., 2009 ; Inouye et al., 1997 ; Luttrell et al., 2001 ; McKay et al., 2009 ; Ory et al., 2003 ; Qu et al., 2021 ) and trigger allosteric kinase activation ( Bhattacharyya et al., 2006 ; Brennan et al., 2011 ; Kahsai et al., 2023 ; Lavoie et al., 2018 ; Zalatan et al., 2012 ; Zang et al., 2021 ). Through these interactions, scaffolds ensure transduction specificity, for instance directing signalling from a MAP3K shared between three cascades to a specific MAPK output in the case of Ste5, or from G-protein-driven GPCR signalling towards MAPK signalling in the case of β-arrestins ( Dhanasekaran et al., 2007 ; Witzel et al., 2012 ). Download figure Open in new tab Figure 1. Sms1 is a novel, essential component of pheromone signalling in fission yeast A. Schematic of MAPK signalling and scaffolds. B. Phospho-Spk1 (arrow) in membrane and soluble fractions from h90 proliferating vegetative cells in MSL+N (veg) and mating cells in MSL-N (mat). Tubulin serves as loading control. C. Phospho-Spk1 (arrow) in membrane fractions from h90 WT and ste4Δ cells in MSL-N. D. Mating efficiency of h90 WT, ste4Δ and byr2Δ cells ( n ≥500 cells) with error bars as s.d. **** P <0.0001. E. Volcano plot representing results from three Ste4-GFP co-immunoprecipitations in mating cells. The grey dotted line indicates p-values <0.05. F. Mating efficiency of heterothallic WT and sms1Δ cells ( n ≥500 cells) with error bars as s.d. **** P <0.0001. G. Phospho-Spk1 (arrow) in membrane fractions from WT, sms1Δ and byr1Δ cells in MSL-N. H. dPSTR analysis of MAPK Spk1 transcriptional output. Images and quantification of nuclear enrichment of the dPSTR reporter in h+ WT, sms1Δ and spk1Δ cells crossed with h- WT after 24 h on MSL-N. Unmated cells were chosen for analysis. Scale bar: 2μm. **** P <0.0001. Second, they localise and recruit the MAPK cascade to the cell surface by direct interaction with phospholipids and with membrane-localised factors, such as the active GPCR or the released Gβγ ( Bell et al., 1999 ; Brennan et al., 2011 ; Eichel et al., 2016 ; Garrenton et al., 2006 ; Inouye et al., 1997 ; Luttrell et al., 2001 ; Pryciak and Huntress, 1998 ; Winters et al., 2005 ; Yin et al., 2009 ; Zalatan et al., 2012 ). Finally, phosphorylation of the MAPK scaffold, in part promoted by MAPK-dependent feedback, negatively regulates the scaffold membrane localisation, preventing untimely activation or terminating signalling ( Cacace et al., 1999 ; Lin et al., 1997 ; Lin et al., 1999 ; McKay et al., 2009 ; Muller et al., 2001 ; Ory et al., 2003 ; Repetto et al., 2018 ; Strickfaden et al., 2007 ; van Drogen et al., 2019 ; Winters and Pryciak, 2019 ; Yu et al., 2008 ). In contrast to the highly conserved and ancient MAPK signalling cascade ( Huang et al., 2025 ), and despite having critical roles, MAPK scaffolds do not appear to be evolutionarily conserved. For instance, KSR and β-arrestins are restricted to Metazoa ( Alvarez, 2008 ; Huang et al., 2025 ). Similarly, Ste5 only exists in Saccharomycotina , where it is thought to have arisen upon MAPK duplication ( Cote et al., 2011 ). This raises the questions whether scaffolds are only required in some organisms, and how MAPK signalling is efficiently transduced in others. For instance, although MAPK signalling plays an essential role in transducing pheromone signalling during fission yeast mating and can be partially replaced by human or budding yeast orthologs ( Hughes et al., 1993 ; Neiman et al., 1993 ; Xu et al., 1994 ), no scaffold has been identified. Given the absence of shared kinases between different MAPK cascades in this organism ( Cote et al., 2011 ), we – and others – have considered for more than 30 years that no MAPK scaffold was involved in fission yeast mating ( Fig. 1A ) ( Cote et al., 2011 ; Davey, 1998 ; Hoffman, 2005 ; Hughes, 1995 ; Merlini et al., 2013 ; Sieber et al., 2023 ). Pheromone-triggered MAPK signalling governs sexual reproduction in fission yeast and is temporally strictly controlled. Pheromones, expressed by each mating type, bind cognate GPCRs on the partner cell, leading to activation of the coupled Gα Gpa1 and signal transmission through a MAPK cascade that comprises the Ras GTPase-binding MAP3K Byr2, the MAP2K Byr1 and the MAPK Spk1 ( Fig. 1A ) ( Sieber et al., 2023 ; Xu et al., 1994 ). Byr2 further binds an essential adaptor Ste4, which acts in parallel to Ras1-GTP, upstream of the MAP3K ( Barr et al., 1996 ; Tu et al., 1997 ). Sexual differentiation is suppressed during cell proliferation in rich environments and initiated by nitrogen starvation, which induces the de-repression of the transcription factor Ste11 that controls the expression of most pheromone cascade components ( Kawamukai, 2024 ; Mata and Bahler, 2006 ). Ste11 is itself a MAPK substrate and its transcriptional activity is further enhanced by pheromone-MAPK signalling, forming a positive feedback loop that drives the cells into the sexual differentiation programme ( Kjaerulff et al., 2005 ). Differentiation underlies the fusion of mating partners to form a diploid zygote, in which mating is actively suppressed and meiosis triggered to form 4 haploid spore progenies ( Vjestica et al., 2021 ; Vjestica et al., 2018 ). In addition to driving transcription, the pheromone signal is also interpreted spatially. Throughout mating, subcellular compartmentalisation of the information is crucial to ensure directional pheromone response resulting in partner selection and cell-cell fusion. Pheromone secretion and reception occur in small, restricted polarity patches that transiently form at the plasma membrane at mating onset ( Bendezu and Martin, 2013 ; Merlini et al., 2016 ), a behaviour conserved in budding yeast ( Dyer et al., 2013 ; Ghose et al., 2021 ; McClure et al., 2015 ; Wang et al., 2019 ). These transient patches, which contain the active form of Cdc42 GTPase, as well as the Gα Gpa1 and active Ras1-GTP, undergo cycles of assembly and disassembly until stabilised by increasing pheromone signalling that drives partner cells to grow towards each other ( Khalili et al., 2018 ; Merlini et al., 2016 ; Merlini et al., 2018 ). Patch proteins, as well as all MAP kinases, further localise at the mature polarity patch, where they promote cell-cell fusion to form the zygote ( Dudin et al., 2016 ). How the activated receptor transduces the pheromone signal and recruits the MAPK cascade to the plasma membrane during fission yeast mating has remained unknown. Here, we discover Sms1 as the essential scaffold of the MAPK signalling pathway in fission yeast mating. Sms1 is recruited to the plasma membrane by its hemi-arrestin domain and restricted to high pheromone signalling patches by specific recognition of the active Gα-GTP. We show that Sms1 is essential to assemble an active MAPK cascade at the membrane, by binding the MAP3K through the adaptor Ste4, as well as the MAP2K and MAPK. Interestingly, Sms1 is required even in presence of hyperactive MAP2K, demonstrating a role beyond kinase activation. Finally, we discovered that Sms1 is negatively regulated by phosphorylation, in part through the MAPK, to prevent pheromone hypersensitivity and untimely mating attempts. The shared features of Sms1 with other MAPK scaffolds – membrane recruitment, MAPK scaffolding, signal transduction and negative feedback regulation – but absence of sequence or structural homology demonstrates convergent evolution of the MAPK scaffolds. Results Sms1 is a novel, essential component of pheromone signalling in fission yeast To verify that pheromone-MAPK signalling occurs at the plasma membrane in fission yeast, we first checked if the active pool of MAPK Spk1 localises at the membrane. To this aim, we performed membrane extraction ( Klug et al., 2021 ; Vitali et al., 2024 ), which we validated by verifying enrichment of the membrane-associated receptor-coupled prenylated Gα Gpa1 protein (Fig. S1A). Activated, phospho-MAPK Spk1 was detected with the cross-reacting phospho-ERK antibody against the conserved TEY motif in ERK-like MAP kinases (Fig. S1B) ( Kelsall et al., 2025 ). This revealed that phosphorylated MAPK Spk1 is enriched in the membrane fraction in mating cells ( Fig. 1B ), demonstrating activation of the ERK-like MAPK Spk1 at the membrane. To identify novel interactors of the MAPK cascade, we focused on the MAP3K Byr2 adaptor Ste4, which belongs to the Ste50 family conserved throughout fungi ( Jung et al., 2011 ; Truckses et al., 2006 ). Ste4 is essential for the mating process as its deletion abrogates Spk1 phosphorylation and prevents mating ( Fig. 1C - 1D ) ( Okazaki et al., 1991 ). To identify novel Ste4 interactors, we immunoprecipitated endogenous Ste4-GFP, or GFP as negative control, from mating cell lysates and analysed the bead eluate by mass spectrometry. We identified 235 significantly enriched proteins, including the MAP3K Byr2 , thus validating the experimental design ( Fig. 1E , Table S1). Amongst Ste4 interactors, we focused on the poorly characterised protein SPAC23E2.03c (UniprotKB Q10136), previously known as Ste7 and shown to promote mating and meiosis ( Matsuyama et al., 2000 ). To avoid any confusion with the homonym MAP2K Ste7 in S. cerevisiae , we changed its name to Sms1 ( s caffold of M APK s ignalling for reasons below). Deletion of sms1 resulted in sterility, even when mated with a wildtype partner ( Fig. 1F ), a phenotype shared by loss of components of the pheromone-MAPK signalling pathway ( Nadin-Davis and Nasim, 1988 ; Obara et al., 1991 ). In agreement with the idea that Sms1 is a core component of MAPK signalling, loss of Sms1 almost fully abrogated MAPK phosphorylation ( Fig. 1G ). To probe its function in MAPK-dependent transcriptional response, we used the dynamic protein synthesis translocation reporter (dPSTR) assay, previously established in budding yeast ( Aymoz et al., 2016 ) ( Fig. 1H ). In the dPSTR assay, nuclear translocation of a constitutively expressed mCherry-SynZip1, which tightly binds a pheromone-induced P.fus1 :NLS-SynZip2, provides a dynamic measure of gene expression. Nuclear translocation in either mating type was abrogated in spk1Δ cells, demonstrating that the assay specifically reports on MAPK-dependent transcriptional response ( Fig. 1H , S1C). In sms1Δ , nuclear translocation was strongly reduced. Thus, Sms1 is an essential component for transduction of the pheromone-MAPK signal. Sms1 interacts with the MAP3K adaptor Ste4 via β-sheet augmentation To verify the interaction of Sms1 with Ste4, we performed co-immunoprecipitation of Sms1 and Ste4, which we expressed in mitotic cells ( Fig. 2A ). As this experiment was performed in proliferating cells that lack Ste11-dependent transcription of the mating-specific proteins ( Anandhakumar et al., 2013 ; Mata and Bahler, 2006 ; Styrkarsdottir et al., 1992 ), this result indicates that the interaction between Sms1 and Ste4 does not require other pheromone-induced factors and may be direct. Download figure Open in new tab Figure 2. Sms1 interacts with the MAP3K cofactor Ste4 via β-sheet augmentation A. Ste4-3xFLAG-mCherry co-precipitates with Sms1-sfGFP. Cell lysates from vegetative cells expressing Ste4-3xFLAG-mCherry and Sms1-sfGFP or GFP under nmt41 promoter were immunoprecipitated with anti-GFP beads and immunoblotted for FLAG or GFP. GFP serves as negative control. B . AlphaFold-Multimer (v2) prediction of the interaction between Ste4 RA domain and Sms1, showing Sms1 residues within 3 Å of Ste4 (aa 420-443). The predicted aligned error (PAE) plot and interface-predicted template modelling (ipTM) score are shown. C . Colocalisation of Pil1-Ste4-mCherry (WT or ΔRA) with Sms1-sfGFP (WT or ΔSBD). Scale bar: 2μm. Pearson correlation coefficient values are shown on the right ( n ≥12 cells) with error bars as s.d. **** P <0.0001. D . Yeast-two-hybrid assay showing that Sms1(400-470) is sufficient for interaction with Ste4-RA. E . Mating efficiency of h90 WT ( sms1-sfGFP ) and sms1 ΔSBD -sfGFP cells ( n ≥500 cells) with error bars as s.d. **** P <0.0001. F . Phospho-Spk1 (arrow) in membrane fractions from WT ( sms1-sfGFP ) and sms1 ΔSBD -sfGFP in MSL-N. Ste4 N-terminus binds the MAP3K Byr2 via SAM-SAM domain interaction, an interaction abolished in the SAM mutant Byr2 N28I (Fig. S2A) ( Barr et al., 1996 ; Ramachander et al., 2002 ; Tu et al., 1997 ). AlphaFold-based predictions indicated that the C-terminal Ras-associated (RA) domain of Ste4 would interact with aa 422-433 of Sms1 via β-sheet augmentation ( Fig. 2B ) ( Jumper et al., 2021 ; Mirdita et al., 2022 ). To test this prediction, we used a co-recruitment assay where an mCherry-tag bait is fused to Pil1 to target it to eisosomes, long invaginations in the plasma membrane of fission yeast, and recruitment of a GFP-tagged prey is assessed by microscopy ( Yu et al., 2021 ). As proof of principle, Pil1-Ste4 recruited MAP3K Byr2 and co-localisation was abolished by the N28I mutation in MAP3K Byr2 , but not by truncation of the Ste4 RA domain (Fig. S2B). Consistent with our co-immunoprecipitation results, Pil1-Ste4 strongly recruited Sms1 to eisosomes ( Fig. 2C ). Truncation of the Ste4 RA domain abolished the interaction with Sms1, as did the replacement of the 12-amino acid long β-strand in Sms1 ( sms1 ΔSBD ; Fig. 2C ). To probe the sufficiency of the predicted regions for Ste4-Sms1 interaction, we further performed yeast two-hybrid assay which confirmed the Ste4 binding domain of Sms1 to the 400-470 amino acid region ( Fig. 2D ). Interestingly, AlphaFold only predicts a β-strand in this region upon interaction with Ste4 and not in monomeric Sms1 ( Fig. 2B ). This interaction therefore represents an example of coupled folding of an intrinsically disordered region ( Holehouse and Kragelund, 2024 ; Wright and Dyson, 2015 ). Taken together, these results validate the AlphaFold structural prediction of interaction through β-sheet augmentation. To test the physiological relevance of the Ste4-Sms1 interaction, we deleted the Ste4 RA domain or the Sms1 Ste4-binding domain (SBD) from the respective endogenous genes. Cells with ste4 ΔRA had nearly absent phospho-Spk1 signal and were fully sterile, indicating this domain is indeed essential for function (Fig. S2C-S2D). The sms1 ΔSBD allele similarly showed strong reduction in Spk1 phosphorylation and prevented mating, confirming the importance of Sms1-Ste4 interaction for function ( Fig. 2E - 2F ). Sms1 localises to membrane-associated polarity patches and fusion site Tagging of Sms1 with sfGFP at the endogenous locus confirmed that Sms1 is expressed upon nitrogen starvation, and its expression is strongly increased by pheromone stimulation due to Ste11-dependent transcription (Fig. S3A) ( Mata and Bahler, 2006 ; Matsuyama et al., 2000 ; Xue-Franzen et al., 2006 ). It also revealed that Sms1-sfGFP localises in discrete membrane-associated patches in mating cells before accumulating at the point of contact between the two mating partners ( Fig. 3A ), where the actin fusion focus assembles to drive cell-cell fusion ( Dudin et al., 2015 ). The membrane-associated Sms1 patches in mating cells colocalised with the Cdc42 scaffold Scd2, indicating that Sms1 is present in mobile polarity patches prior to mating partner selection ( Fig. 3B ), like Ras1 and Gα Gpa1 ( Merlini et al., 2016 ; Merlini et al., 2018 ). Sms1 localisation mirrored that of Ste4, which also localised to mobile polarity patches ( Fig. 3C , 3D). Upon cell-cell fusion, Sms1 dissociated from its membrane bound domains and was cleared from the zygote (Fig. S3B) consistent with a previous report ( Matsuyama et al., 2000 ). A decrease in protein level upon cell-cell fusion was not observed for Ste4 (Fig. S3B), suggesting a distinct regulation on Sms1 in the zygote. Together, these results identify Sms1 as a novel, essential member of the pheromone signalling pathway that polarises to the cell surface during the mating process. Download figure Open in new tab Figure 3. Sms1 localises to membrane-associated polarity patches and the fusion site A. Mating of h-sms1-sfGFP . Arrowheads and arrow indicate localisation of Sms1 in polarity patches and fusion site, respectively. B-D. Colocalisation of (B) Sms1-sfGFP and Scd2-mCherry, (C) Sms1-sfGFP and Ste4-mCherry and (D) Ste4-GFP and Scd2-mCherry in polarity patches (arrowheads) in h90 mating cells. E. Endogenous Sms1-sfGFP in membrane and soluble fractions from h90 cells grown in MSL-N. Arrow indicates the size of full-length Sms1-sfGFP. F. Localisation of Sms1-sfGFP in h90 cells grown in MSL-N with or without expression of P.tdh1-NLS-GST-GBP to retain it in the nucleus, as shown on bottom scheme. Brightness and contrast in the presence of NLS-GBP is reduced to prevent signal saturation. G. Mating efficiency of cells as in (F). n ≥500 cells with error bars as s.d. **** P 0.05). H. Endogenous Sms1-sfGFP in membrane and soluble fractions from h90 cells expressing or not P.tdh1-NLS-GST-GBP grown in MSL-N. Scale bars: 2µm. Whole-cell extraction of mating cells expressing Sms1-sfGFP only showed low molecular weight fragments, in contrast to the strong full-length band of Ste4-GFP, suggesting that Sms1 is an unstable protein (Fig. S3C). Immunoprecipitation of Sms1-sfGFP fragments showed that the protein is ubiquitinated (Fig. S3D). Similarly, Sms1 overexpression in mitotic cells showed some full-length product, but a large fraction was also degraded (Fig. S3E). By contrast, expression of Sms1 in the hypomorphic mts3-1 proteasome mutant ( Gordon et al., 1996 ) strongly stabilised the full-length band of Sms1 (Fig. S3E), demonstrating that Sms1 is degraded by the ubiquitin-proteasome system. Since Sms1 is unstable yet essential for mating, we hypothesised that Sms1 might be differentially regulated at the membrane (polarity patches and fusion site) and the cytosol. Indeed, membrane extraction of endogenous Sms1-sfGFP revealed more abundant full-length protein, in contrast with the low-molecular weight degradation fragments present in the soluble fraction ( Fig. 3E ). High-molecular weight bands further suggested post-translational modifications of Sms1 ( Fig. 3E ). These results demonstrate that membrane-associated Sms1 is mostly full-length and thus largely protected from proteolysis. We noted that Sms1-sfGFP also localises to the nucleus (Fig. S3B), in contrast to the nuclear exclusion of Ste4, suggesting that a pool of Sms1 transits through the nucleus. To determine the functional importance of the different pools of Sms1 during mating, we engineered a system to retain Sms1 in the nucleus. Co-expression of GFP-binding protein (GBP) ( Rothbauer et al., 2006 ) tagged with a nuclear localisation sequence (NLS) retained Sms1-sfGFP in the nucleus, decreasing protein stability and drastically hindering mating efficiency ( Fig. 3F - 3H ). We conclude that nuclear export of Sms1 is necessary its function. This is consistent with a functional role at the plasma membrane, where the protein is largely protected from proteolysis. The arrestin domain of Sms1 interacts with the plasma membrane, where it is stabilised by active Gα Sms1 is predicted to be highly disordered, and its only folded domain is a highly unusual arrestin fold (Fig. S4A, S4B). Whereas canonical arrestin proteins require the interaction between their two arrestin lobes to prevent untimely activation ( Chen et al., 2018 ), Sms1 is predicted to have a single arrestin C-domain. However, this arrestin domain is remarkably similar in structure with the C-domain of the well-characterised classical arrestin proteins such as mammalian β-arrestin 2 ( Fig. 4A ). Interestingly, the Sms1 arrestin domain expressed in vegetative cells localised with the plasma membrane marker LactC2 ( Fig. 4B ) ( Yeung et al., 2008 ). Based on the recent identification of hydrophobic residues in the C-edge of β-arrestins-2 that function together with positively charged PIP-binding residues for insertion in the lipid bilayer ( Grimes et al., 2023 ), we mutated the corresponding amino acids in Sms1 arrestin domain to create a lipid-binding deficient mutant ( sms1 LBM ) ( Fig. 4A ), which failed to localise at the cell surface ( Fig. 4B ). Sms1 arrestin domain localisation to the plasma membrane was also abrogated in its3-1 , a temperature sensitive mutant of the enzyme converting PI4P into PI(4,5)P2 ( Zhang et al., 2000 ) ( Fig. 4C ), indicating this is the major phosphoinositide species required for Sms1 membrane interaction. The lipid binding affinity of the arrestin domain is thus conserved in Sms1, and this property is essential for Sms1 function as shown by the sterility of a sms1 LBM allele lacking the lipid-binding residues ( Fig. 4D ). Download figure Open in new tab Figure 4. The arrestin domain of Sms1 interacts with the plasma membrane, where it is stabilised by active Gα A. Sequence-independent structural alignment of Sms1 arrestin domain (blue) and human β-arrestin 2 (ARRB2; grey) AlphaFold2 predictions using PyMol-cealign with a root mean square deviation (RMSD) of 3.305 Å. Basic and hydrophobic residues mutated in the LBM mutant are highlighted in red. B . Colocalisation of sfGFP-tagged Sms1 arrestin domain (WT or with LBM mutation) with the plasma membrane (PM) marker mCherry-LactC2 expressed in vegetative cells. C . Localisation of Sms1 arrestin domain expressed in WT or its3-1 vegetative cells. D . Mating efficiency of h90 cells with indicated sms1 alleles at endogenous locus ( n ≥500 cells) with error bars as s.d. **** P 0.05). E . Sms1-sfGFP localisation in h-ste6Δ, ras1Δ and gpa1Δ cells mated with WT h+ Scd2-mCherry. Arrowheads indicate the localisation of Sms1 in polarity patches. Brightness and contrast of GFP in ste6Δ and in ras1Δ is reduced to prevent signal saturation. F . Localisation of Sms1 arrestin domain expressed under nmt41 promoter and native full-length Sms1-mCherry in mating cells. G-H . Gα-GTP co-precipitates with Sms1-sfGFP. Membrane fractions from vegetative cells expressing Gpa1-3xFLAG-mCherry and Sms1-sfGFP under the nmt41 promoter were immunoprecipitated with anti-GFP beads and immunoblotted for FLAG or GFP. In (G), Sms1 full-length, arrestin domain and middle fragment (aa 158-397) were used. In (H), Gpa1 was either WT, GDP-locked (G242A; GA) or GTP-locked (Q244L; QL). No GFP construct (G), GFP or the unrelated transmembrane protein GFP-Tna1 (H) were used as negative control. Scale bars: 2μm. The targeting of Sms1 arrestin domain to the entire plasma membrane of mitotic cells raised the question of its restriction at the polarity patches and fusion site. A possible explanation would be an indirect recruitment by Ras1 because this GTPase binds the MAP3K Byr2 ( Barr et al., 1996 ; Gronwald et al., 2001 ), localises to the polarity patches ( Merlini et al., 2018 ) and was proposed to recruit the MAP3K Byr2 to the membrane ( Bauman et al., 1998 ). However, upon deletion of Ras1 or its activator GEF Ste6, which strongly impair mating ( Fukui et al., 1986 ; Hughes et al., 1990 ; Nadin-Davis et al., 1986 ), Sms1 nevertheless polarised at the plasma membrane, forming unstable patches oriented towards WT partner cells ( Fig. 4E ). By contrast, Sms1 polarisation was abolished in absence of the Gα Gpa1 ( Fig. 4E ). Thus, Sms1 polarises to the plasma membrane upon pheromone signalling activation but independently of Ras1 activity. As an alternative hypothesis, we investigated whether Sms1 might bind the receptor-coupled Gα Gpa1 . When expressed in mating cells, Sms1 arrestin domain, but no other Sms1 fragment, not only localised to the plasma membrane but was enriched at the fusion site, suggesting that the arrestin domain contains further localisation information ( Fig. 4F , S4C). Co-immunoprecipitation showed that Sms1 interacts with Gpa1 in the membrane fraction, and that this interaction occurs through the arrestin domain ( Fig. 4G ). Furthermore, using active GTP-locked Gpa1 Q244L and inactive GDP-locked Gpa1 G242A ( Obara et al., 1991 ), we found that Sms1 preferentially binds active Gα ( Fig. 4H ). Together, these results demonstrate that Sms1 is targeted by its arrestin domain to the plasma membrane, where it recognises the GPCR-activated Gα, thus restricting Sms1 to high pheromone signalling domains. Sms1 is the MAPK scaffold of the pheromone signalling pathway The interactions of Sms1 with phospholipids, Gα Gpa1 and Ste4 and their essential role in sexual reproduction indicate that Sms1 functions at the top of the pheromone-MAPK signalling pathway. Given the function of Sms1 in transducing the MAPK signal, we also tested its possible interaction with the MAP2K Byr1 and MAPK Spk1 . To avoid expression of other mating signalling components, we overexpressed tagged proteins in vegetative cells, and further deleted the transcription factor Ste11 to prevent sexual differentiation by constitutively active MAP2K Byr1 , in which the conserved MAP2K dual phosphorylation sites are mutated to aspartic acid (Byr1 DD ; ( Ozoe et al., 2002 )). Co-immunoprecipitation experiments demonstrated that Sms1 binds active Byr1 DD in the membrane fraction ( Fig. 5A ). Similarly, we found that Spk1 co-immunoprecipitates with Sms1-GFP but not GFP alone ( Fig. 5B ). Thus, Sms1 associates with both MAP2K and MAPK. Download figure Open in new tab Figure 5. Sms1 is the MAPK scaffold of the pheromone signalling pathway A. V5-Byr1 DD co-precipitates with Sms1-sfGFP. Membrane and soluble fractions from vegetative ste11Δ cells expressing V5-Byr1 DD and Sms1-sfGFP under the nmt41 promoter were immunoprecipitated with anti-GFP beads and immunoblotted for FLAG or GFP. GFP serves as negative control. B. Spk1-3xFLAG-mCherry co-precipitates with Sms1-sfGFP. Whole cell extracts from vegetative cells expressing Spk1-3xFLAG-mCherry and Sms1-sfGFP or GFP under nmt41 promoter were immunoblotted for FLAG and GFP. C. Phospho-Spk1 co-precipitates with endogenous Sms1-3xFLAG-mCherry. Membrane fractions from endogenous Sms1-3xFLAG-mCherry h90 cells grown in MSL+N(veg) or MSL-N(mat) were immunoprecipitated with anti-FLAG beads and immunoblotted for phosphorylated Spk1 (anti-pERK1/2) and FLAG. D. Endogenous Sms1-3xFLAG-mCherry co-precipitates with phospho-Spk1. Membrane and soluble fractions from h90 cells in mating conditions (MSL-N) were immunoprecipitated with anti-pERK1/2 beads and immunoblotted for FLAG and pERK1/2. E. Phospho-Spk1 (arrow) in membrane fractions from h90 byr1 DD and h90 byr1 DD sms1Δ cells in MSL-N. F. Quantification of nuclear enrichment of the dPSTR reporter in h90 byr1 DD with spk1Δ , sms1Δ or otherwise WT (control) after 24 h on MSL-N. **** P <0.0001. G. DIC images and shmooing efficiency of h90 byr1 DD with sms1Δ , ste4Δ , spk1Δ or otherwise WT (control) in MSL-N. Cell wall accumulation was present in all conditions. Error bars represent s.d. **** P 0.05). Scale bars: 2μm. To probe for the complex formed during mating, we used the phospho-specific pERK antibody. Endogenous FLAG-tagged Sms1 co-immunoprecipitated the endogenous, active phopho-MAPK Spk1 in the membrane fraction ( Fig. 5C ). Conversely, pERK beads pulled down endogenous Sms1 – remarkably only the full-length protein – from the membrane fraction ( Fig. 5D ). Thus, the active MAPK Spk1 specifically interacts with full-length Sms1, further supporting the stabilisation of the active Sms1 complex at the plasma membrane to ensure local MAPK activation. Together, these data demonstrate that Sms1 is a novel MAPK scaffold, which promotes pheromone signalling by associating with each single component of the signalling cascade, including the Gα Gpa1 , the MAP3K Byr2 through its adaptor Ste4, the active MAP2K Byr1 and MAPK Spk1 . We thus renamed the protein as S caffold for M APK S ignalling 1 (Sms1), which also avoids confusion with S. cerevisiae MAP2K. The scaffold is stabilised at the plasma membrane in association with the active kinase cascade. To probe whether Sms1 is solely required for MAPK activation, we used byr1 DD strains, in which the native byr1 gene encodes a constitutively active MAP2K Byr1 . In these conditions, we expected the MAPK to remain constitutively activated by Byr1 DD irrespective of the presence of the scaffold. Indeed, loss of Sms1 only moderately decreased pSpk1 level ( Fig. 5E ) and MAPK-dependent transcriptional response measured by the dPSTR assay ( Fig. 5F ). Phenotypically, byr1 DD at the endogenous locus causes a fus (fusion-defective) phenotype characterised by constitutive growth of mating projections (known as shmooing; Fig. 5G ) ( Dudin et al., 2016 ; Ozoe et al., 2002 ). These cells also show accumulation of cell wall. In byr1 DD cells , shmooing was abrogated by deletion of downstream signalling components, such as the MAPK Spk1 , but not of upstream ones, such as ste4 ( Fig. 5G ). Unexpectedly, in absence of Sms1, byr1 DD cells showed a total loss of the shmooing phenotype ( Fig. 5G ). This indicates that, in addition to acting as a scaffold that promotes MAPK signal transduction, Sms1 is also required for local MAPK output to induce the formation of the mating projection. Negative feedback by Spk1 removes Sms1 from the plasma membrane to prevent untimely mating attempts The presence of high-molecular weight bands suggested that Sms1 is regulated by phosphorylation ( Fig. 3E ). Indeed, these high-molecular weight bands were largely abrogated by phosphatase treatment ( Fig. 6A ). Sms1 phosphorylation was further supported by its interaction in vegetative cells with the phospho-binding proteins 14-3-3, Rad24 (Fig. S5A). To test if Sms1 might be regulated by proline-directed kinases such as the MAPK, we mutated to alanine all Sms1 serine and threonine residues followed by a proline (Sms1 22A ) ( Fig. 6A ). High-molecular weight bands were not observed for Sms1 22A and phosphatase treatment did not change its migration pattern, demonstrating that Sms1 expressed in vegetative cells undergoes proline-directed phosphorylation ( Fig. 6A ). Download figure Open in new tab Figure 6. Negative feedback by phosphorylation removes Sms1 from the plasma membrane to prevent untimely mating attempts A . Membrane fractions from Sms1-sfGFP and Sms1 22A -sfGFP expressed under nmt41 promoter in vegetative ste11Δ cells before and after alkaline phosphatase (AP) treatment. A schematic of Sms1 with all SP and TP sites, indicating those mutated in Sms1 22A/E and Sms1 4A/E , is shown at the top. B . Localisation of native Sms1 22A -sfGFP or Sms1-sfGFP in h+ starved WT and spk1Δ cells. Quantification of the intensity of membrane patches is shown at the bottom ( n >40 cells). **** P <0.0001. Quantification of the number of patches is shown in Fig. S5B. C . Time points and kymographs from timelapse imaging of Sms1-sfGFP and Sms1 22A -sfGFP in h-sxa2Δ cells treated with 10 nM P-factor for 60 min. Arrowheads indicate polarity patches. Asterisks indicate cell poles. Contrasts are identical in the kymographs of both strains to illustrate the different amounts of protein at the membrane, but reduced for Sms1 22A -sfGFP time points to prevent signal saturation. Additional examples are shown in Fig. S5C. D . Quantification of shmoo length in h-sxa2Δ sms1+ or sms1 22A cells upon treatment with 0-1000 nM P-factor for 24 hrs (n>350 cells). **** P 0.05) with error bars as s.d. E . Phospho-Spk1 in membrane fractions from h90 sms1+ or sms1 22A grown in MSL+Glutamate. F. Mating efficiency of strains as in (E). G-H . Timelapse imaging of (G) sms1 22A and sms1 22E , and (H) sms1 4A and sms1 4E h90 mutant cells during mating. WT and mutant forms of Sms1 are tagged with sfGFP at the endogenous locus. Arrowheads point to ectopic polarisation in the zygote leading to polarised growth towards other mating partners. Brightness and contrast are reduced for Sms1 22A -sfGFP to prevent signal saturation. The strains’ mating efficiency is shown on the right ( n ≥500 cells) with error bars as s.d. **** P 0.05). Scale bars: 2μm. To probe the functional role of Sms1 phosphorylation, we expressed Sms1 22A -sfGFP as sole copy under the endogenous promoter, similarly as for Sms1 WT -sfGFP. While Sms1 formed patches at the membrane in mating cells ( Fig. 3 ), it remained mostly cytosolic upon its starvation-induced expression in cells not exposed to pheromones ( Fig. 6B ). By contrast, we found that, upon starvation-induced expression, Sms1 22A -sfGFP was strongly enriched in membrane localised patches ( Fig. 6B , S5B). Remarkably, deletion of MAPK Spk1 was sufficient to induce a similar, though less extensive, increase in plasma membrane accumulation of Sms1 WT ( Fig. 6B , S5B). We conclude that MAPK Spk1 likely phosphorylates Sms1 and prevents its accumulation at the plasma membrane. Sms1 22A exhibited gain-of-function phenotypes at all stages of the mating process. First, we focused on the response to synthetic pheromone of a single mating partner. For these assays, we used M cells lacking the P-factor protease Sxa2, preventing degradation of the synthetic pheromone. In response to 10 nM P-factor, these cells exhibit dynamic polarity patches, to which Sms1-sfGFP localises ( Fig. 6C ) ( Bendezu and Martin, 2013 ; Merlini et al., 2016 ). By contrast, sms1 22A cells formed stable, higher-intensity patches that were largely constrained to cell poles, often simultaneously at both cell poles ( Fig. 6C , S5C, Video 1). These stable patches induced polarised growth, and sms1 22A cells formed shmoos of increasing length at all P-factor dosages tested, even when WT cells did not ( Fig. 6D ). This hypersensitivity of sms1 22A cells to pheromone indicates that Sms1 phosphorylation normally limits the pheromone response. Second, we examined mating initiation. Mating is induced by nitrogen starvation in fission yeast and the presence of glutamate as nitrogen source largely suppresses mating in WT cells ( Berard et al., 2024 ; Egel, 1971 ). By contrast, sms1 22A mutant cells presented hyperactivation of Spk1 during growth in glutamate-containing medium, leading to high mating in these conditions ( Fig. 6E - 6F ). This indicates that the very low levels of Sms1 during mitotic growth are normally phosphorylated, consistent with its association with 14-3-3 proteins, and that this prevents mating activation. Finally, we looked at mating termination in zygotes. In mating assays sms1 22A cells formed pairs and fused efficiently but exhibited a striking phenotype upon cell-cell fusion. In contrast to the rapid signal disappearance of Sms1-sfGFP in zygotes, Sms1 22A -sfGFP remained polarised at the plasma membrane, leading to growth of the zygote towards other mating partners ( Fig. 6G , Video 2). Thus, phospho-regulation of Sms1 is required to switch off signalling and to prevent inappropriate mating responses in zygotes. Conversely, a phospho-mimetic allele, sms1 22E , abolished plasma membrane recruitment and led to a sterility phenotype ( Fig. 6G ). Sms1 22E also blocked the fus phenotype of constitutively active MAP2K byr1 DD cells (Fig. S5D), further supporting a phosphorylation-dependent inhibition of Sms1. While we did not systematically map which of the 22 sites cause the described phenotypes, we found that the four phosphorylation sites in the arrestin domain play a key role, as Sms1 4A showed localisation to the membrane of the zygote, while Sms1 4E was sterile ( Fig. 6A , 6H). We note that other phosphorylation sites also contribute to Sms1 function, as sms1 4A was less potent than sms1 22A in inducing persistent growth in zygotes. Together, our results show that the Sms1 scaffold undergoes a negative feedback loop where its associated MAPK counteracts its excessive surface accumulation, preventing untimely mating attempts. Discussion In this paper, we identify Sms1 as a structurally novel MAPK scaffold essential for fission yeast mating. Sms1 exhibits key features of a scaffold protein: i) it interacts with all three MAP kinases, ii) it localises to the plasma membrane, where it assembles the MAPK cascade for local signalling, and iii) it is negatively regulated by phosphorylation to promote membrane detachment and signalling arrest. Our data support the following model of the regulatory steps of the MAPK scaffold Sms1 ( Fig. 7A ). Transient interactions of Sms1 hemi-arrestin domain with phospholipids at the plasma membrane allows it to survey the cell surface. Upon pheromone perception in proximity of a mating partner, the now activated Gα-GTP stabilises Sms1 at the membrane, forming a polarity patch. Sms1 recruits the MAP3K adaptor, the MAP2K and the MAPK, assembling the MAPK cascade. On Sms1, spatial proximity of the kinases facilitates their sequential activation, transducing the signal to trigger downstream global signalling, and brings the activated MAPK in proximity to local substrates to promote morphogenesis. To limit signalling, the MAPK in turn phosphorylates Sms1, triggering its release from the plasma membrane. This negative feedback loop promotes exploratory patch dynamics and restricts signalling to prevent untimely mating behaviours. Download figure Open in new tab Figure 7. Model of Sms1 scaffold function A. Sms1 is recruited to sites of pheromone receptor activation through PI(4,5)P2 and Gα binding, where it assembles the MAPK cascade. This promotes local activation of the MAPK Spk1 , which induces sexual differentiation and local signalling for pheromone-directed growth. MAPK Spk1 also phosphorylates Sms1, promoting its detachment from the membrane and signal arrest. This negative feedback is critical to reduce pheromone sensitivity and promote patch dynamics during mating, and to prevent untimely mating of vegetative cells and zygotes. B. Although sharing essential roles in MAPK binding and membrane recruitment, and negatively regulated by phosphorylation, MAPK scaffolds across phyla have diverse evolutionary origins. Roles of the Sms1 scaffold Because Sms1 binds the active Gα-GTP at the plasma membrane and all MAPKs, it constitutes a key adaptor between pheromone-receptor engagement and downstream signal transmission, thus ensuring the MAPK cascade is activated at sites of pheromone perception. Localised membrane-binding occurs upon coincidence detection of phospholipids and the activated Gα-GTP, which is conceptually similar to Ste5 binding phospholipids and free Gβγ ( Garrenton et al., 2006 ; Inouye et al., 1997 ). Membrane recruitment is essential for Sms1 function, in absence of which there is no MAPK activation. Mechanistically, a key step to activate the MAP3K Byr2 is its binding by Ras1-GTP, similar to Raf activation by Ras GTPase in mammalian cells ( Masuda et al., 1995 ; Nussinov et al., 2019 ; Tu et al., 1997 ; Van Aelst et al., 1993 ). A second MAP3K Byr2 activation event may involve phosphorylation by p21-activated kinase ( Tu et al., 1997 ), similar to MAP3K activation in S. cerevisiae ( Drogen et al., 2000 ). Both reactions take place at the plasma membrane, to which Sms1 recruits MAP3K Byr2 via Ste4. This is consistent with previous genetic epistasis showing that Ste4 and Ras1 form two additive inputs to MAP3K Byr2 activation ( Barr et al., 1996 ; Tu et al., 1997 ). Thus, one role of Sms1 is as an adaptor that recruits and concentrates the MAPK cascade to sites of receptor engagement in vicinity of Ras1 at the membrane. This raises the question whether Sms1 “simply” acts as a membrane adaptor to initiate signalling or whether it is also important to transduce the signal through the consecutive kinases. We have shown that, when the MAP2K is constitutively activated, Sms1 only plays a minor role in the activation of the MAPK Spk1 . While this result suggests that Sms1 facilitates MAPK Spk1 phosphorylation by the MAP2K, perhaps by bringing them in proximity to favour their direct interaction ( Yang et al., 1998 ), it speaks against a major role of Sms1 in allosteric MAPK activation. This is different from the allosteric role of the budding yeast scaffold Ste5, which is required for phosphorylation of MAPK Fus3 (but not the second MAPK Kss1) even in presence of activated MAP2K ( Bhattacharyya et al., 2006 ; Flatauer et al., 2005 ; Good et al., 2009 ). Ste5 evolution and allostery were proposed to coincide with MAPK duplication and re-use of MAPK in distinct cascades in budding yeast ( Cote et al., 2011 ; Coyle et al., 2013 ). The mechanistic difference we observe in Sms1 is in line with the observations that there is no MAPK duplication in fission yeast, nor re-use of the same MAPK in distinct cascades, simplifying the specificity of signal transmission. Whether Sms1 is necessary for activation of the MAP2K Byr1 through allosteric effects, as observed for KSR ( Brennan et al., 2011 ), awaits future exploration. One interesting observation is that, even though Sms1 is not essential for MAPK Spk1 activation by the hyperactive MAP2K Byr1 DD nor for global transcriptional output, the scaffold is essential for the local polarised response. Thus, Sms1 has a critical function in promoting local signalling. This function could arise from positioning the cascade at the right place, thus ensuring local activation of relevant substrates at the cell cortex to drive polarised growth. Sms1 may also directly promote substrate specificity by recruiting substrates to the MAPK. One question this raises is whether Sms1 may be instructive in linking receptor activation to the polarity machinery. In principle, given the known interactions of Sms1-Ste4-MAP3K Byr2 -Ras1 ( Barr et al., 1996 ; Tu et al., 1997 ), Sms1 could recruit Ras1 via Ste4-MAP3K Byr2 , but this hypothesis is inconsistent with the observation that byr1 DD shmoo formation is not impaired by ste4 deletion. Furthermore, Sms1 forms restricted patches towards mating partners even in ras1Δ cells, indicating spatial signal interpretation independently of Ras1 GTPase. In budding yeast, two distinct scaffolds with similar domain composition, Ste5 and Far1, link the Gβγ to the MAPK cascade and the Cdc42 polarity module, respectively. However, neither exists in S. pombe ( Cote et al., 2011 ). Future experiments should establish the roles of Sms1 scaffold in spatial interpretation of the pheromone signal. A negative feedback loop on Sms1 prevents MAPK hyperactivation Negative regulation of plasma membrane association by phosphorylation is a recurring feature in MAPK scaffolds ( Witzel et al., 2012 ). In keeping with this theme, phosphorylation of proline-directed serine and threonine residues strongly inhibits Sms1 membrane association. Reflecting the major role of the hemi-arrestin domain in binding the membrane, this domain contains up to four critical phosphorylation sites. Future work should evaluate how phosphorylation interferes with membrane binding as these sites are not in immediate proximity to the phospholipid-binding basic residues on the concave side of the arrestin domain. Because Sms1 membrane localisation is antagonised by the MAPK Spk1 , a likely scenario is that MAPK Spk1 targets (some of) these sites to promote Sms1 membrane detachment. Thus, a negative feedback loop – where Sms1 promotes the activation of MAPK Spk1 at the cortex, which in turn induces Sms1 membrane detachment – regulates MAPK output. Negative regulation of the MAPK scaffold is critical to prevent activation by spurious noise signal and to prevent response saturation ( Locasale et al., 2007 ). Mating in fission yeast relies on the formation of a dynamic polarity patch that undergoes assembly-disassembly cycles at the cortex to sample potential mating partners ( Bendezu and Martin, 2013 ; Merlini et al., 2016 ). We show that the Sms1-MAPK Spk1 negative feedback contributes to patch instability by destabilising the patch. Indeed, in non-phosphorylatable sms1 22A cells, patches are long-lived and more intense, resulting in pheromone hypersensitivity. This negative feedback likely combines with other negative regulations of patch components, including inactivation of Ras1 GTPase by its GAP Gap1 ( Merlini et al., 2018 ) and of Cdc42 GTPase by its GAP Rga3 ( Gallo Castro and Martin, 2018 ). One open question is how the patch stabilises despite negative feedback on Sms1 at the incipient fusion site. One possible scenario could be the pheromone-dependent recruitment of an antagonist phosphatase, similar to the stimulated recruitment of PP2A that stabilises KSR1 at the membrane (Ory 2003). Remarkably, Sms1 phosphorylation is also crucial to prevent untimely activation of the pathway in rich medium, and to terminate signalling at the end of mating. Low-level Spk1 expression may help repress stochastic pathway activation during mitotic growth in nitrogen-rich medium. More likely, however, additional kinases active in these conditions contribute to Sms1 phosphorylation. This would be similar to the regulation of Ste5, which is phosphorylated by both CDK1 and the MAPK Fus3 on partially overlapping sites to antagonise membrane binding ( Choudhury et al., 2018 ; Repetto et al., 2018 ; Strickfaden et al., 2007 ; Winters and Pryciak, 2019 ). Establishing how Sms1 is rapidly phosphorylated post-fusion to prevent zygote mating is a key open question. In summary, Sms1 is both an essential component of the mating MAPK cascade and a central regulatory hub that ensures timely mating behaviours. Sms1 hemi-arrestin and IDR as key determinants of its scaffolding function Sms1 presents a single arrestin domain, which can be considered a hemi-arrestin as opposed to the canonical two-domain arrestin fold of the arrestin family. This hemi-arrestin domain provides affinity for phosphoinositides at the membrane via the presence of a basic patch on the concave side, a characteristic conserved throughout the arrestin family (Fig. S5E). The Metazoa-specific β-arrestin subgroup emerged from the more ancient α-arrestins, which are present throughout eukaryotes and typically recruit ubiquitin ligases through a PY motif to promote the ubiquitination and subsequent degradation of receptors and other membrane proteins ( Alvarez 2008 ; Zbieralski 2022). Our work defining Sms1 as MAPK scaffold, the observation that it does not contain a PY motif, and our inability to detect interaction with the pheromone receptor by co-immunoprecipitation or two-hybrid assay, indicate that Sms1 does not play a typical α-arrestin function. Because β-arrestins are only found in Metazoa, Sms1 and β-arrestins likely independently acquired MAPK interfaces to function as signalling scaffolds ( Fig. 7B ). The similarity may even extend further, as suggested by the presence on the convex side of Sms1 hemi-arrestin of a DEF MAPK-docking motif, which structurally closely aligns with the lariat loop of β-arrestin 1 recently identified as a MAP kinase interface (Fig. S5E) ( Qu et al., 2021 ). Sms1 scaffolding function also requires its extensive intrinsically disordered regions (IDRs) in the C-terminus, notably for Ste4-MAP3K interaction. IDRs may facilitate evolutionary convergence as they provide structural and conformational plasticity for the appearance of multivalent interactions with the kinases, for example upon coupled binding. In addition, IDRs are particularly prone to be regulated by multisite phosphorylation, thus favouring feedback regulation by the kinases and leading to the formation of signalling switches ( Wright and Dyson, 2015 ). Convergent evolution of the MAPK scaffolds Beyond the evolutionary re-use of the arrestin fold in a MAPK scaffold, Sms1 displays extensive functional and regulatory similarities with the RING-containing budding yeast Ste5 and the MAP3K-derived mammalian KSR1/2 proteins, with whom it shares no sequence or structural homology. This discovery further establishes evolutionary convergence as a principle for the emergence of MAPK scaffolds ( Fig. 7B ) ( DiRusso et al., 2022 ; Witzel et al., 2012 ). We want to emphasise that the convergent evolution of Sms1, Ste5 and KSR1 does not only concern their affinity for the MAP kinases, but also specific features of these proteins such as their translocation to the plasma membrane and their inhibition by phosphorylation. This similarity is particularly striking between Sms1 and Ste5, which both scaffold a MAPK module that transduces pheromone signalling, which elicits highly similar physiological processes in divergent yeasts of the ascomycete lineage. Yet another MAPK scaffold, HAM-5 with a Gβ-like fold, is proposed to link a functionally similar MAPK cascade underlying hyphal fusion in filamentous ascomycetes ( Dettmann et al., 2014 ; Jonkers et al., 2014 ). Remarkably, although not evolutionarily conserved, each of these proteins has become indispensable for signalling through the MAPK cascade they scaffold. This raises a fundamental conundrum about their evolution. Did ancestral cells contain scaffolds, which were lost and supplanted by new ones in extant species? Or did scaffolds simultaneously appear in various lineages? In either case, under what evolutionary pressures were these new, essential components co-opted? Limitations of the study Our model is based on a combination of microscopy of endogenously tagged proteins and co-immunoprecipitation of proteins ectopically expressed in mitotic cells, which lack activation of the mating pathway, allowing to conclude that these interactions are likely direct. The Sms1-Ste4 interaction was also confirmed by mutational analysis. However, in vitro reconstitution of the biochemical complex formed by Sms1 and the MAP kinases will be informative to map protein interfaces, quantify interaction strength and probe kinetics and mechanism of kinase activation. We have shown that phosphorylation of Sms1 is essential to prevent pheromone hypersensitivity and spurious induction of mating in rich medium and in the zygote. While the MAPK Spk1 contributes to this phosphorylation thus forming a negative feedback loop on Sms1, we have not examined the possible function of other proline-directed kinases to negatively regulate Sms1 membrane binding. Material and Methods Resource tables View this table: View inline View popup Download powerpoint Table S1: List of the 20 most enriched Ste4 interaction partners identified by mass spectrometry. View this table: View inline View popup Table S2: List of strains All strains are prototroph except otherwise indicated. View this table: View inline View popup Download powerpoint Table S3. List of antibodies and magnetic beads View this table: View inline View popup Download powerpoint Table S4. List of software and algorithms AlphaFold predictions and structural alignments Interaction between Sms1 C-terminus (aa 350-569) and Ste4 RA domain (aa 176-264) was predicted using AlphaFold-multimer on ColabFold with 20 recycles ( Mirdita et al., 2022 ). Sequence independent alignments between the arrestin domain of Sms1 and β-arrestin 1 or 2 was performed by incremental combinatorial extension (CEAlign) ( Shindyalov and Bourne, 1998 ) on PyMol (Schrödinger). Strains, media and growth conditions Strains used in this study are listed in Table S2. Transformations of S. pombe and S. cerevisiae cells were done according to standard genetic manipulation methods. Cells were grown in MSL supplemented with appropriate amino acid and nitrogen source (ammonium (N) or glutamate (E)) for at least 18 hrs at 30°C to reach optical density at 600 nm (OD600) between 0.5 and 1. For mating experiments, cells grown in MSL+E were washed three times with MSL-N and resuspended in MSL-N at OD600 of 1.6. For stimulation with P-factor cells were pre-grown in MSL+N and similarly washed with MSL-N Heterothallic strains were grown separately and mixed at 1:1 ratio just before MSL-N washes. To avoid flocculation in MSL+N, byr1 DD expressing strains were grown in YE until being washed and resuspended in MSL-N. The temperature-sensitive its3-1 cells were grown in MSL+N at 25°C, mounted on MSL+N 2% agarose pad and incubated at 25°C or 32°C for 5 hrs before imaging. Yeast two-hybrid assay All plasmids were generated using standard molecular biology techniques. Two-hybrid assays were performed by co-transformation of the pGAD and pGBD plasmids containing the Ste4, Sms1 and Byr2 variants in AH109 host strain (Clontech). Interactions were assessed by growth on selective SD medium lacking histidine and supplemented with 3-amino-1,2,4-triazole (3AT). Mating assays Cells were grown for 36 hrs in MSL+E at 30°C to reach optical density at 600 nm (OD600) between 0.5 and 1. Cells were washed three times with MSL-N and resuspended in MSL-N at OD600 of 1.6. Cells were mounted on an MSL-N 2% agarose pad before incubation at 30°C for 24 hrs or at 25°C for 48 hrs. Mating efficiency was measured as the number of mating pairs and zygotes multiplied by two and divided by the total number of cells, as previously described in ( Vjestica et al., 2016 ). Mating efficiency in MSL+E ( Fig. 6E , 6F) was performed similarly without MSL-N washes and imaging was performed directly after mounting the cells on an MSL+E 2% agarose pad. Shmooing efficiency was measured as the number of cells exhibiting polarised growth divided by the total number of cells. Microscopy All images in Figs 1 , 3 to 6 and Figs S1, S3 to S5 (except Figs 4B , 4C , 4F , 6B and S4C) were acquired on a DeltaVision platform (Applied Precision) composed of a customised inverted microscope (Olympus IX-71), a UPlan Aporchromat 100x/1.4 NA oil objective), a camera (Photometrics CoolSNAP HQ2) and a colour combined unit illuminator (Insight SSI 7, Social Science Insights). Images were acquired using softWoRx v4.1.2 software (Applied Precision). Super resolution imaging in Figs 2C , 4B , 4C , 4F, 6B, S2B and S4C were performed using a Zeiss LSM 980 scanning confocal microscope fitted on an inverted Axio Observer 7 microscope with Airyscan2 detector optimised for a 63x/1.40 NA oil objective and 488 nm and 561 nm lasers. Imaging was set in super resolution mode with a maximum of 7 frame scanning speed. Laser power was used at <2.5% with pixel time below 2.3 μs. Images were acquired by the ZEN Blue software (Zeiss). Image analysis dPSTR For quantification of dPSTR signal in Fig. 1H , S1C and 5F, three-channel images (DIC, mCherry and DAPI) were acquired on a DeltaVision microscope. The DIC channel was used to determine the cell contour, the mCherry channel labels the dPSTR signal and the DAPI channel allows visualisation of the nucleus by detecting NLS-mtagBFP2. For experiments in Fig. 1H and S1C, heterothallic WT, spk1Δ or sms1Δ cells expressing the dPSTR construct ( P.pom1 :mCherry-SynZip1 P.fus1 :NLS-SynZip2) together with NLS-mtagBFP2, were mixed with untagged heterothallic cells of opposite mating type. For experiment in Fig. 5F homothallic byr1 DD , byr1 DD spk1 Δ or byr1 DD sms1Δ cells expressing the dPSTR construct ( P.pom1 :mCherry-SynZip1 P.fus1 :NLS-SynZip2) together with NLS-mtagBFP2 were used. Images were acquired 24h after MSL-N washes. Individual cell masks were semi-automatically generated from the DIC image using the Segment Anything Model (SAM) ( Kirillov et al., 2023 ). To identify nuclei within these cells, candidate regions were extracted from the BFP channel. Nuclear detection was then performed using an automated pipeline combining convolutional filtering, percentile-based thresholding, and morphological post-processing, ensuring robust segmentation of nuclear areas. The resulting nuclear mask was compared with the cell mask to define both nuclear and cytoplasmic compartments, with the cytoplasm defined as the cell mask excluding the nucleus. These masks were overlaid with the dPSTR fluorescence signal in the mCherry image, enabling quantitative extraction of mean fluorescence intensities. To normalise for background signal, the nucleus-to-cytoplasm ratio (N/C) was computed as follow: (Inuc - Ibg) / (Icyto - Ibg), where Inuc, Icyto and Ibg represent the mean fluorescence intensities of the nucleus, cytoplasm, and background, respectively. Background signal was estimated as the signal excluding tagged cells. Colocalisation in co-recruitment assay The Pearson correlation coefficient was used to quantify colocalisation in the Pil1 co-recruitment assay in Fig. 2C and S2B as previously described in ( Yu et al., 2021 ). In brief, background and cytosolic signals were subtracted in each channel. For each condition, at least 10 individual cells were manually selected as regions of interests (ROIs) using the freehand selection tool. Pearson’s R values (no threshold) for each ROI were obtained using Fiji’s Coloc 2 plugin. Measure of fluorescence zygotic intensity of Sms1 and Ste4 To quantify Sms1 and Ste4 fluorescence intensities in zygotes, movies were aligned using the Fiji MutliStackReg plugin ( Thevenaz et al., 1998 ) to minimise stage drift. In both channels, background subtraction was performed using a Rolling Ball algorithm with a radius equal to the width of a cell. An elliptic region of interest (ROI) was defined to measure cytoplasmic fluorescence in the zygote, in a region that does not overlap with the nuclei at any of the timepoint. Mean grey values of the ROIs were measured for each timeframe from the first frame after cell-cell fusion (defined as the loss of GFP signal at the fusion site). For each channel, fluorescence intensities were normalised by the average signal of the first frame after cell-fusion. Fluorescence intensity of Sms1-sfGFP membrane patches Quantification of membrane patches of Sms1-sfGFP or Sms1 22A -sfGFP in Fig. 6B was performed on images acquired on an LSM980 Zeiss confocal microscope with Airyscan2 with 16x averaging. For quantification of single cells, a 3-pixel wide segmented line was designed around the total cell contour to quantify the signal at the membrane, and an additional 3-pixel wide segmented line was created to measure internal signal. Normalisation was performed by dividing the perimetral fluorescence intensity at each point by the average internal signal of each cell. Normalised intensity of the membrane patches was performed for at least 40 cells per strain. The area under the curve of the fluorescence intensity was determined for each strain with baseline of 1.5 using GraphPad Prism. The membrane patches (total peak area per cell) were plotted in Fig. 6B , and their frequency (number of peaks per cell) were plotted in Fig. S5B. Shmoo length Quantification of shmoo length in Fig. 6D was performed on binary cell masks from DIC images acquired on a DeltaVision microscope. The masks were first skeletonised using a previously described approach ( Lee et al., 1994 ). To increase robustness, secondary skeleton branches were removed, leaving only the main trunk. The endpoints of this trunk were then identified, and the connecting path was extended until it intersected with the cell boundary at both tips, ensuring that the axis spanned the full cell length. Finally, spline interpolation was applied to the extended path to generate a smooth continuous curve representing the major axis of the cell. Total cell length was defined as the length of this major axis. For each strain, the shmoo length for each condition was determined by subtracting the average cell length of untreated cells (0 nM P-factor).. SDS-PAGE and western blotting Lysis buffer (LB, 50 mM Tris-HCl pH7.4, 200 mM NaCl, 1mM EDTA) was prepared by supplementing it with protease inhibitors (LBi, protease inhibitor cocktail (Roche 11836145001), 0.7 μM Antipain, 0.08 μM Aprotinin, 0.8 μM Chymostatin, 1μM Leupeptin, 0.7 μM Pepstatin A, 5μM 1,10 Phenanthroline, 3.5μM Benzamidine-HCl, 100 μM PMSF (Phenylmethylsulfonyl fluoride)). PhosSTOP (Roche, 4906837001) was also added in LBi for detection of phospho-Spk1. 100 ODs of cells were lysed in 400 μl of LBi + 1% LMNG (Lauryl Maltose Neopentyl Glycol) + 0.1% CHS (Cholesteryl Hemisuccinate) by 4 cycles of 30 sec of bead-beating at 4°C with 2 min on ice between cycles. Cell lysates were eluted into collection tubes by centrifuging pierced tubes at 400 g for 20 min at 4°C. Lysates were cleared by centrifuging at 13’000 g for 20 min at 4°C. Protein concentration was determined by Bradford assay. Equalised protein amounts were denatured at 65°C for 15 min in 4x NuPAGE LDS sample buffer containing 5% β-mercaptoethanol. Samples were run in 4-12% and 4-20% Bis-Tris SurePAGE gradient gels (Genscript) and MOPS running buffer (Genscript M00138). Proteins were transferred onto a 0.45 μm nitrocellulose membrane (Cytiva). 5% milk in TBST (0.5% Tween-20) was used for blocking and antibody incubation, and TBST was used for washing. The list of the antibodies used in this study is available in Table S3. Phospho-ERK1/2 (Thr202/Tyr204) antibody cross-reacting with phospho-MAPK Spk1 ( Kelsall et al., 2025 ) (Fig. S1B) was used to detect its phosphorylated conserved TEY motif. Membrane fractionation experiments Membrane fractionation was performed as previously described ( Klug et al., 2021 ; Vitali et al., 2024 ). In brief, 200 ODs cells were lysed in LBi (without detergent) as described above. After low-speed elution, crude membrane and soluble fractions were obtained by centrifugation for 1 hr at 21’000 g at 4°C. The membrane pellets were resuspended in LBi + 1% LMNG + 0.1 % CHS. Insoluble material was cleared by centrifugation at 13’000 g for 20 min at 4°C. The cleared extracts were snap frozen at -80°C until being used for co-immunoprecipitation or denatured to be loaded into SDS gels. Co-immunoprecipitation 200 ODs of cells were lysed in LBi + 1% LMNG + 0.1% CHS as described above. Equalised protein amounts were added to prewashed magnetic beads and incubated for 3 hrs on a rotor at 4°C. Beads were washed three times with LBi + 1% NP-40 and eluted for 15 min at 65°C in 2x NuPAGE LDS sample buffer containing 5% of β-mercaptoethanol. The list of the magnetic beads used in this study is available in Table S3. Protein dephosphorylation 200 ODs of cells were lysed in EDTA-free lysis buffer (50 mM Tris-HCl pH7.4, 200 mM NaCl) with protease inhibitors (EDTA-free protease inhibitor cocktail (Roche, 4693159001), 1.5 mM sodium orthovanadate, 10 mM sodium fluoride, 100 μM PMSF) and processed for membrane fractionation as described above. 80 units of alkaline phosphatase (Roche 11097075001) were added to 100 μg of total protein when indicated and all samples were incubated at 37°C for 1 hr, before being denatured and run in SDS-PAGE gels. Mass spectrometry Sample preparation and protein digestion miST (General affinity beads, on-beads digestion): Samples were digested following a modified version of the iST method ( Kulak et al., 2014 ) (named miST method). 25 ul of miST lysis buffer (1% Sodium deoxycholate, 100mM Tris pH 8.6, 10 mM DTT), were added to the beads. After mixing and dilution 1:1 (v:v) with H 2 O, samples were heated 5 min at 75°C. Reduced disulfides were alkylated by adding 13 ul of 160 mM chloroacetamide (33 mM final) and incubating for 45min at 25°C in the dark. After digestion with 1.0 ug of Trypsin/LysC mix (Promega #V5073) for 2h at 25°C, sample supernatants were transferred in new tubes. To remove sodium deoxycholate, two sample volumes of isopropanol containing 1% TFA were added to the digests, and the samples were desalted on a strong cation exchange (SCX) plate (Oasis MCX; Waters Corp., Milford, MA) by centrifugation. After washing with isopropanol/1%TFA, peptides were eluted in 200ul of 60% MeCN, 39% water, 1% (v/v) ammonia, and dried by centrifugal evaporation. Liquid Chromatography-Mass Spectrometry analyses TIMS-TOF DDA (Ultimate): LC-MS/MS analyses were carried out on a TIMS-TOF Pro (Bruker, Bremen, Germany) mass spectrometer interfaced through a nanospray ion source (“captive spray”) to an Ultimate 3000 RSLCnano HPLC system (Dionex). Peptides were separated on a reversed-phase custom packed 45 cm C18 column (75 μm ID, 100Å, Reprosil Pur 1.9 um particles, Dr. Maisch, Germany) at a flow rate of 250 nl/min with a 2-27% acetonitrile gradient in 93 min followed by a ramp to 45% in 15 min and to 90% in 5 min (total method time: 140 min, all solvents contained 0.1% formic acid). Data-dependent acquisition was carried out using a standard TIMS PASEF method ( Meier et al., 2018 ) with ion accumulation for 100 ms for each the survey MS1 scan and the TIMS-coupled MS2 scans. Duty cycle was kept at 100%. Up to 10 precursors were targeted per TIMS scan. Precursor isolation was done with a 2 or 3 m/z windows below or above m/z 800, respectively. The minimum threshold intensity for precursor selection was 2500. If the inclusion list allowed it, precursors were targeted more than one time to reach a minimum target total intensity of 20’000. Collision energy was ramped linearly based uniquely on the 1/k0 values from 20 (at 1/k0=0.6) to 59 eV (at 1/k0=1.6). Total duration of a scan cycle including one survey and 10 MS2 TIMS scans was 1.16 s. Precursors could be targeted again in subsequent cycles if their signal increased by a factor 4.0 or more. After selection in one cycle, precursors were excluded from further selection for 60s. Mass resolution in all MS measurements was approximately 35’000. Data processing MaxQuant (DDA): Data files were analysed with MaxQuant 1.6.14.0 ( Cox and Mann, 2008 ) incorporating the Andromeda search engine ( Cox et al., 2011 ). Cysteine carbamidomethylation was selected as fixed modification while methionine oxidation and protein N-terminal acetylation were specified as variable modifications. The sequence databases used for searching were the S. pombe reference proteome based on the UniProt database ( www.uniprot.org , version of 1. February 2021 containing 5140 sequences), and a “contaminant” database containing the most usual environmental contaminants and enzymes used for digestion (keratins, trypsin, etc). Mass tolerance was 4.5 ppm on precursors (after recalibration) and 20 ppm on MS/MS fragments. The “match between runs” feature was not activated. Both peptide and protein identifications were filtered at 1% FDR relative to hits against a decoy database built by reversing protein sequences. Data analysis Perseus (DDA, DIA, TMT): All subsequent analyses were done with the Perseus software package (version 1.6.15.0) ( Tyanova et al., 2016 ). Contaminant proteins were removed, and intensity iBAQ values ( Schwanhausser et al., 2011 ) were log2-transformed and normalised based on the median. After assignment to groups, only proteins quantified in at least 3 samples of one group were kept. After missing values imputation (based on normal distribution using Perseus default parameters), t-tests were carried out among all conditions, with Benjamini-Hochberg FDR correction for multiple testing (adjusted p-value threshold <0.05). Imputed values were later removed. The difference of means obtained from the tests were used for 1D enrichment analysis on associated GO/KEGG annotations as described ( Cox and Mann, 2012 ). The enrichment analysis was also FDR-filtered (Benjamini-Hochberg, Q-val<0.02). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD068764 Statistical analysis Two-tailed Student t -tests were performed to compare two groups in Figs. 2E , 6F and figs. S2C and S2D. For comparisons of more than two groups, initial Brown-Forsythe tests were performed to assess for equality of group variances. One-way ANOVA with Tukey multiple comparison test was performed for Figs. 1D , 1F , 3G , 4D , 5G, 6G, 6H. Kruskal-Wallis with Dunn’s multiple comparison test was performed for Figs. 1H , S1C, 2C , 5F , 6B and figs. S2A and S3A. A two-way ANOVA with Šídák multiple comparisons test was performed for Fig. 6D . Author contributions Conceptualisation, B.S., S.G.M.; methodology, B.S., L.Me.; validation, B.S., L.Me., M.B., W. Li, M.B.; software: W.L.; formal analysis, B.S., L.Me., W.L.; investigation, B.S., L.Me., M.B.; resources, L.Mi, S.G-L.; writing – original draft, B.S.; writing – review & editing, B.S., L.Me., S.G.M.; visualisation, B.S.; supervision, S.G.M.; funding acquisition, S.G.M.; project administration, B.S., S.G.M. Download figure Open in new tab Figure S1: Controls for membrane fractionation, phospho-Spk1 antibody specificity and dPSTR assay A. Membrane and soluble fractions from cells over-expressing Gpa1-3xFLAG-mCherry under the nmt41 promoter immunoblotted for FLAG. The arrow marks the position of the Gα Gpa1 . B. Membrane fractions from h90 cells deleted for spk1 ( spk1Δ ) or expressing spk1 as2 ( spk1+ ) in rich MSL+N medium (vegetative) and MSL-N starvation medium (induced to mate; mat) immunoblotted for phosphorylated ERK1/2. The arrow marks the specific phospho-Spk1 signal. C. Nuclear enrichment of the dPSTR reporter in h- WT, sms1Δ and spk1Δ cells crossed with h+ WT and imaged after 24 hrs. Unmated cells were chosen for analysis. **** P <0.0001. Scale bar: 2µm. Download figure Open in new tab Figure S2: Controls for Pil1 and two-hybrid assays, and analysis of ste4 ΔRA mutant A. Yeast two-hybrid assay of Ste4 with Byr2, blocked by Byr2 N28I ( Tu et al., 1997 ). B. Colocalisation of Pil1-Ste4-mCherry (WT or ΔRA) with Byr2-GFP (WT or N28I). Pearson correlation coefficient values are shown on the right ( n ≥11 cells) with error bars as s.d. *** P <0.001; **** P <0.0001. Scale bar: 2μm. C. Phospho-Spk1 (arrow) in membrane fractions of Ste4-GFP and Ste4 ΔRA -GFP cells (both with also tagged Sms1-sfGFP) in MSL-N starvation induced conditions. Tubulin serves as loading control. D. Mating efficiency of h90 WT and ste4 ΔRA -GFP cells ( n ≥500 cells) with error bars as s.d. **** P <0.0001. Download figure Open in new tab Figure S3: Sms1 is an unstable protein expressed specifically during mating A. Whole-cell fluorescence intensity of Sms1-sfGFP upon nitrogen starvation ( h- and h+ ) and mating ( h- x h+ ) after subtraction of the autofluorescence ( n >30 cells) with error bars as s.d. **** P <0.0001. B. Timelapse of Sms1-sfGFP and Ste4-mCherry in h90 mating cells and early zygote. Note the nuclear depletion of Ste4 (arrowheads) but not of Sms1, and the rapid decrease in Sms1 (but not Ste4) levels in the zygote. Whole-zygote fluorescence intensity of Sms1-sfGFP and Ste4-mcherry, normalised to t0, is shown on the right ( n =14 cells), with error bars as s.d. C. Whole cell extracts from h90 cells expressing endogenously tagged Ste4-GFP or Sms1-sfGFP in MSL-N starvation conditions immunoblotted for GFP. D. GFP-based immunoprecitipates from h90 endogenously tagged Sms1-sfGFP or untagged cells immunoblotted for ubiquitin. Arrowheads indicate ubiquitinated fragments of Sms1-sfGFP. E. Increased stability of Sms1-sfGFP expressed under nmt41 promoter in hypomorphic mts3-1 proteasome mutant at the indicated temperature during 3h. In (A.B), scale bar: 2μm. In (C-E), arrows show the position of full-length proteins. Download figure Open in new tab Figure S4: Sms1 predicted structure and fragment localization A. Structural model of Sms1 predicted by AlphaFold2 with predicted local distance difference test (pLDDT) score as confidence measure. B. Disorder scores of Sms1 generated using IUPred3. C. Colocalisation of sfGFP-tagged Sms1 fragments expressed under nmt41 promoter with endogenous full-length Sms1-mCherry in mating cells. Scale bar: 2μm Download figure Open in new tab Figure S5: Phosphorylation of Sms1 inhibits its function A. 14-3-3 co-precipitates with Sms1-sfGFP. Whole-cell extracts from vegetative cells expressing Rad24-3xMyc and Sms1-sfGFP or GFP as a control under nmt41 promoter were immunoprecipitated with anti-GFP beads and immunoblotted for Myc and GFP. B. Quantification strategy and histogram distribution of the number of native Sms1 22A -sfGFP or Sms1-sfGFP membrane patches in h+ starved cells ( n >40 cells) defined as the number of peaks per cell above the baseline level in cells as in Fig. 6B . C. Additional kymographs from timelapse imaging of Sms1-sfGFP and Sms1 22A -sfGFP in h-sxa2Δ cells stimulated with 10nM P-factor for 60 min, as in Fig. 6C . Identical contrasting parameters were used for both conditions, with asterisks indicating cell poles. D. DIC pictures and shmooing efficiency of h90 byr1 DD with sms1+ or sms1 22E -sfGFP at endogenous locus as in Fig. 5G . Error bars represent s.d. **** P <0.0001. Scale bar: 2μm. E. Sequence-independent structural alignment of the Alphafold2 prediction of Sms1 arrestin domain (blue) with the X-ray crystal structure of bovine β-arrestin 1 (ID: 1G4R) with a root mean square deviation (RMSD) of 3.406 Å. MAP kinase interfaces identified in ( Qu et al., 2021 ) are shown in black. On the convex side of Sms1 arrestin domain the canonical DEF MAPK-docking motif is highlighted in green, whereas the basic residues on the concave side are in red. Video S1: Sms1 patch dynamics and stabilisation in Sms1 22A Timelapse imaging of Sms1-sfGFP and Sms1 22A -sfGFP in h-sxa2Δ cells treated with 10 nM P-factor for 60 min. Arrowheads indicate polarity patches. Contrasts are reduced for Sms1 22A -sfGFP time points to prevent signal saturation. Video S2: Mating behaviour of sms1 22A zygotes Timelapse imaging of h90 WT and sms1 22A mutant cells during mating. Sms1 is tagged with sfGFP at the endogenous locus. Note the ectopic polarisation of Sms1 22A in the zygote leading to polarised growth towards other mating partners. Brightness and contrast are reduced for Sms1 22A -sfGFP to prevent signal saturation. Acknowledgments We thank Prof Li-Lin Du (NIBS, Beijing, China) for the Pil1 plasmids, Dr Serge Pelet (UNIL, Lausanne, Switzerland) for dPSTR reagents, Dr Manfredo Quandroni (UNIL, Lausanne, Switzerland) and the UNIL Protein Analysis Facility for mass spectrometry, Dr Yoel Klug (Oxford University, UK) for the membrane extraction protocol, Prof Aleksandar Vjestica (UNIL, Lausanne, Switzerland) for strains and comments on the manuscript, and Prof Omaya Dudin (UNIGE, Geneva, Switzerland), Dr Fangfang Lu (Broad Institute, Cambridge, United States) and members of the Martin lab for comments on the manuscript. This work was supported by grants from the Swiss National Science Foundation (#176396 and # 191990) and the European Research Council (SexYeast) to SGM. 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Share Negative feedback regulation of the hemi-arrestin MAPK scaffold Sms1 prevents untimely mating Boris Sieber , Laura Merlini , Wanlan Li , Maëlys Besomi , Laetitia Michon , Sushila Gordon-Lennox , Sophie G Martin bioRxiv 2025.09.24.678297; doi: https://doi.org/10.1101/2025.09.24.678297 Share This Article: Copy Citation Tools Negative feedback regulation of the hemi-arrestin MAPK scaffold Sms1 prevents untimely mating Boris Sieber , Laura Merlini , Wanlan Li , Maëlys Besomi , Laetitia Michon , Sushila Gordon-Lennox , Sophie G Martin bioRxiv 2025.09.24.678297; doi: https://doi.org/10.1101/2025.09.24.678297 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17645) Bioengineering (13867) Bioinformatics (41873) Biophysics (21420) Cancer Biology (18550) Cell Biology (25447) Clinical Trials (138) Developmental Biology (13361) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24289) Genetics (15587) Genomics (22473) Immunology (17707) Microbiology (40322) Molecular Biology (17144) Neuroscience (88457) Paleontology (666) Pathology (2826) Pharmacology and Toxicology (4815) Physiology (7634) Plant Biology (15111) Scientific Communication and Education (2042) Synthetic Biology (4285) Systems Biology (9813) Zoology (2268)