Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.     

Targeting of Chitin Synthase 3 to Polarized Growth Sites in Yeast Requires Chs5p and Myo2p

Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycledependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p.     

The complex interactions of Chs5p,the ChAPs,and the cargo Chs3p

The exomer complex is a putative vesicle coat required for the direct transport of a subset of cargoes from the trans-Golgi network (TGN) to the plasma membrane. Exomer comprises Chs5p and the ChAPs family of proteins (Chs6p, Bud7p, Bch1p, and Bch2p), which are believed to act as cargo receptors. In particular, Chs6p is required for the transport of the chitin synthase Chs3p to the bud neck. However, how the ChAPs associate with Chs5p and recognize cargo is not well understood. Using domain-switch chimeras of Chs6p and Bch2p, we show that four tetratricopeptide repeats (TPRs) are involved in interaction with Chs5p. Because these roles are conserved among the ChAPs, the TPRs are interchangeable among different ChAP proteins. In contrast, the N-terminal and the central parts of the ChAPs contribute to cargo specificity. Although the entire N-terminal domain of Chs6p is required for Chs3p export at all cell cycle stages, the central part seems to predominantly favor Chs3p export in small-budded cells. The cargo Chs3p probably also uses a complex motif for the interaction with Chs6, as the C-terminus of Chs3p interacts with Chs6p and is necessary, but not sufficient, for TGN export.     

The Syntaxin Tlg1p Mediates Trafficking of Chitin Synthase III to Polarized Growth Sites in Yeast

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the α factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.     

Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi

In Saccharomyces cerevisiae, the synthesis of chitin is temporally and spatially regulated through the transport of Chs3p (chitin synthase III) to the plasma membrane in the bud neck region. Traffic of Chs3p from the trans-Golgi network (TGN)/early endosome to the plasma membrane requires the function of Chs5p and Chs6p. Chs6p belongs to a family of four proteins that we have named ChAPs for Chs5p-Arf1p-binding Proteins. Here, we show that all ChAPs physically interact not only with Chs5p but also with the small GTPase Arf1p. A short sequence at the C-terminus of the ChAPs is required for protein function and the ability to bind to Chs5p. Simultaneous disruption of two members, Deltabud7 and Deltabch1, phenocopies a Deltachs6 or Deltachs5 deletion with respect to Chs3p transport. Moreover, the ChAPs interact with each other and can form complexes. In addition, they are all at least partially localized to the TGN in a Chs5p-dependent manner. Most importantly, several ChAPs can interact physically with Chs3p. We propose that the ChAPs facilitate export of cargo out of the Golgi.     

Retention of Chs2p in the ER requires N-terminal CDK1-phosphorylation sites

In budding yeast, the secretory pathway is constitutively transporting cargoes such as invertase and α-factor throughout the cell division cycle. However, chitin synthase 2 (Chs2p), another cargo of the secretory pathway, is retained at the endoplasmic reticulum (ER) during mitosis when the mitotic kinase activity is high. Chs2p is exported from the ER to the mother-daughter neck only upon mitotic kinase destruction, indicating that the mitotic kinase activity is critical for the ER retention of Chs2p. However, a key question is whether the mitotic kinase acts directly upon Chs2p to prevent its ER export. We report here that mutation of Ser residues to Glu at 4 perfect CDK1-phosphorylation sites at the N-terminus of Chs2p leads to its retention in the ER when the mitotic kinase activity is absent. Conversely, Ser-to-Ala mutations result in the loss of Chs2p ER retention even when mitotic kinase activity is high. The mere over-expression of the non-destructible form of the mitotic cyclin in G1 cells can confine the wild-type Chs2p but not the Ser-to-Ala mutant in the ER. Furthermore, over-expression of the Ser-to-Ala mutant kills cells. Time-lapsed imaging revealed that Chs2p is exported from the ER rapidly and synchronously to the Golgi upon metaphase release. Our data indicate that direct phosphorylation of Chs2p by the mitotic CDK1 helps restrain it in the ER during mitosis to prevent its rapid export in an untimely manner until after sister chromatid occurs and mitotic exit executed.     

A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the “neck filaments” that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.     

Exit from mitosis triggers Chs2p transport from the endoplasmic reticulum to mother-daughter neck via the secretory pathway in budding yeast

Budding yeast chitin synthase 2 (Chs2p), which lays down the primary septum, localizes to the mother-daughter neck in telophase. However, the mechanism underlying the timely neck localization of Chs2p is not known. Recently, it was found that a component of the exocyst complex, Sec3p-green fluorescent protein, arrives at the neck upon mitotic exit. It is not clear whether the neck localization of Chs2p, which is a cargo of the exocyst complex, was similarly regulated by mitotic exit. We report that Chs2p was restrained in the endoplasmic reticulum (ER) during metaphase. Furthermore, mitotic exit was sufficient to cause Chs2p neck localization specifically by triggering the Sec12p-dependent transport of Chs2p out of the ER. Chs2p was "forced" prematurely to the neck by mitotic kinase inactivation at metaphase, with chitin deposition occurring between mother and daughter cells. The dependence of Chs2p exit from the ER followed by its transport to the neck upon mitotic exit ensures that septum formation occurs only after the completion of mitotic events.     

Dependence of Chs2 ER export on dephosphorylation by cytoplasmic Cdc14 ensures that septum formation follows mitosis

Cytokinesis, which leads to the physical separation of two dividing cells, is normally restrained until after nuclear division. In Saccharomyces cerevisiae, chitin synthase 2 (Chs2), which lays down the primary septum at the mother-daughter neck, also ensures proper actomyosin ring constriction during cytokinesis. During the metaphase-to-anaphase transition, phosphorylation of Chs2 by the mitotic cyclin-dependent kinase (Cdk1) retains Chs2 at the endoplasmic reticulum (ER), thereby preventing its translocation to the neck. Upon Cdk1 inactivation at the end of mitosis, Chs2 is exported from the ER and targeted to the neck. The mechanism for triggering Chs2 ER export thus far is unknown. We show here that Chs2 ER export requires the direct reversal of the inhibitory Cdk1 phosphorylation sites by Cdc14 phosphatase, the ultimate effector of the mitotic exit network (MEN). We further show that only Cdc14 liberated by the MEN after completion of chromosome segregation, and not Cdc14 released in early anaphase by the Cdc fourteen early anaphase release pathway, triggers Chs2 ER exit. Presumably, the reduced Cdk1 activity in late mitosis further favors dephosphorylation of Chs2 by Cdc14. Thus, by requiring declining Cdk1 activity and Cdc14 nuclear release for Chs2 ER export, cells ensure that septum formation is contingent upon chromosome separation and exit from mitosis.     

Septins,under Cla4p regulation,and the chitin ring are required for neck integrity in budding yeast

CLA4, encoding a protein kinase of the PAK type, and CDC11, encoding a septin, were isolated in a screen for synthetic lethality with CHS3, which encodes the chitin synthase III catalytic moiety. Although Ste20p shares some essential function with Cla4p, it did not show synthetic lethality with Chs3p. cla4 and cdc11 mutants exhibited similar morphological and septin localization defects, including aberrant and ectopic septa. Myo1p, which requires septins for localization, formed abnormally wide rings in cla4 mutants. In cultures started with unbudded cells, an inhibitor of Chs3p activity, nikkomycin Z, aggravated the abnormalities of cla4 and cdc11 mutants and gave rise to enlarged necks at the mother-bud junction, leading to cell death. It is concluded that Cla4p is required for the correct localization and/or assembly of the septin ring and that both the septin ring and the Chs3p-requiring chitin ring at the mother-bud neck cooperate in maintaining the neck constricted throughout the cell cycle, a vital function in budding yeast.     

Oligomerization of the chitin synthase Chs3 is monitored at the Golgi and affects its endocytic recycling

Chs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP‐1‐dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N‐terminal cytosolic region. However, the truncated ^Δ126Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI‐ and Rer1‐dependent manner. A subset of the oligomerization‐deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N‐terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly.     

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III activity and chitin chain length

Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a approximately 60% decrease in CSIII activity, which is correlated with a approximately 30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.     

Chs6p-dependent Anterograde Transport of Chs3p from the Chitosome to the Plasma Membrane in Saccharomyces cerevisiae

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Δ strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4–1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.     

Ypt32p regulates the translocation of Chs3p from an internal pool to the plasma membrane

The transport of the chitin synthase III, Chs3p, to the plasma membrane is temporally and spatially regulated. Chs3p is delivered to the plasma membrane at the beginning of the cell cycle, forming chitin rings, and at the end of the cell cycle, forming the primary septum. During the rest of the cell cycle, it is maintained in intracellular compartments, termed chitosomes that share characteristics with the late Golgi and the early endosomes. Chs5p and Chs6p are required for the cell cycle-dependent delivery of Chs3p to the cell surface, but the mechanisms underlying the temporal regulation are still unknown. The Rab proteins, Ypt31/32p, are required for exit of secretory vesicles from the late Golgi and for recycling of proteins between the late Golgi and early endosomes. Either gain of Ypt32p function, by overexpression, or loss-of-function mutations alter the localization of Chs3p-GFP. Moreover, cells overexpressing Ypt32p accumulate chitin at the cell surface independent of Chs5p. Overexpression of Ypt32p also disrupts the localization of the late Golgi protein Sec7. We propose that Ypt31/32p have a role in regulating the delivery of Chs3p to the plasma membrane and deposition of chitin at the cell surface.     

The chaperone Chs7 forms a stable complex with Chs3 and promotes its activity at the cell surface

The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane‐localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the endoplasmic reticulum (ER). Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. Here, we show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments. We identified mutations in the Chs7 C‐terminal cytosolic domain that do not affect its chaperone function, but cause it to dissociate from Chs3 at a post‐ER transport step. Mutations that prevent the continued association of Chs7 with Chs3 do not block delivery of Chs3 to the cell surface, but dramatically reduce its catalytic activity. This suggests that Chs7 engages in functionally distinct interactions with Chs3 to first promote its folding and ER exit, and subsequently to regulate its activity at the plasma membrane.      

Chs5/6 complex: a multiprotein complex that interacts with and conveys chitin synthase III from the trans-Golgi network to the cell surface

In Saccharomyces cerevisiae, the polysaccharide chitin is deposited at the mother bud junction by an integral membrane enzyme, chitin synthase 3 (Chs3p). The traffic of Chs3p to the cell surface from the trans-Golgi network (TGN) depends on two proteins, Chs5p and Chs6p, which sort selected cargo proteins into secretory vesicles. We have found that Chs5p forms a large higher-order complex of around 1 MDa with Chs6p and three Chs6 paralogs: Bch1p, Bud7p, and Bch2p. The Chs5/6 complex transiently interacts with its cargo, Chs3p, and the presence of Chs3p in the complex is dependent on every subunit. Chs5p and Chs6p have unique and crucial roles in Chs3p transport because either a chs5delta or chs6delta mutant drastically reduces the level of Chs3p bound to the remaining subunits of the complex. Bch1p and Bud7p appear to have a redundant function in Chs3p transport because deletion of both is necessary to displace Chs3p from the complex. The role of Bch2p in Chs3p binding is the least important. Chs5p is essential for structural integrity of the Chs5/6 complex and may act as a scaffold through which the other subunits assemble. Our results suggest a model of protein sorting at the TGN that involves a peripheral, possibly coat, complex that includes multiple related copies of a specificity determining subunit.     

Neck compartmentalization as the molecular basis for the different endocytic behaviour of Chs3 during budding or hyperpolarized growth in yeast cells

Yeast cells normally grow by budding, but under certain specific conditions they are also able to grow in hyperpolarized forms reminiscent of hyphal growth. During vegetative growth, the synthesis of the septum that physically separates yeast cells during cytokinesis depends on the correct assembly of the septin ring. Septins and actin patches are assembled at the neck, forming two concentric rings where the actin patch ring occupies the external-most part. This specific positioning defines a plasma membrane region at the neck from which other lateral membrane compartments are excluded. In this scenario, correct assembly of the chitin ring is dependent on the anchoring of Chs3 to the septin ring through Chs4. The anchoring of Chs3 to septins through Chs4 prevents the arrival of this protein at endocytic sites, thus reducing the endocytosis of Chs3. This allows an equilibrium to be set up between the antero- and retrograde transport of Chs3, facilitating the synthesis of the chitin ring at the neck. In contrast, hyperpolarized growth is characterized by a reduced endocytic turnover of Chs3, which in turn lead to the accumulation of Chs3 at the plasma membrane and a concomitant increase in chitin synthesis.     

Cell cycle-regulated trafficking of Chs2 controls actomyosin ring stability during cytokinesis

Cytokinesis requires the coordination of many cellular complexes, particularly those involved in the constriction and reconstruction of the plasma membrane in the cleavage furrow. We have investigated the regulation and function of vesicle transport and fusion during cytokinesis in budding yeast. By using time-lapse confocal microscopy, we show that post-Golgi vesicles, as well as the exocyst, a complex required for the tethering and fusion of these vesicles, localize to the bud neck at a precise time just before spindle disassembly and actomyosin ring contraction. Using mutants affecting cyclin degradation and the mitotic exit network, we found that targeted secretion, in contrast to contractile ring activation, requires cyclin degradation but not the mitotic exit network. Analysis of cells in late anaphase bearing exocyst and myosin V mutations show that both vesicle transport and fusion machineries are required for the completion of cytokinesis, but this is not due to a delay in mitotic exit or assembly of the contractile ring. Further investigation of the dynamics of contractile rings in exocyst mutants shows these cells may be able to initiate contraction but often fail to complete the contraction due to premature disassembly during the contraction phase. This phenotype led us to identify Chs2, a transmembrane protein targeted to the bud neck through the exocytic pathway, as necessary for actomyosin ring stability during contraction. Chs2, as the chitin synthase that produces the primary septum, thus couples the assembly of the extracellular matrix with the dynamics of the contractile ring during cytokinesis.     

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.     

Specific protein targeting during cell differentiation: polarized localization of Fus1p during mating depends on Chs5p in Saccharomyces cerevisiae

In budding yeast, chs5 mutants are defective in chitin synthesis and cell fusion during mating. Chs5p is a late-Golgi protein required for the polarized transport of the chitin synthase Chs3p to the membrane. Here we show that Chs5p is also essential for the polarized targeting of Fus1p, but not of other cell fusion proteins, to the membrane during mating.