Phosphoregulation of the novel hemi-arrestin MAPK scaffold Sms1 prevents untimely mating

Domain: biology
Published: 2026-03-17
Editorial outcome: accept

Abstract
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 a 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 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 reproduction1. 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 mammals2, 3, 4 and likely already existed in the last eukaryotic common ancestor5. 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 activity6. 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 signalling7, 8, 9, 10. KSR1/2 is critical for Ras-induced tumorigenesis11. β-arrestins, beyond their canonical roles in binding phosphorylated G-protein coupled receptors (GPCR) to arrest signalling12, also scaffold MAPK signalling for cytosolic ERK activation13, 14, 15. Interestingly, despite their divergent structures, these MAPK scaffolds display the same three critical features. First, they bind all three MAP kinases7, 13, 14, 16, 17, 18, 19, 20 and trigger allosteric kinase activation21, 22, 23, 24, 25, 26. 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-arrestins27, 28. 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 Gβγ14, 16, 21, 23, 29, 30, 31, 32, 33, 34. Finally, phosphorylation of the MAPK scaffold, in part promoted by MAPK-dependent feedback, negatively regulates the scaffold membrane localisation, preventing untimely activation or terminating signalling17, 20, 35, 36, 37, 38, 39, 40, 41, 42, 43.

In contrast to the highly conserved and ancient MAPK signalling cascade5, and despite having critical roles, MAPK scaffolds do not appear to be evolutionarily conserved. For instance, KSR and β-arrestins are restricted to Metazoa5, 44. Similarly, Ste5 only exists in Saccharomycotina, where it is thought to have arisen at the same time as MAPK duplication45. 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 orthologs46, 47, 48, no scaffold has been identified. Given the absence of shared kinases between different MAPK cascades in this organism45, we – and others – have considered for more than 30 years that no MAPK scaffold was involved in fission yeast mating (Fig. 1A)45, 49, 50, 51, 52, 53.

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)48, 50. Byr2 further binds an essential adaptor Ste4, which acts in parallel to Ras1-GTP, upstream of the MAP3K54, 55. 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 components56, 57. 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 programme58. Differentiation underlies the fusion of mating partners to form a diploid zygote, in which the Mi-Pi bipartite transcription factor is assembled and triggers the activation of the RNA-binding protein Mei2. In turn, Mei2 signalling represses mating and initiates meiosis to form 4 haploid spore progenies 59, 60.

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 onset61, 62, a behaviour conserved in budding yeast63, 64, 65, 66. 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 other62, 67, 68. Patch proteins, as well as all MAP kinases, further localise at the mature polarity patch, where they promote cell-cell fusion to form the zygote69. How the activated receptor transduces the pheromone signal and recruits the MAPK cascade to the plasma membrane during fission yeast mating remains an open question.

Here, we discover Sms1, which was previously described as essential for mating and inhibitory for meiosis, and known as Ste770, 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 pheromone signalling patches by specific recognition of the Gα. 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 the 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 MAPKSpk1 localises at the membrane. To this aim, we performed membrane extraction71, 72, which we validated by verifying enrichment of the membrane-associated receptor-coupled prenylated GαGpa1 protein (Fig. S1A). Activated, phospho-MAPKSpk1 was detected with the cross-reacting phospho-ERK antibody against the conserved TEY motif in ERK-like MAP kinases (Fig. S1B)73. This revealed that phosphorylated MAPKSpk1 is enriched in the membrane fraction in mating cells (Fig. 1B), demonstrating activation of the ERK-like MAPKSpk1 at the membrane.

To identify novel interactors of the MAPK cascade, we focused on the MAP3KByr2 adaptor Ste4, which belongs to the Ste50 family conserved throughout fungi74, 75. Ste4 is essential for the mating process as its deletion abrogates Spk1 phosphorylation and prevents mating (Fig. 1C-1D)76. 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 MAP3KByr2, thus validating the experimental design (Fig. 1E, Table S1). Amongst Ste4 interactors, we focused on the poorly characterised protein SPAC23E2.03c (UniprotKB Q10136), which was previously identified in sterile screens and known as Ste770. This protein was shown to be a critical component of cell conjugation that represses meiosis onset, likely through interaction with Mei2 and its kinase inhibitor Pat1, and is unstable in zygotes 70. To avoid any confusion with the homonym MAP2K Ste7 in S. cerevisiae, and upon approval by the PomBase Gene Naming committee, we changed its name to Sms1 (scaffold of MAPK signalling for reasons below).

Deletion of sms1 resulted in sterility, even when mated with a wildtype partner (Fig. 1F), consistent with previous report70. This phenotype is shared by loss of components of the pheromone-MAPK signalling pathway77, 78. 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 yeast79 (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 spk1∆ cells, demonstrating that the assay specifically reports on MAPK-dependent transcriptional response (Fig. 1H, S1C). In sms1∆, 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 proteins57, 80, 81, this result indicates that the interaction between Sms1 and Ste4 does not require other pheromone-induced factors and may be direct.

Ste4 N-terminus binds the MAP3KByr2 via SAM-SAM domain interaction, an interaction abolished in the SAM mutant Byr2N28I (Fig. S2A)54, 55, 82. 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)83, 84. 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 microscopy85. As proof of principle, Pil1-Ste4 recruited MAP3KByr2 and co-localisation was abolished by the N28I mutation in MAP3KByr2, 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∆SBD; 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 region86, 87. Taken together, these results are consistent with 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∆RA had nearly absent phospho-Spk1 signal and were fully sterile, indicating this domain is indeed essential for function (Fig. S2C-S2D). The sms1∆SBD 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 (Fig. S3A). As Ste11 is de-repressed in these conditions, this is consistent with Ste11-dependent transcription of sms1 57, 70, 88. 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 fusion89. 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 GαGpa1 62, 67. 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 report70. 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.

Immunoblotting after 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). Optimization of the extraction buffer and protocol (see methods) allowed to recover some full-length Sms1, though a large fraction was still degraded (Fig. 3E, soluble fraction). 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 mutant90 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) versus 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)91 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 the 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 activation92, 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)93. 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 bilayer94, we mutated the corresponding amino acids in Sms1 arrestin domain to create a lipid-binding deficient mutant (sms1LBM) (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)P295 (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 sms1LBM allele lacking the lipid-binding residues (Fig. 4D).

The targeting of Sms1 arrestin domain to the entire plasma membrane of mitotic cells raised the question of its restriction to the polarity patches and fusion site. A possible explanation would be an indirect recruitment by Ras1 because this GTPase binds the MAP3KByr2 54, 96, localises to the polarity patches67 and was proposed to recruit the MAP3KByr2 to the membrane97. However, upon deletion of Ras1 or its activator GEF Ste6, which strongly impair mating98, 99, 100, Sms1 nevertheless polarised at the plasma membrane, forming unstable patches oriented towards WT partner cells (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 be recruited by the receptor-coupled GαGpa1. 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). Sms1 similarly interacted with active GTP-locked Gpa1Q244L and inactive GDP-locked Gpa1G242A (ref 77) (Fig. 4H, S4D). In agreement with Gpa1 recruiting Sms1, Sms1 polarisation was abolished in absence of the GαGpa1 (Fig. 4E). Together, these results demonstrate that Sms1 is targeted by its arrestin domain to the plasma membrane, where it recognises the Gα, thus restricting Sms1 to pheromone signalling domains.

Sms1 is the MAPK scaffold of the pheromone signalling pathway
The interactions of Sms1 with phospholipids, GαGpa1 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 MAP2KByr1 and MAPKSpk1. 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 MAP2KByr1, in which the conserved MAP2K dual phosphorylation sites are mutated to aspartic acid (Byr1DD; 101). Co-immunoprecipitation experiments demonstrated that Sms1 binds the MAP2KByr1 (Fig S5A). We also detected a strong interaction of Sms1 with active Byr1DD 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.

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-MAPKSpk1 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 MAPKSpk1 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 GαGpa1, the MAP3KByr2 through its adaptor Ste4, the active MAP2KByr1 and MAPKSpk1. We thus renamed the protein as Scaffold for MAPK Signalling 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 byr1DD strains, in which the native byr1 gene encodes a constitutively active MAP2KByr1. In these conditions, we expected the MAPK to remain constitutively activated by Byr1DD irrespective of the presence of the scaffold. Indeed, loss of Sms1 did not significantly affect pSpk1 level (Fig. 5E, S5B) and only moderately decreased MAPK-dependent transcriptional response measured by the dPSTR assay (Fig. 5F). Phenotypically, byr1DD at the endogenous locus causes a fus (fusion-defective) phenotype characterised by constitutive growth of mating projections (known as shmooing; Fig. 5G)69, 101. These cells also show accumulation of cell wall. In byr1DD cells , shmooing was abrogated by deletion of downstream signalling components, such as the MAPKSpk1, but not of upstream ones, such as ste4 (Fig. 5G). Unexpectedly, in absence of Sms1, byr1DD 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 protein 14-3-3, Rad24 (Fig. S6A). 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 (Sms122A) (Fig. 6A). High-molecular weight bands were not observed for Sms122A and phosphatase treatment did not change its migration pattern, demonstrating that Sms1 expressed in vegetative cells undergoes proline-directed phosphorylation (Fig. 6A).

To probe the functional role of Sms1 phosphorylation, we expressed Sms122A-sfGFP as sole copy under the endogenous promoter, similarly as for Sms1WT-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, Sms122A-sfGFP was strongly enriched in membrane localised patches (Fig. 6B, S6B). Remarkably, deletion of MAPKSpk1 was sufficient to induce a similar, though less extensive, increase in plasma membrane accumulation of Sms1WT (Fig. 6B, S6B). We conclude that MAPKSpk1 likely phosphorylates Sms1 and prevents its accumulation at the plasma membrane.

Sms122A 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)61, 62. By contrast, sms122A cells formed stable, higher-intensity patches that were largely constrained to cell poles, often simultaneously at both cell poles (Fig. 6C, S6C, Supplementary Movie 1). These stable patches induced polarised growth, and sms122A cells formed shmoos of increasing length at all P-factor dosages tested, even when WT cells did not (Fig. 6D). This hypersensitivity of sms122A 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 cells102, 103. By contrast, sms122A 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 sms122A 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, Sms122A-sfGFP remained polarised at the plasma membrane, leading to growth of the zygote towards other mating partners (Fig. 6G, Supplementary Movie 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, sms122E, abolished plasma membrane recruitment and led to a sterility phenotype (Fig. 6G). Sms122E also blocked the fus phenotype of constitutively active MAP2K byr1DD cells (Fig. S6D), 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 Sms14A showed localisation to the membrane of the zygote, while Sms14E was sterile (Fig. 6A, 6H). We note that other phosphorylation sites also contribute to Sms1 function, as sms14A was less potent than sms122A 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.

Methods
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 recycles84. Sequence independent alignments between the arrestin domain of Sms1 and β-arrestin 1 or 2 was performed by incremental combinatorial extension (CEAlign)120 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 1 102, 121. 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, byr1DD 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).

Pil1 co-recruitment assay
All plasmids were generated using standard molecular biology techniques and stably integrated in the genome. Fusion constructs are expressed under nmt41 promoter and comprise the gene of interest fused in frame with the pil1 ORF at the 3’ end. Expression was induced by growth of cells in MSL+N and the bottom z-section of cells is shown.

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 described121. 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. For mating and shmooing efficiencies, for each condition, cells were counted from microscopy images until reaching 500 cells and including the remaining cells in the last image (n≥500 cells per condition). This quantification was repeated in at least three biological experiments. Mating and shmooing efficiencies are shown as bar graphs for the mean of the independent experiments (dots), and the error bars as the standard deviation.

Microscopy
All images in Figs 1, 3 to 6 and Fig. S1, S3 to S6 (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 AporchromAat 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). Softwares used for data acquisition are listed in Table S4.

Super resolution imaging in Figs 2C, 4B, 4C, 4F, 6B, Fig. 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 v.3.3 Blue software (Zeiss).

Image analysis
dPSTR
For quantification of dPSTR signal in Fig. 1H, 5F, and S1C 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, spk1∆ or sms1∆ 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 byr1DD, byr1DD spk1∆ or byr1DD sms1∆ 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)122. 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 described85. 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 123 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 Sms122A-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. S6B.

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 124. 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
For experiments shown in Fig. S3C-D, RIPA buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 0.1% SDS, 0.4% sodium deoxycholate, 1% NP-40) was supplemented with protease inhibitors (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, 1 mM PMSF (Phenylmethylsulfonyl fluoride)). 100 ODs of cells were lysed in 500 μl of RIPA buffer by 8 cycles of 30 sec of bead-beating at 4°C with 30 sec between cycles. Lysates were cleared by centrifuging at 13’000 g for 20 min at 4°C before transferring the supernatants into collection tubes.

For all other experiments, lysis buffer (LB, 50 mM Tris-HCl pH7.4, 200 mM NaCl, 1 mM EDTA) was prepared by supplementing it with the protease inhibitors described above (LBi). 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.

For all experiments, 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 (0.05% 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-MAPKSpk1 73 (Fig. S1B) was used to detect its phosphorylated conserved TEY motif.

Membrane fractionation experiments
Membrane fractionation was performed as previously described 71, 72. 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
For Fig. S3D, 100 ODs of cells were lysed in RIPA buffer 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 RIPA buffer and eluted for 10 min at 95°C in 2x NuPAGE LDS sample buffer containing 5% of β-mercaptoethanol. For all other experiments, 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 method125 (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 H2O, 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 method126 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 Max Quant 1.6.14.0 127 incorporating the Andromeda search engine128. 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)129. Contaminant proteins were removed, and intensity iBAQ values130 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 described131. 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, S2C, S2D and S5B. 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 and S4D. Kruskal-Wallis with Dunn’s multiple comparison test was performed for Figs. 1H, S1C, 2C, 5F, 6B, S2A and S3A. A two-way ANOVA with Šídák multiple comparisons test was performed for Fig. 6D. For boxplots, the central line indicates the median, the bottom and top edges of the box indicate the 25th and 75th percentiles, and the whiskers extend to the smallest and largest values. No statistical method was used to predetermine sample size. The experiments were not randomized and measurements were taken from distinct samples.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes the characterization of the S. pombe protein Ste7 (renamed Sms1in this study) as “the” scaffold protein for the S. pombe pheromone response MAP kinase cascade. In this context it is defined as formally equivalent to the Ste5 scaffold of the S. cerevisiae pathway, but although providing the same function, the S. pombe protein is structurally totally distinct.\n\nThe authors initially select the protein of interest, SPAC23E2.03c, from a pool of 235 candidates identified in a pull-down assay that identified interactors with the “specialized” adapter protein Ste4. It became the primary focus in part because previous studies had shown the protein, named Ste7, to be a critical component of the mating pathway. The current authors suggest the protein is poorly characterized, but the paper identifying the KO phenotype involves solid analysis. The current manuscript proposes renaming the gene Sms1 from Ste7 to prevent confusion with the MAPK of the S. cerevisiae pathway, but the field has already lost that fight (the SpSte4 adapter vs the ScSte4 G protein beta subunit is a clear example). Therefore, I think the name should remain Ste7 as it fully describes the phenotype (mutants are sterile) and has primacy in the literature, but in any event I hope the S. pombe nomenclature committee will be consulted prior to any publication defining this gene as Sms1.\n\nInitial experiments confirm the mating failure of the mutant, the loss of MAPK phosphorylation, and loss of pheromone-induced gene activation. Subsequent analysis directed at the role of the Ste7/Sms1 element starts by confirming the interaction with Ste4 – the assay used to by these investigators to identify Ste7/Sms1 in the first place. All these experiments are solidly informative and competently done but are not really moving the story forward. If space is any issue, some (or all) of these results could be moved to supplementary figures.\n\nThe authors now investigate the characteristics of the association of the Ste7/Sms1 protein with the Ste4 adapter in more depth. Alpha-fold modeling suggests beta sheet augmentation could link the arrestin fold module of Ste7/Sms1 to the Ras domain of the Ste4 adaptor. Previous S. cerevisiae analysis of the equivalent part of the circuit has the Ras domain (of Ste50) linking to proteins like Opy2 and small GTPases while the SAM domain links to the SAM domain of the MAPKK Ste11. This puts the Ste7/Sms1 protein apparently in a similar position to membrane association elements like Cdc42 and Opy2. Direct test of the association between the arrestin fold region of Ste7/Sms1and Ste4 involved an eisosome-defined association assay to investigate the requirement of the arrestin and RA domains in the formation of the protein complex. The wild-type domains bring the two fusion proteins together in the eisosomes; mutation of the RA domain or the arrestin fold region block the interaction. The data presentation of this experiment is incomplete – we are shown the GFP signal for the Ste7/Sms1 fusion protein, and the merge, but the mCherry signal for the Pil1-Ste4 fusion is not independently presented. This should be done – we need to see the structure of the signal for the eisosome-localized binding target. The functional analysis supports the picture provided by the structural study – constructs that are not associating do not allow normal signaling. The manuscript claims that these results support an association through a beta sheet augmentation, and that AlphaFold establishes that the interaction represents an example of protein association directing the folding of an unordered protein region. This is an over-interpretation of the data – the AlphaFold work is suggestive and not conclusive. It would be better to say the results are “consistent with” rather than “validate” the AlphaFold model.\n\nAt this point the Ste7/Sms1 protein has essentially been characterized as behaving like the Opy2 protein in S. cerevisiae – interacting with the RA domain of an adapter that also connects to an MPKK through its SAM domain. The next set of experiments investigates where the Ste7/Sms1 protein localizes in vegetative and signaling cells using GFP fusion constructs. The protein signal is increased under nitrogen starvation and further enhanced by pheromone treatment of the cells. Localization is in membrane patches, and ultimately sites of cell fusion in mating cells. The patches colocalize with Scd2, and are thus, along with Ste4, part of polarity patches in the membrane. Intriguingly, after cell fusion the fates of Ste4 and Ste7/Sms1 are different – with Ste4 remaining, and Ste7/Sms1 disappearing. When the structure of the protein was investigated in cell extracts the protein was found to be unstable and ubiquitinated – assessment of the influence of protein localization on stability found that the membrane associated version of the protein was both more stable and post-translationally modified. Nuclear localization of the GFP fusion protein was detected, but trapping the protein in the nucleus impacted protein stability and compromised mating, suggesting that nuclear export is essential for proper functioning, and supporting membrane localization as a key element in Ste7/Sms1 function.\n\nTo look directly at membrane association of the Ste7/Sms1 protein the authors mutate residues shown to be important for directing the structurally-similar mammalian beta arrestins to the plasma membrane. These modifications blocked cell surface localization of the S. pombe protein and caused sterility. Temperature sensitive inactivation of Its3, a PI4P convertase, also compromised Ste7/Sms1 cell surface localization. The structural similarities of the arrestin domains, and the involvement of phosphoinositides and lipid associating hydrophobic residues are taken to support a role for Ste7/Sms1 in interacting with the plasma membrane – this argument seems solid. Further assessment of the polarity-patch-linking element found that loss of the G protein alpha subunit blocked the polarization, while loss of Ras1 did not, leading the authors to propose the pheromone pathway G protein linkage was critical for the association Ste7/Sms1into pheromone-induced-polarization and for mating. Additional testing suggests the linkage involves the activated form of the alpha subunit and requires the arrestin domain of the putative adapter protein.\n\nWhile all these investigations provide convincing evidence of the Ste7/Sms1 protein playing a critical role in the pheromone pathway that involves directed membrane association and G protein binding, the authors needed to show critically-required-binding of the other pheromone pathway kinases to claim a general scaffold function for the protein. To investigate these bindings and their critical role in the pheromone response, they first looked into direct intracellular binding using immunoprecipitation in cells compromised for mating due to missing signaling components. CoIP shows interaction between the arrestin protein and both Byr1 and Spk1. This experiment is poorly described - as I understand it the Byr1 protein, which is mutated to be constitutively activated, is tagged with V5, but the legend says the protein was identified by the anti-FLAG antibody? The logic of the activation of Byr1 for the assay is not explained – why not use tagged wild type protein in the assay? These experiments are the most critical to establish the central point of the manuscript – the arrestin-like protein Ste7/Sms1 is a MAP kinase cascade scaffold. Therefore, the story here needs to be clear and unambiguous.\n\nFurther assessment of complex formation in mating competent cells involves FLAG-tagged Ste7/Sms1co-immunoprecipitating phospho-Spk1as assessed using a phospho-ERK antibody. MAP kinase coated beads could pull out full length Ste7/Sms1 from the membrane fraction of the cell. These assays don’t ensure that all the proteins are complexed at the same time, but do provide solid evidence for interaction of the arrestin protein with all the kinase members of the mating kinase cascade.\n\nFunctional assays established that in the absence of the scaffold constitutively active MEK (activated Byr1) the MAPK Spk1 remained phosphorylated, but the constitutive shmooing phenotype characteristic of the activated MEK kinase is blocked, suggesting the role of the scaffold is not simply to facilitate the kinase phosphorylation cascade.\n\nThe final classes of experiments relate to negative feedback removing the arrestin protein from the membrane to keep mating off under non-mating-conducive conditions. This is shown to be phosphorylation controlled, and proteins mutated for “all” candidate MAP kinase Spk1 sites lack any phosphorylation-directed gel shifts. This non-phosphorylatable protein shows improper localization, and directs hyper response to pheromone, while generating pseudophosphorylated residues at the candidate phosphorylation sites abolished membrane localization. These experiments generally establish a role of phosphorylation, at least in part likely from Spk1, as playing an important role in Ste7/Sms1 function. More detailed analysis of site specificity and the role of other non-proline directed sites remain to be done.\n\nAt the end of the discussion the authors wonder if the scaffolds are evolving independently. That was the prediction for the Ste5/Far1 development in C. albicans - the Ste5 vWA domain was structurally distinct from the Far1 vWA domain, suggesting independent development, not simply duplication and divergence (ref 45).\n\nMinor points\nIn the abstract and the start of the discussion the Sms1 protein is defined as a “structurally novel” scaffold. The arrestin fold is not “structurally novel”, and this protein, while representing a new scaffold, is not characterized in a way that highlights much about its structure being of dramatic interest. I think novel should not be used in this context.\nThe phrase “Because Sms1 binds the active Gα-GTP at the plasma membrane and all MAPKs,” is awkward\nReference 45 is not discussing MAPK duplication, but rather the potential duplication of Far1 into the Far1/Ste5 pair.\n“…spurious noise signal…” is awkward", "reviewer_2": "This very nice manuscript reports the identification and characterization of the previously unknown scaffold for the MAPK signaling cascade that controls mating in the fission yeast, Schizosaccharomyces pombe. The data convincingly demonstrate that the previously understudied protein Sms1 acts as the missing membrane-bound scaffold required for transducing the mating signal, and interacts with all components of the MAPK signaling pathway. The paper further describes how this new scaffold is regulated – by upregulation during mating, nuclear export, lipid binding, partner binding, ubiquitination, and negative feedback via phosphorylation. In my opinion, it is a very comprehensive work with no significant weaknesses. The data are strong and support the conclusions. The paper is also well-written and will be of wide interest to the protein kinase signaling field.\n\nI do have some suggestions for changes to the text/figures. These are all relatively minor and listed below.\n\nAbstract:\na) Add an “a” to “scaffolds that recruit kinases to subcellular locations and promote signal transduction have only been described in few species” between in and few.\n\nIntro and discussion:\na) Please add a \"the\" between in and presence \"even in presence of hyperactive MAP2K\"\nb) Rather short shrift is given to the fact that Sms1 was previously known as Ste7 and some information was known about Ste7. I think this information should be included in the final paragraph of the introduction in addition to mentioning it in the results section.\nc) The discussion would also benefit from describing how the new information regarding Sms1/Ste7 function can explain/expand on or contrasts with what was previously known about Ste7.\n\nResults:\n1) “Amongst Ste4 interactors, we focused on the poorly characterised protein SPAC23E2.03c (UniprotKB Q10136), previously known as Ste7\nand shown to promote mating and meiosis.” I recommend replacing “and” in this sentence with “because it was previously”.\n2) A more detailed explanation for why the observed results in Fig S3 are due to Ste11-dependent transcription would be helpful.\n3) “Whole-cell extraction of mating cells expressing Sms1-sfGFP only showed low molecular weight fragments”. This sentence confused me for a couple of reasons. First, low molecular weight fragments were observed by immunoblotting of whole cell lysates, not by extraction. Second, the sentence/paragraph would benefit from further explanation and contrasting the conditions with previous co-IP experiments/blots shown earlier and blots shown later (e.g. Fig. 3E) in the paper. These other blots showed more than low molecular weight fragments. Expanding the explanation of exactly what the differences in conditions are would avoid confusion and promote a better understanding of these variable blotting results.\n4) “differentially regulated at the membrane (polarity patches and fusion site) and the cytosol.” The meaning would be clearer if the second “and” in this sentence were replaced with “versus”.\n5) In “its restriction at the polarity patches and fusion site”, please replace “at” with “to”.\n6) The sentence “ By contrast, Sms1 polarisation was abolished in absence of the GαGpa1 (Fig. 4E)” seems to be in the wrong place in the text. For a more logical flow and better understanding, I suggest re-working this sentence and those in the following paragraph.\n7) The preferential binding of Sms1 to the GTP-locked version of Gpa1 is difficult to substantiate without quantification of the blots.\n8) “loss of Sms1 only moderately decreased pSpk1 level (Fig. 5E)”. This statement also requires quantification of the blots.\n9) “the phospho-binding proteins 14-3-3, Rad24”; please delete “s” from proteins\n\nFigure/figure legends:\n1) In figure 1, inconsistency with Sexual capitalized and cell not.\n2) Throughout the figures, tubulin is used as a control for the amount of soluble protein but included elsewhere (although not always) as if it is a loading control for the membrane fractions (example Fig. 1C) and of course, it is not a loading control for the membrane fractions. Therefore, it seems that the blue banner of “soluble” should be added above all the tubulin blots and/or an explanation of this control and its appearance under membrane fraction blots should be provided.\n3) In the legend for Fig. 1E, it says the volcano plot represents the results of immunoprecipitations. This description is incomplete because it is a representation of the MS results and this could be expanded.\n4) Legend to figures 1H and S1Cshould include the number of cells analyzed – looks like many cells were analyzed.\n5) Legend to Figure 4, last sentence should end in controls rather than control.\n6) The input and IP panels of Fig. 5C. are not well-aligned in that pSpk1 bands in the two panels are at different distances from the markers.\n7) Please clarify whether or not the Sms1 blots are from lysates or IPs in Fig 6A.\n\nMethods:\n1. Yeast media composition and standard methods should be referenced.\n2. Please describe constructs for the eisosome experiments.", "reviewer_3": "Review of Negative feedback regulation of the hemi-arrestin MAPK scaffold Sms1\nprevents untimely mating, by Boris Sieber et al.\n\nIn this manuscript the authors make the surprising discovery of a scaffold protein of the Sch. pombe pheromone response\npathway. After 30 years of investigation in which the community thought there was no scaffold, the group of Dr. Sophie\nMartin present clear evidence that the protein they rebaptized Sms1 (formerly known as SPAC23E2.03c or Ste7) is actually\nan essential component working as a scaffold.\n\nThey show convincing evidence all along the manuscript.\nSms1 binds the Galpha subunit Gpa1, the cofactor Ste4 (which brings along the Map3K Byr2), the MapK2 Byr1 and the\nMapK Spk1.\nSms1 associates with the lipids of the plasma membrane, and both interactions (Gpa1 and lipids) are necessary for Sms1 to\nbe recruited to the PM. It’s association to the PM stabilizes it and is essential for shmoo formation.\nSms1 seems be negatively regulated by multiple S/T-P phosphorylations, since the 22A mutant is hyperactive and the 22E\nis not active and cannot associate with the PM and they suggest it is negative feedback from the MAPK.\n\nAlthough Spk1 needs Sms1 localization to be activated by Byr1, Sms1 is not necessary if Byr1 is constitutively active; that\nis, Sms1 is not an allosteric modulator of Spk1.\n\nThis is a remarkable paper, with data well supported by multiple experiments. It is quite amazing that Sms1 performs almost\nall the functions and operates quite similarly to the budding yeast Ste5 scaffold, but it shares no common evolutionary origin\nwith it.\n\nMain comments:\nComment 1- There is no direct evidence that Sms1 is regulated by Spk1-dependent negative feedback. The strong evidence\nis that it is regulated by phosphorylation in S/T-P sites, but experiments are needed to establish that Spk1 is the kinase.\nGiven the sheer number of new results, I’m somewhat puzzled that this particular aspect was chosen as the main result of\nthe paper, appearing in the title itself.\nThe current evidence is this:\n1- The 22A Sms1 mutant yeast show a hyperactive pathway and Sms1 highly localized at the membrane. The exact\nopposite happens to the 22E Sms1 yeast. This means that phosphorylation of Sms1 negatively regulates it.\n2- Deletion of Spk1 causes a similar but milder phenotype as the 22A Sms1 mutant.\nThat is all. To establish that it is a negative feedback one would need results of this sort (these are only examples):\na) Deletion of Spk1 results in complete loss (or extensive reduction) of the high MW bands that are show in Fig6A\nb) Inhibition of Spk1-as mutants with PP1 analogs causes spontaneous recruitment of Sms1 to the membrane\nc) Byr1-dd suppresses Sms1 PM localization in a Spk1 dependent fashion\nd) In vitro phosphorylation assays that show that Spk1 can phosphorylate Sms1 (or fragments of it).\nI don’t think this soft of experiments need to be done for the paper to be accepted, but to claim that Spk1 acts on Sms1 in a\nnegative feedback loop, yes. So, I strongly encourage the authors to change the title accordingly.\n\nComment 2. It is not clear if they tested the direct interaction between Sms1 and Byr2. Please add that information."}, "author_response": "Response to reviewers\nReviewer 1\nThis manuscript describes the characterization of the S. pombe protein Ste7 (renamed Sms1in\nthis study) as “the” scaffold protein for the S. pombe pheromone response MAP kinase cascade.\nIn this context it is defined as formally equivalent to the Ste5 scaffold of the S. cerevisiae pathway,\nbut although providing the same function, the S. pombe protein is structurally totally distinct.\nThe authors initially select the protein of interest, SPAC23E2.03c, from a pool of 235 candidates\nidentified in a pull-down assay that identified interactors with the “specialized” adapter protein\nSte4. It became the primary focus in part because previous studies had shown the protein, named\nSte7, to be a critical component of the mating pathway. The current authors suggest the protein\nis poorly characterized, but the paper identifying the KO phenotype involves solid analysis. The\ncurrent manuscript proposes renaming the gene Sms1 from Ste7 to prevent confusion with the\nMAPK of the S. cerevisiae pathway, but the field has already lost that fight (the SpSte4 adapter\nvs the ScSte4 G protein beta subunit is a clear example). Therefore, I think the name should\nremain Ste7 as it fully describes the phenotype (mutants are sterile) and has primacy in the\nliterature, but in any event I hope the S. pombe nomenclature committee will be consulted prior\nto any publication defining this gene as Sms1.\nThe reviewer can be reassured. The name change was performed with agreement from the\nfirst author of the original Ste7 paper and received the approval of the PomBase Gene naming\ncommittee (https://www.pombase.org/submit-data/gene-naming-committee-members). Upon\ntheir validation, the gene name has already been changed on PomBase\n(https://www.pombase.org/gene/SPAC23E2.03c) with Ste7 appearing as a synonym. We have\nnow specified in the manuscript that the name change was approved by PomBase.\nInitial experiments confirm the mating failure of the mutant, the loss of MAPK phosphorylation,\nand loss of pheromone-induced gene activation. Subsequent analysis directed at the role of the\nSte7/Sms1 element starts by confirming the interaction with Ste4 – the assay used to by these\ninvestigators to identify Ste7/Sms1 in the first place. All these experiments are solidly informative\nand competently done but are not really moving the story forward. If space is any issue, some (or\nall) of these results could be moved to supplementary figures.\nThe authors now investigate the characteristics of the association of the Ste7/Sms1 protein with\nthe Ste4 adapter in more depth. Alpha-fold modeling suggests beta sheet augmentation could\nlink the arrestin fold module of Ste7/Sms1 to the Ras domain of the Ste4 adaptor. Previous S.\ncerevisiae analysis of the equivalent part of the circuit has the Ras domain (of Ste50) linking to\nproteins like Opy2 and small GTPases while the SAM domain links to the SAM domain of the\nMAPKK Ste11. This puts the Ste7/Sms1 protein apparently in a similar position to membrane\nassociation elements like Cdc42 and Opy2. Direct test of the association between the arrestin\nfold region of Ste7/Sms1and Ste4 involved an eisosome-defined association assay to investigate\nthe requirement of the arrestin and RA domains in the formation of the protein complex. The wild-\ntype domains bring the two fusion proteins together in the eisosomes; mutation of the RA domain\nor the arrestin fold region block the interaction. The data presentation of this experiment is\nincomplete – we are shown the GFP signal for the Ste7/Sms1 fusion protein, and the merge, but\nthe mCherry signal for the Pil1-Ste4 fusion is not independently presented. This should be done\n– we need to see the structure of the signal for the eisosome-localized binding target.\nWe have added the pictures of the mCherry channel in Figure 2C and S2B as suggested.\nThe functional analysis supports the picture provided by the structural study – constructs that are\nnot associating do not allow normal signaling. The manuscript claims that these results support\nan association through a beta sheet augmentation, and that AlphaFold establishes that the\ninteraction represents an example of protein association directing the folding of an unordered\nprotein region. This is an over-interpretation of the data – the AlphaFold work is suggestive and\nnot conclusive. It would be better to say the results are “consistent with” rather than “validate” the\nAlphaFold model.\nWe have changed the wording as suggested.\nAt this point the Ste7/Sms1 protein has essentially been characterized as behaving like the Opy2\nprotein in S. cerevisiae – interacting with the RA domain of an adapter that also connects to an\nMPKK through its SAM domain. The next set of experiments investigates where the Ste7/Sms1\nprotein localizes in vegetative and signaling cells using GFP fusion constructs. The protein signal\nis increased under nitrogen starvation and further enhanced by pheromone treatment of the cells.\nLocalization is in membrane patches, and ultimately sites of cell fusion in mating cells. The\npatches colocalize with Scd2, and are thus, along with Ste4, part of polarity patches in the\nmembrane. Intriguingly, after cell fusion the fates of Ste4 and Ste7/Sms1 are different – with Ste4\nremaining, and Ste7/Sms1 disappearing. When the structure of the protein was investigated in\ncell extracts the protein was found to be unstable and ubiquitinated – assessment of the influence\nof protein localization on stability found that the membrane associated version of the protein was\nboth more stable and post-translationally modified. Nuclear localization of the GFP fusion protein\nwas detected, but trapping the protein in the nucleus impacted protein stability and compromised\nmating, suggesting that nuclear export is essential for proper functioning, and supporting\nmembrane localization as a key element in Ste7/Sms1 function. To look directly at membrane\nassociation of the Ste7/Sms1 protein the authors mutate residues shown to be important for\ndirecting the structurally-similar mammalian beta arrestins to the plasma membrane. These\nmodifications blocked cell surface localization of the S. pombe protein and caused sterility.\nTemperature sensitive inactivation of Its3, a PI4P convertase, also compromised Ste7/Sms1 cell\nsurface localization. The structural similarities of the arrestin domains, and the involvement of\nphosphoinositides and lipid associating hydrophobic residues are taken to support a role for\nSte7/Sms1 in interacting with the plasma membrane – this argument seems solid. Further\nassessment of the polarity-patch-linking element found that loss of the G protein alpha subunit\nblocked the polarization, while loss of Ras1 did not, leading the authors to propose the pheromone\npathway G protein linkage was critical for the association Ste7/Sms1into pheromone-induced-\npolarization and for mating. Additional testing suggests the linkage involves the activated form of\nthe alpha subunit and requires the arrestin domain of the putative adapter protein.\nWhile all these investigations provide convincing evidence of the Ste7/Sms1 protein playing a\ncritical role in the pheromone pathway that involves directed membrane association and G protein\nbinding, the authors needed to show critically-required-binding of the other pheromone pathway\nkinases to claim a general scaffold function for the protein. To investigate these bindings and their\ncritical role in the pheromone response, they first looked into direct intracellular binding using\nimmunoprecipitation in cells compromised for mating due to missing signaling components. CoIP\nshows interaction between the arrestin protein and both Byr1 and Spk1. This experiment is poorly\ndescribed - as I understand it the Byr1 protein, which is mutated to be constitutively activated, is\ntagged with V5, but the legend says the protein was identified by the anti-FLAG antibody?\nWe apologize for the confusion. The coIP was indeed done with V5-tagged constitutively active\nByr1DD and immunoblotted with anti-V5 antibodies. The FLAG was a typo and has now been\ncorrected with V5 in the figure legend.\nThe logic of the activation of Byr1 for the assay is not explained – why not use tagged wild type\nprotein in the assay?\nWe performed the experiment with both wildtype Byr1 and Byr1DD. The interaction is clear,\nthough seems a bit weaker with wildtype Byr1. We have now added the coIP of Sms1 with\nwildtype Byr1 as Figure S5A.\nThese experiments are the most critical to establish the central point of the manuscript – the\narrestin-like protein Ste7/Sms1 is a MAP kinase cascade scaffold. Therefore, the story here needs\nto be clear and unambiguous. Further assessment of complex formation in mating competent\ncells involves FLAG-tagged Ste7/Sms1co-immunoprecipitating phospho-Spk1as assessed using\na phospho-ERK antibody. MAP kinase coated beads could pull out full length Ste7/Sms1 from\nthe membrane fraction of the cell. These assays don’t ensure that all the proteins are complexed\nat the same time, but do provide solid evidence for interaction of the arrestin protein with all the\nkinase members of the mating kinase cascade. Functional assays established that in the absence\nof the scaffold constitutively active MEK (activated Byr1) the MAPK Spk1 remained\nphosphorylated, but the constitutive shmooing phenotype characteristic of the activated MEK\nkinase is blocked, suggesting the role of the scaffold is not simply to facilitate the kinase\nphosphorylation cascade. The final classes of experiments relate to negative feedback removing\nthe arrestin protein from the membrane to keep mating off under non-mating-conducive\nconditions. This is shown to be phosphorylation controlled, and proteins mutated for “all”\ncandidate MAP kinase Spk1 sites lack any phosphorylation-directed gel shifts. This non-\nphosphorylatable protein shows improper localization, and directs hyper response to pheromone,\nwhile generating pseudophosphorylated residues at the candidate phosphorylation sites\nabolished membrane localization. These experiments generally establish a role of\nphosphorylation, at least in part likely from Spk1, as playing an important role in Ste7/Sms1\nfunction. More detailed analysis of site specificity and the role of other non-proline directed sites\nremain to be done.\nIndeed, site specificity, and the possible role of other proline-directed kinases in regulating\nSms1 will be probed in future studies.\nAt the end of the discussion the authors wonder if the scaffolds are evolving independently. That\nwas the prediction for the Ste5/Far1 development in C. albicans - the Ste5 vWA domain was\nstructurally distinct from the Far1 vWA domain, suggesting independent development, not simply\nduplication and divergence (ref 45).\nThe case here is a different from the Ste5/Far1 example. The domain organisation of Ste5 and\nFar1 is very similar, though their vWA are structurally distinct. The cited study examined the\nevolution of these proteins and showed that emergence of Ste5 predates the whole-genome\nduplication event in Saccharomycotina, but still hypothesized it arose by duplication of part of\nthe Far1 gene. By contrast, Sms1 is structurally completely distinct from Ste5, and we discuss\nhow evolutionary forces may have led to co-option of distinct proteins that converge to similar\nscaffolding functions.\nMinor points\nIn the abstract and the start of the discussion the Sms1 protein is defined as a “structurally novel”\nscaffold. The arrestin fold is not “structurally novel”, and this protein, while representing a new\nscaffold, is not characterized in a way that highlights much about its structure being of dramatic\ninterest. I think novel should not be used in this context.\nWe agree that the arrestin fold is not per se novel. The function of the protein as a scaffold is.\nWe changed the wording from “structurally novel scaffold” to “novel scaffold”. We point out the\ndistinct structural fold at the end of the abstract.\nThe phrase “Because Sms1 binds the active Gα-GTP at the plasma membrane and all MAPKs,”\nis awkward\nAgreed. We changed the sentence to “Because Sms1 binds Gα and all MAPKs at the plasma\nmembrane,…”\nReference 45 is not discussing MAPK duplication, but rather the potential duplication of Far1 into\nthe Far1/Ste5 pair.\nWhile the main message of that paper is indeed on the Far1/Ste5 function, the phylogenetic\nanalysis shows that Ste5 arose simultaneously with the MAPK duplication observed in\nSaccharomycotina, so we feel this is an appropriate citation.\n“…spurious noise signal…” is awkward\nWe have changed this wording to “…spurious environmental noise…”\nReviewer 2\nThis very nice manuscript reports the identification and characterization of the previously unknown\nscaffold for the MAPK signaling cascade that controls mating in the fission yeast,\nSchizosaccharomyces pombe. The data convincingly demonstrate that the previously\nunderstudied protein Sms1 acts as the missing membrane-bound scaffold required for\ntransducing the mating signal, and interacts with all components of the MAPK signaling pathway.\nThe paper further describes how this new scaffold is regulated – by upregulation during mating,\nnuclear export, lipid binding, partner binding, ubiquitination, and negative feedback via\nphosphorylation. In my opinion, it is a very comprehensive work with no significant weaknesses.\nThe data are strong and support the conclusions. The paper is also well-written and will be of\nwide interest to the protein kinase signaling field.\nI do have some suggestions for changes to the text/figures. These are all relatively minor and\nlisted below.\nAbstract:\na) Add an “a” to “scaffolds that recruit kinases to subcellular locations and promote signal\ntransduction have only been described in few species” between in and few.\nDone\nIntro and discussion:\na) Please add a \"the\" between in and presence \"even in presence of hyperactive MAP2K\"\nDone\nb) Rather short shrift is given to the fact that Sms1 was previously known as Ste7 and some\ninformation was known about Ste7. I think this information should be included in the final\nparagraph of the introduction in addition to mentioning it in the results section.\nc) The discussion would also benefit from describing how the new information regarding\nSms1/Ste7 function can explain/expand on or contrasts with what was previously known about\nSte7.\nWe agree. We have added mention that Sms1 was previously known as Ste7 at the end of the\nintroduction and highlighted that Sms1 was known both for its role in cell conjugation and in\nmeiosis repression. We have also added in the discussion that Pat1, found as an Sms1\ninteractor by (Matsuyama et al., 2000), represents an interesting candidate for repression of\nSms1 in nitrogen-rich conditions.\nResults:\n1) “Amongst Ste4 interactors, we focused on the poorly characterised protein SPAC23E2.03c\n(UniprotKB Q10136), previously known as Ste7 and shown to promote mating and meiosis.” I\nrecommend replacing “and” in this sentence with “because it was previously”.\nWe have changed that sentence.\n2) A more detailed explanation for why the observed results in Fig S3 are due to Ste11-dependent\ntranscription would be helpful.\nSte11 is de-repressed upon nitrogen starvation and further enhanced upon pheromone\nstimulation. Previous -omics studies had also shown that sms1 transcription is Ste11-\ndependent. We have rephrased the text to make it clearer that it is an inference from our data,\nconsistent with previously published literature.\n3) “Whole-cell extraction of mating cells expressing Sms1-sfGFP only showed low molecular\nweight fragments”. This sentence confused me for a couple of reasons. First, low molecular\nweight fragments were observed by immunoblotting of whole cell lysates, not by extraction.\nSecond, the sentence/paragraph would benefit from further explanation and contrasting the\nconditions with previous co-IP experiments/blots shown earlier and blots shown later (e.g. Fig.\n3E) in the paper. These other blots showed more than low molecular weight fragments. Expanding\nthe explanation of exactly what the differences in conditions are would avoid confusion and\npromote a better understanding of these variable blotting results.\nThe blots shown in Fig S3C and S3D were obtained before optimisation of the protein\nextraction protocol. In these early experiments, we could not recover any full-length Sms1\nprotein. We then tested multiple parameters in the extraction buffer and protocol to attempt to\nimprove recovery of full-length protein. The main important change was to perform membrane\nextraction but change of the detergent also helped. In the early experiments, we used RIPA\nbuffer, which contains SDS and NP-40. We then substituted these detergents for LMNG. All\nother experiments were performed in these improved solubilization conditions.\nWe have added a sentence in the main text to explain this optimization, which refers to the\nmethods, where this is described in detail: “Optimization of the extraction buffer and protocol\n(see methods) allowed to recover some full-length Sms1, though a large fraction was still\ndegraded (Fig. 3E, soluble fraction).”\n4) “differentially regulated at the membrane (polarity patches and fusion site) and the cytosol.”\nThe meaning would be clearer if the second “and” in this sentence were replaced with “versus”.\nDone\n5) In “its restriction at the polarity patches and fusion site”, please replace “at” with “to”.\nDone\n6) The sentence “ By contrast, Sms1 polarisation was abolished in absence of the GαGpa1 (Fig.\n4E)” seems to be in the wrong place in the text. For a more logical flow and better understanding,\nI suggest re-working this sentence and those in the following paragraph.\nWe have moved this information to the next paragraph as suggested.\n7) The preferential binding of Sms1 to the GTP-locked version of Gpa1 is difficult to substantiate\nwithout quantification of the blots.\nThank you for noticing this omission. Upon quantification of the experimental replicates, it\nbecame apparent that there is too much variability to conclude on a preferential binding of\nSms1 for GTP-locked Gpa1. The quantification of the three independent experiments is now\nadded as Fig. S4D. We also modified the manuscript accordingly by only mentioning the\ninteraction of Sms1 with Gpa1, irrespective of its activation status.\n8) “loss of Sms1 only moderately decreased pSpk1 level (Fig. 5E)”. This statement also requires\nquantification of the blots.\nWe have modified this statement as the loss of Sms1 does not significantly affect pSpk1 level\nin the quantification of the three biological replicates (added as Fig. S5B).\n9) “the phospho-binding proteins 14-3-3, Rad24”; please delete “s” from proteins\nDone\nFigure/figure legends:\n1) In figure 1, inconsistency with Sexual capitalized and cell not.\nCorrected\n2) Throughout the figures, tubulin is used as a control for the amount of soluble protein but\nincluded elsewhere (although not always) as if it is a loading control for the membrane fractions\n(example Fig. 1C) and of course, it is not a loading control for the membrane fractions. Therefore,\nit seems that the blue banner of “soluble” should be added above all the tubulin blots and/or an\nexplanation of this control and its appearance under membrane fraction blots should be provided.\nIndeed, the tubulin was detected in the soluble fraction. We have added to the figure: tubulin\n(soluble).\n3) In the legend for Fig. 1E, it says the volcano plot represents the results of immunoprecipitations.\nThis description is incomplete because it is a representation of the MS results and this could be\nexpanded.\nThe legend has been expanded to be more precise.\n4) Legend to figures 1H and S1Cshould include the number of cells analyzed – looks like many\ncells were analyzed.\nN values are now provided in the legend\n5) Legend to Figure 4, last sentence should end in controls rather than control.\nCorrected\n6) The input and IP panels of Fig. 5C. are not well-aligned in that pSpk1 bands in the two panels\nare at different distances from the markers.\nThank for noticing this alignment issue, we have now adjusted the orientation of the phospho-\nERK1/2 IP panel to ensure alignment of the pSpk1 band.\n7) Please clarify whether or not the Sms1 blots are from lysates or IPs in Fig 6A.\nThese are from lysates. The information is now in the legend.\nMethods:\n1. Yeast media composition and standard methods should be referenced.\nWe have added reference for the composition of the MSL medium, which is the critical one for\nthis study.\n2. Please describe constructs for the eisosome experiments.\nThanks for spotting this omission. The constructs and experiments are now described in the\nmethods.\nReviewer #3:\nIn this manuscript the authors make the surprising discovery of a scaffold protein of the\nSch. pombe pheromone response pathway. After 30 years of investigation in which the\ncommunity thought there was no scaffold, the group of Dr. Sophie Martin present clear\nevidence that the protein they rebaptized Sms1 (formerly known as SPAC23E2.03c or\nSte7) is actually an essential component working as a scaffold. They show convincing\nevidence all along the manuscript. Sms1 binds the Galpha subunit Gpa1, the cofactor\nSte4 (which brings along the Map3K Byr2), the MapK2 Byr1 and the MapK Spk1.\nSms1 associates with the lipids of the plasma membrane, and both interactions (Gpa1\nand lipids) are necessary for Sms1 to be recruited to the PM. It’s association to the PM\nstabilizes it and is essential for shmoo formation. Sms1 seems be negatively regulated\nby multiple S/T-P phosphorylations, since the 22A mutant is hyperactive and the 22E is\nnot active and cannot associate with the PM and they suggest it is negative feedback\nfrom the MAPK.\nAlthough Spk1 needs Sms1 localization to be activated by Byr1, Sms1 is not necessary\nif Byr1 is constitutively active; that is, Sms1 is not an allosteric modulator of Spk1.\nThis is a remarkable paper, with data well supported by multiple experiments. It is quite\namazing that Sms1 performs almost all the functions and operates quite similarly to the\nbudding yeast Ste5 scaffold, but it shares no common evolutionary origin with it.\nMain comments:\nComment 1- There is no direct evidence that Sms1 is regulated by Spk1-dependent\nnegative feedback. The strong evidence is that it is regulated by phosphorylation in S/T-\nP sites, but experiments are needed to establish that Spk1 is the kinase. Given the sheer\nnumber of new results, I’m somewhat puzzled that this particular aspect was chosen as\nthe main result of the paper, appearing in the title itself. The current evidence is this: 1-\nThe 22A Sms1 mutant yeast show a hyperactive pathway and Sms1 highly localized at\nthe membrane. The exact opposite happens to the 22E Sms1 yeast. This means that\nphosphorylation of Sms1 negatively regulates it. 2- Deletion of Spk1 causes a similar but\nmilder phenotype as the 22A Sms1 mutant.\nThat is all. To establish that it is a negative feedback one would need results of this sort (these are only examples): a) Deletion of\nSpk1 results in complete loss (or extensive reduction) of the high MW bands that are\nshow in Fig6A b) Inhibition of Spk1-as mutants with PP1 analogs causes spontaneous\nrecruitment of Sms1 to the membrane c) Byr1-dd suppresses Sms1 PM localization in a\nSpk1 dependent fashion d) In vitro phosphorylation assays that show that Spk1 can\nphosphorylate Sms1 (or fragments of it).\nI don’t think this soft of experiments need to be done for the paper to be accepted, but to\nclaim that Spk1 acts on Sms1 in a negative feedback loop, yes. So, I strongly encourage\nthe authors to change the title accordingly.\nWe agree with this comment. Although it is very likely that Spk1 functions in negative feedback\nregulation of Sms1, the experiments we have done so far do not directly demonstrate this point.\nWe have changed the title accordingly. Future work will address whether Spk1 indeed directly\nphosphorylates Sms1.\nComment 2. It is not clear if they tested the direct interaction between Sms1 and Byr2.\nPlease add that information.\nWe have not tested whether Sms1 directly binds Byr2. This is now mentioned in the discussion.\nResponse to reviewers\nReviewer #2:\nThe majority of my comments have been addressed satisfactorily. However, I found the\nlevel of response to three points a bit perfunctory and therefore insufficient. Specifically,\nI am not yet satisfied with how previous knowledge regarding Ste7 is introduced in the\nintroduction. William Wells, a JCB editor, wrote a piece regarding a rising tendency (way\nback in 2006)to put what should be in an introduction into the discussion, sometimes in\nan effort to make work seem more novel than it is. To avoid this interpretation of motive\nby others, I refer the authors to PMID: 16954351 and ask that they take the\nrecommendations in this perspective more seriously. What was known previously about\nSte7 should be in the introduction to help the readers put the new work in context. This\nwould have helped me better appreciate the conceptual advance of the present work. I\nalso still think that the reason effort was put into Sms1/Ste7 from the list of proteomics\nhits was BECAUSE of what was known previously about this gene product. Finally, I\ndidn't really understand still from a couple of comments in the discussion what was\nknown previously about Ste7.\nThe reviewer is right that the known sterility of sms1/ste7∆ oriented our decision to investigate\nthis protein in more depth, and we have never hidden this information. The only previous work\non Sms1/Ste7 cloned the gene, thus attributing molecular information to the sterility phenotype,\nand showed that Sms1/Ste7 inhibits meiosis upon overexpression. It further showed that the\nprotein is unstable in zygotes and that it interacts (in two-hybrid assays) with the RNA-binding\nprotein Mei2, the master regulator of meiosis, and with its Pat1 kinase inhibitor.\nWe have introduced the previous knowledge on the sterility of sms1/ste7∆ in the introduction\nand have now slightly expanded on it. We then bring the further information on Sms1/Ste7 at\nthe time we first describe it in the results, rather than in the introduction. This is not to hide\ninformation, but to make the flow clearer. Sms1 was discovered in our biochemical screen, the\nsetup of which was not based on any knowledge about Sms1/Ste7. Indeed, the importance of\nthe previous work on Sms1/Ste7 was clearly not appreciated by the field (and we have to\nadmit, we were not aware of it before encountering the protein). The Matsuyama et al paper\nhas been cited 11 times since 2000, 6 times for technical points (not even mentioning\nSms1/Ste7), 3 times by listing Sms1/Ste7 as an example of an arrestin domain-containing\nprotein, once as mis-citation of Ste7 MAP2K and only once for the biology it reported. In no\nway does this reduce its importance, but clearly the work was not a centre piece of our\nunderstanding of the yeast mating process.\nWe have also added a sentence in the discussion on the role of its degradation in zygotes, as\nadditional regulation to the phosphorylation we propose, to promote zygotic events. It is difficult\nto write more, as there is currently no understanding on how the reported interactions with\nMei2 and Pat1 may link or not with its role as pheromone-MAPK scaffold."}]

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