Pck2 association with the plasma membrane and efficient response of the cell integrity pathway require regulation of PI4P homeostasis by exomer.

Exomer is a protein complex that facilitates trafficking between the Golgi and the plasma membrane (PM). exomer is composed of Cfr1 and Bch1, and we have found that full activation of the cell integrity pathway (CIP) in response to osmotic stress requires exomer. In the wild-type, the CIP activators Rgf1 (Rho1 GEF) and Pck2 (PKC homologue) and the MEK kinase Mkh1 localize in the PM, internalize after osmotic shock and re-localize after adaptation.

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 -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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.