Exomer Is Part of a Hub Where Polarized Secretion and Ionic Stress Connect.

Plasma membrane and membranous organelles contribute to the physiology of the Eukaryotic cell by participating in vesicle trafficking and the maintenance of ion homeostasis. Exomer is a protein complex that facilitates vesicle transport from the trans-Golgi network to the plasma membrane, and its absence leads to the retention of a set of selected cargoes in this organelle. However, this retention does not explain all phenotypes observed in exomer mutants. The Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and cfr1Δ and bch1Δ were sensitive to high concentrations of potassium salts but not sorbitol, which showed sensitivity to ionic but not osmotic stress. Additionally, the activity of the plasma membrane ATPase was higher in exomer mutants than in the wild-type, pointing to membrane hyperpolarization, which caused an increase in intracellular K+ content and mild sensitivity to Na+, Ca2+, and the aminoglycoside antibiotic hygromycin B. Moreover, in response to K+ shock, the intracellular Ca2+ level of cfr1Δ cells increased significantly more than in the wild-type, likely due to the larger Ca2+ spikes in the mutant. Microscopy analyses showed a defective endosomal morphology in the mutants. This was accompanied by an increase in the intracellular pools of the K+ exporting P-type ATPase Cta3 and the plasma membrane Transient Receptor Potential (TRP)-like Ca2+ channel Pkd2, which were partially diverted from the trans-Golgi network to the prevacuolar endosome. Despite this, most Cta3 and Pkd2 were delivered to the plasma membrane at the cell growing sites, showing that their transport from the trans-Golgi network to the cell surface occurred in the absence of exomer. Nevertheless, shortly after gene expression in the presence of KCl, the polarized distribution of Cta3 and Pkd2 in the plasma membrane was disturbed in the mutants. Finally, the use of fluorescent probes suggested that the distribution and dynamics of association of some lipids to the plasma membrane in the presence of KCl were altered in the mutants. Thus, exomer participation in the response to K+ stress was multifaceted. These results supported the notion that exomer plays a general role in protein sorting at the trans-Golgi network and in polarized secretion, which is not always related to a function as a selective cargo adaptor.

Analysis of the SNARE Stx8 recycling reveals that the retromer-sorting motif has undergone evolutionary divergence.

Fsv1/Stx8 is a Schizosaccharomyces pombe protein similar to mammalian syntaxin 8. stx8Δ cells are sensitive to salts, and the prevacuolar endosome (PVE) is altered in stx8Δ cells. These defects depend on the SNARE domain, data that confirm the conserved function of syntaxin8 and Stx8 in vesicle fusion at the PVE. Stx8 localizes at the trans-Golgi network (TGN) and the prevacuolar endosome (PVE), and its recycling depends on the retromer component Vps35, and on the sorting nexins Vps5, Vps17, and Snx3. Several experimental approaches demonstrate that Stx8 is a cargo of the Snx3-retromer. Using extensive truncation and alanine scanning mutagenesis, we identified the Stx8 sorting signal. This signal is an IEMeaM sequence that is located in an unstructured protein region, must be distant from the transmembrane (TM) helix, and where the 133I, 134E, 135M, and 138M residues are all essential for recycling. This sorting motif is different from those described for most retromer cargoes, which include aromatic residues, and resembles the sorting motif of mammalian polycystin-2 (PC2). Comparison of Stx8 and PC2 motifs leads to an IEMxx(I/M) consensus. Computer-assisted screening for this and for a loose Ψ(E/D)ΨXXΨ motif (where Ψ is a hydrophobic residue with large aliphatic chain) shows that syntaxin 8 and PC2 homologues from other organisms bear variation of this motif. The phylogeny of the Stx8 sorting motifs from the Schizosaccharomyces species shows that their divergence is similar to that of the genus, showing that they have undergone evolutionary divergence. A preliminary analysis of the motifs in syntaxin 8 and PC2 sequences from various organisms suggests that they might have also undergone evolutionary divergence, what suggests that the presence of almost-identical motifs in Stx8 and PC2 might be a case of convergent evolution.

Ent3 and GGA adaptors facilitate diverse anterograde and retrograde trafficking events to and from the prevacuolar endosome.

Carboxypeptidases Y (Cpy1) and S (Cps1), the receptor Vps10, and the ATPase subunit Vph1 follow the carboxypeptidase Y (CPY) pathway from the trans-Golgi network (TGN) to the prevacuolar endosome (PVE). Using Schizosaccharomyces pombe quantitative live-cell imaging, biochemical and genetic analyses, we extended the previous knowledge and showed that collaboration between Gga22, the dominant Golgi-localized Gamma-ear-containing ARF-binding (GGA) protein, and Gga21, and between Gga22 and the endosomal epsin Ent3, was required for efficient: i) Vps10 anterograde trafficking from the TGN to the PVE; ii) Vps10 retrograde trafficking from the PVE to the TGN; iii) Cps1 exit from the TGN, and its sorting in the PVE en route to the vacuole; and iv) Syb1/Snc1 recycling to the plasma membrane through the PVE. Therefore, monomeric clathrin adaptors facilitated the trafficking of Vps10 in both directions of the CPY pathway, and facilitated trafficking events of Cps1 in different organelles. By contrast, they were dispensable for Vph1 trafficking. Thus, these adaptors regulated the traffic of some, but not all, of the cargo of the CPY pathway, and regulated the traffic of cargoes that do not follow this pathway. Additionally, this collaboration was required for PVE organization and efficient growth under stress.

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain.

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8-10 aa)C signature motif. Structure-function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.

Traffic Through the Trans-Golgi Network and the Endosomal System Requires Collaboration Between Exomer and Clathrin Adaptors in Fission Yeast.

Despite its biological and medical relevance, traffic from the Golgi to the plasma membrane (PM) is one of the least understood steps of secretion. Exomer is a protein complex that mediates the trafficking of certain cargoes from the trans-Golgi network/early endosomes to the PM in budding yeast. Here, we show that in Schizosaccharomyces pombe the Cfr1 and Bch1 proteins constitute the simplest form of an exomer. Cfr1 co-immunoprecipitates with Assembly Polypeptide adaptor 1 (AP-1), AP-2, and Golgi-localized, gamma-adaptin ear domain homology, ARF-binding (GGA) subunits, and cfr1+ interacts genetically with AP-1 and GGA genes. Exomer-defective cells exhibit multiple mild defects, including alterations in the morphology of Golgi stacks and the distribution of the synaptobrevin-like Syb1 protein, carboxypeptidase missorting, and stress sensitivity. S. pombe apm1Δ cells exhibit a defect in trafficking through the early endosomes that is severely aggravated in the absence of exomer. apm1Δ cfr1Δ cells exhibit a dramatic disorganization of intracellular compartments, including massive accumulation of electron-dense tubulovesicular structures. While the trans-Golgi network/early endosomes are severely disorganized in the apm1Δ cfr1Δ strain, gga21Δ gga22Δ cfr1Δ cells exhibit a significant disturbance of the prevacuolar/vacuolar compartments. Our findings show that exomer collaborates with clathrin adaptors in trafficking through diverse cellular compartments, and that this collaboration is important to maintain their integrity. These results indicate that the effect of eliminating exomer is more pervasive than that described to date, and suggest that exomer complexes might participate in diverse steps of vesicle transport in other organisms.

The long life of an endocytic patch that misses AP-2.

Endocytosis is the process by which cells regulate extracellular fluid uptake and internalize molecules bound to their plasma membrane. This process requires the generation of protein-coated vesicles. In clathrin-mediated endocytosis (CME) the assembly polypeptide 2 (AP-2) adaptor facilitates rapid endocytosis of some plasma membrane receptors by mediating clathrin recruitment to the endocytic site and by connecting cargoes to the clathrin coat. While this adaptor is essential for early embryonic development in mammals, initial results suggested that it is dispensable for endocytosis in unicellular eukaryotes. The drastic effect of depleting AP-2 in metazoa and the mild effect of deleting AP-2 subunits in Saccharomyces cerevisiae have prevented a detailed analysis of the dynamics of endocytic patches in the absence of this adaptor. Using live-cell imaging of Schizosaccharomyces pombe endocytic sites we have shown that eliminating AP-2 perturbs the dynamics of endocytic patches beyond the moment of coat assembly. These perturbations affect the cell growth pattern and cell wall synthesis. Our results highlight the importance of using different model organisms to address the study of conserved aspects of CME.

The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast.

In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution.

Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion.

Regulation of cell wall synthesis by the clathrin light chain is essential for viability in Schizosaccharomyces pombe.

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, ß(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.

The integrity of the cytokinesis machinery under stress conditions requires the glucan synthase Bgs1p and its regulator Cfh3p.

In yeast, cytokinesis requires coordination between nuclear division, acto-myosin ring contraction, and septum synthesis. We studied the role of the Schizosaccharomyces pombe Bgs1p and Cfh3p proteins during cytokinesis under stress conditions. Cfh3p formed a ring in the septal area that contracted during mitosis; Cfh3p colocalized and co-immunoprecipitated with Cdc15p, showing that Cfh3p interacted with the contractile acto-myosin ring. In a wild-type strain, a significant number of contractile rings collapsed under stress conditions and this number increased dramatically in the cfh3Δ, bgs1cps1-191, and cfh3Δ bgs1/cps1-191. Our results show that after osmotic shock Cfh3p is essential for the stability of the (1,3) glucan synthase Bgs1p in the septal area, but not at the cell poles. Finally, cells adapted to stress; they repaired their contractile rings and re-localized Bgs1p to the cell surface some time after osmotic shock. A detailed analysis of the cytokinesis machinery in the presence of KCl revealed that the actomyosin ring collapsed before Bgs1p was internalized, and that it was repaired before Bgs1p re-localized to the cell surface. In the cfh3Δ, bgs1/cps1-191, and cfh3Δ bgs1/cps1-191 mutants, which have reduced glucan synthesis, the damage produced to the ring had stronger consequences, suggesting that an intact primary septum contributes to ring stability. The results show that the contractile actomyosin ring is very sensitive to stress, and that cells have efficient mechanisms to remedy the damage produced in this structure.

The FN3 and BRCT motifs in the exomer component Chs5p define a conserved module that is necessary and sufficient for its function.

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi--including Schizosaccharomyces pombe, which has no detectable amounts of chitin--have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.

The tetraspan protein Dni1p is required for correct membrane organization and cell wall remodelling during mating in Schizosaccharomyces pombe.

In fungi, success of mating requires that both cells agglutinate, modify their extracellular envelopes, and fuse their plasma membranes and nuclei to produce a zygote. Here we studied the role of the Schizosaccharomyces pombe Dni1 protein in the cell fusion step of mating. Dni1p is a tetraspan protein bearing a conserved cystein motif similar to that present in fungal claudin-related proteins. Dni1p expression is induced during mating and Dni1p concentrates as discrete patches at the cell-cell contact area and along the mating bridge. Proper Dni1p localization depends on Fus1p, actin and integrity of lipid rafts. In dni1Delta mutants, cell differentiation and agglutination are as efficient as in the wild-type strain, but cell fusion is significantly reduced at temperatures above 25 degrees C. We found that the defect in cell fusion was not associated with an altered cytoskeleton, with an abnormal distribution of Fus1p, or with a defect in calcium accumulation, but with a severe disorganization of the plasma membrane and cell wall at the area of cell-cell contact. These results show that Dni1p plays a relevant role in co-ordinating membrane organization and cell wall remodelling during mating, a function that has not been described for other proteins in the fission yeast.

The fission yeast SEL1 domain protein Cfh3p: a novel regulator of the glucan synthase Bgs1p whose function is more relevant under stress conditions.

In Schizosaccharomyces pombe, Bgs1/Cps1p is a beta(1,3)-glucan synthase required for linear beta(1,3)-glucan synthesis and primary septum formation. Here, we have studied the regulation of Bgs1p by Cfh3/Chr4p, a member of a family of conserved adaptor proteins, which resembles the chitin synthase regulator Chs4p from Saccharomyces cerevisiae and Candida albicans. cfh3Delta cells showed a genetic interaction with cps1-191, and Cfh3p co-immunoprecipitated with Bgs1/Cps1p. In the absence of cfh3(+), cells were more sensitive to digestion by glucanases, and both Calcofluor staining and glucan synthesis were reduced. We found that in a wild-type strain, beta(1,3)-glucan synthesis was reduced under stress conditions. In the cfh3Delta, cps1-191, and cfh3Delta cps1-191 strains, beta(1,3)-glucan synthesis was further reduced, and growth was impaired under stress conditions, suggesting that Cfh3p and Bgs1p might play a role in ensuring growth in unfavorable environments. In a cfh3Delta mutant, Bgs1p was delocalized when the cells were distressed, but a blockade in endocytosis prevented this delocalization. Finally, we found that the SEL1 repeats are required for Cfh3p function. These results show that Cfh3p is a regulatory protein for Bgs1p and that its function is particularly necessary when the cells are undergoing stress.

The Schizosaccharomyces pombe Map4 adhesin is a glycoprotein that can be extracted from the cell wall with alkali but not with beta-glucanases and requires the C-terminal DIPSY domain for function.

SUMMARY: In fungi, cell adhesion is required for flocculation, mating and virulence, and it is mediated by covalently bound cell wall proteins termed adhesins. Map4, an adhesin required for mating in Schizosaccharomyces pombe, is N-glycosylated and O-glycosylated, and is an endogenous substrate for the mannosyl transferase Oma4p. Map4 has a modular structure with an N-terminal signal peptide, a serine and threonine (S/T)-rich domain that includes nine repeats of 36 amino acids (rich in serine and threonine residues, but lacking glutamines), and a C-terminal DIPSY domain with no glycosylphosphatidyl inositol (GPI)-anchor signal. Map4 can be extracted from cell walls with SDS/mercaptoethanol sample buffer or with mild alkali solutions. After extensive extraction with hot sample buffer, no more protein can be released by beta-glucanases or alkali. Additionally, none of the cysteine residues of the protein is required for its retention at the cell wall. These results show that Map4 is not directly bound to beta-glucans and point to the existence of alkali- and SDS/mercaptoethanol-sensitive linkages between cell wall proteins. The N-terminal S/T-rich regions are required for cell wall attachment, but the C-terminal DIPSY domain is required for agglutination and mating in liquid and solid media.

The fission yeast Chs2 protein interacts with the type-II myosin Myo3p and is required for the integrity of the actomyosin ring.

In Schizosaccharomyces pombe cytokinesis requires the function of a contractile actomyosin ring. Fission yeast Chs2p is a transmembrane protein structurally similar to chitin synthases that lacks such enzymatic activity. Chs2p localisation and assembly into a ring that contracts during division requires the general system for polarised secretion, some components of the actomyosin ring, and an active septation initiation network. Chs2p interacts physically with the type-II myosin Myo3p revealing a physical link between the plasma membrane and the ring. In chs2Delta mutants, actomyosin ring integrity is compromised during the last stages of contraction and it remains longer in the midzone. In synchronous cultures, chs2Delta cells exhibit a delay in septation with respect to the control strain. All these results show that Chs2p participates in the correct functioning of the medial ring.

The Schizosaccharomyces pombe cfr1+ gene participates in mating through a new pathway that is independent of fus1+.

Conjugation is a complex event directed to ensure the transfer of genetic material, which is achieved by the union of two cells. In fungi, success of this relevant process requires digestion of the cell wall at the point where both cells have agglutinated and, later, the union of the plasma membranes and nuclei from the mating partners. In order to gain information about cell fusion, we have cloned and disrupted the cfr1+ gene from the fission yeast Schizosaccharomyces pombe. cfr1+ gene is slightly induced at the beginning of mating but Cfr1p protein is degraded soon after the cells are transferred to nitrogen-lacking medium. cfr1Delta mutants present a defect in cell fusion owing to a failure in the digestion of the cell walls between the two parental cells. Finally, cytological and genetic analyses show that cfr1+ acts in a new pathway involved in conjugation that is independent of fus1+, the only gene that has been found to be specifically required for cell fusion during mating in the fission yeast.

In Schizosaccharomyces pombe chs2p has no chitin synthase activity but is related to septum formation.

Chitin synthesis occurs in most fungi through the action of different chitin synthase (CS) isoenzymes. In Schizosaccharomyces pombe the chs2(+) gene codes for a protein with significant similarity to CS enzymes, but lacking most of the residues considered to be essential for activity, including the QRRRW domain. Here we show that chs2p is a functional protein that localises to the growing edge of the septum but is not a CS enzyme. Strong over-expression is lethal, while moderate expression leads to a severe defect in septum formation. These results suggest that chs2p has remained through evolution to play an alternative role in septation.

Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization.

Chitin synthase III is essential for the increase in chitin level and for cell integrity in cells lacking Gas1p, a beta(1,3)-glucanosyltransferase. In order to discover whether the upregulation of chitin synthesis proceeds through the canonical transport and activation pathway of Chs3p or through an alternative one, here we studied the effects of the inactivation of the GAS1 and CHS4-5-6-7 genes. All the double-null mutants showed a temperature-sensitive cell lysis phenotype that could be suppressed by the presence of an osmotic stabilizer. In liquid YEPD at 30 degrees C, chs4 delta gas1 delta, chs5 delta gas1 delta, chs6 deltagas1 delta and chs7 delta gas1 delta mutants were unable to grow, whereas they grew very slowly in minimal medium and showed low viability. High osmolarity suppressed the defective phenotype and restored growth. In chs4 gas1, chs5 gas1 and chs7 gas1, chitin levels did not increase and were reduced to only 10%, while in chs6 gas1 the value of gas1 was reduced to 20-40%. To investigate at which level the upregulation of chitin synthesis could occur, mRNA levels were monitored. The expression of CHS4-5-6-7 did not change significantly in gas1 delta. In strains expressing HA-tagged forms, the localization of Chs3p and Chs5p was examined. In the gas1 mutant the fluorescence pattern was affected and the proteins appeared abnormally present in the bud. The results indicate that: (a) the function of the CHS4-7 genes is required for chitin hyperaccumulation in gas1 mutant and for cell integrity; (b) homologous genes do not replace their function; (c) the regulation of CHS4-7 genes does not occur at transcriptional level. Control of the position of chitin synthesis could be important in protecting the bud from lysis.