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1.
Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.  相似文献   

2.
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.  相似文献   

3.
Saccharomyces cerevisiae Chs2 (chitin synthase 2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and is expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an active form at high levels. Both constructs show a pH and cation dependence similar to the wild-type enzyme, as well as increased activity after trypsin treatment. Using further biochemical analysis, we have identified two mechanisms of chitin synthase regulation. First, it is hyperactivated by a soluble yeast protease. This protease is expressed during exponential growth phase, when budding cells require Chs2 activity. Secondly, LC-MS/MS (liquid chromatography tandem MS) experiments on purified Chs2 identify 12 phosphorylation sites, all in the N-terminal domain. Four of them show the perfect sequence motif for phosphorylation by the cyclin-dependent kinase Cdk1. As we also show that phosphorylation of the N-terminal domain is important for Chs2 stability, these sites might play an important role in the cell cycle-dependent degradation of the enzyme, and thus in cell division.  相似文献   

4.
Previous genetic evidence led to the conclusion that proteinase B of yeast was not involved in the function of chitin synthetase 1 (Chs1), based on the demonstration of normal septum formation, cell division and chitin deposition in mutants devoid of the proteinase (Zubenko, G.S., Mitchell, A.P., and Jones, E.W. (1979) Proc. Natl. Acad. Sci. USA 76, 2395-2399). Later, however, it was found that the essential enzyme for septum formation is chitin synthetase 2, whereas Chs1 acts as an auxiliary enzyme, whose absence results in daughter cell lysis under acidic conditions (Cabib, E., Sburlati, A., Bowers, B. and Silverman, S.J. (1989) J. Cell Biol. 108, 1665-1672). By using the lytic behavior as a criterion, we have now found that prb1 strains are not defective in Chs1 function. Certain strains contain a recessive suppressor of lysis which could mask the Chs1 defect. However, appropriate crosses and transformation experiments showed that the prb1 mutants do not harbor the suppressor. It may now be concluded with confidence that proteinase B is not required for chitin synthetase 1 function.  相似文献   

5.
Echinocandins are a new generation of novel antifungal agent that inhibit cell wall beta(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca(2+)-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
9.
Swm1p, a subunit of the APC cyclosome, was originally identified for its role in the later stages of the sporulation process and is required for spore wall assembly. In addition, this protein is required to maintain cell wall integrity in vegetative cells during growth at high temperature. Electron microscopy analyses of mutant cells grown at the restrictive temperature in the absence of osmotic support show that the cell wall is clearly abnormal, with large number of discontinuities that may be responsible for the observed lysis. The mutant cells show a 7-fold reduction in glucan synthase activity during growth at 38 degrees C and a 3.5-fold increase in the chitin content of the cell wall. The chitin is deposited in a delocalized manner all over the cell wall, where it accumulates in patches in abnormal regions. The excess chitin is mainly synthesized by the action of chitin synthase III (Chs3p), since it disappears in the swm1 chs3 double-mutant.  相似文献   

10.
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.  相似文献   

11.
The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor. To be effective, the interaction of Calcofluor with growing cells had to occur at around pH 6. Treatment of growing cells from these strains with the fluorochrome led to an increase in the total levels of Chs1 and Chs2 activities measured on permeabilized cells. During treatment, basal levels (activities expressed in the absence of exogenous proteolytic activation) of Chs1 and Chs2 increased nine- and fourfold, respectively, through a mechanism dependent on protein synthesis, since the effect was abolished by cycloheximide. During alpha-factor treatment, both Chs1 and Chs2 levels increased; however, as opposed to what occurred during the mitotic cell cycle, there was no further increase in Chs1 or Chs2 activities by Calcofluor treatment.  相似文献   

12.
The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small- budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal- Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.  相似文献   

13.
The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.  相似文献   

14.
To explore the function of chitin in Schizosaccharomyces pombe, we have cloned chs1+ and chs2+, encoding putative chitin synthases, based on sequences in the Sanger Centre database. The synthetic lethal phenotype of the S. cerevisiae chs1 chs2 chs3 mutant was complemented by expression of S. pombe chs1+ or chs1+, indicating that both chs1+ and chs2+ in fact encode chitin synthase. The homothallic Deltachs1 strain formed abnormal asci that contained 1, 2, or 3 spores, while the Deltachs2 strain had no noticeable phenotype. The chs1 chs2 double disruptant looked similar phenotypically to the Deltachs1 strain. The Chs2-GFP fusion protein predominantly localized at the septum after the septum was formed during vegetative growth. The level of chs2+ mRNA increased just before the septum was formed. Levels of Chs2-13Myc synthesis also changed during the cell cycle. Thus, chs1+ is required for proper spore formation, and chs2+ is perhaps involved in septum formation.  相似文献   

15.
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.  相似文献   

16.
Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense.  相似文献   

17.
Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role.  相似文献   

18.
It is shown that the deletion of BGL2 gene leads to increase in chitin content in the cell wall of Saccharomyces cerevisiae. A part of the additional chitin can be removed from the bgl2Δ cell wall by alkali or trypsin treatment. Chitin synthase 1 (Chs1) activity was increased by 60 % in bgl2Δ mutant. No increase in chitin synthase 3 (Chs3) activity in bgl2Δ cells was observed, while they became more sensitive to Nikkomycin Z. The chitin level in the cell walls of a strain lacking both BGL2 and CHS3 genes was higher than that in chs3Δ and lower than that in bgl2Δ strains. Together these data indicate that the deletion of BGL2 results in the accumulation and abnormal incorporation of chitin into the cell wall of S. cerevisiae, and both Chs1 and Chs3 take part in a response to BGL2 deletion in S. cerevisiae cells. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

19.
Actomyosin ring contraction and chitin primary septum deposition are interdependent processes in cell division of budding yeast. By fusing Myo1p, as representative of the contractile ring, and Chs2p for the primary septum, to different fluorescent proteins we show herein that the two processes proceed essentially at the same location and simultaneously. Chs2p differs from Myo1p in that it reflects the changes in shape of the plasma membrane to which it is attached and in that it is packed after its action into visible endocytic vesicles for its disposal. To ascertain whether this highly coordinated system could function independently of other cell cycle events, we reexamined the septum-like structures made by the septin mutant cdc3 at various sites on the cell cortex at the nonpermissive temperature. With the fluorescent fusion proteins mentioned above, we observed that in cdc3 at 37 degrees C both Myo1p and Chs2p colocalize at different spots of the cell cortex. A contraction of the Myo1p patch could also be detected, as well as that of a Chs2p patch, with subsequent appearance of vesicles. Furthermore, the septin Cdc12p, fused with yellow or cyan fluorescent protein, also colocalized with Myo1p and Chs2p at the aberrant locations. The formation of delocalized septa did not require nuclear division. We conclude that the septation apparatus, composed of septins, contractile ring, and the chitin synthase II system, can function at ectopic locations autonomously and independently of cell division, and that it can recruit the other elements necessary for the formation of secondary septa.  相似文献   

20.
In yeast, certain resident trans-Golgi network (TGN) proteins achieve steady-state localization by cycling through late endosomes. Here, we show that chitin synthase III (Chs3p), an enzyme involved in the assembly of the cell wall at the mother-bud junction, populates an intracellular reservoir that is maintained by a cycle of transport between the TGN and early endosomes. Traffic of Chs3p from the TGN/early endosome to the cell surface requires CHS5 and CHS6, mutant alleles of which trap Chs3p in the TGN/early endosome. Disruption of the clathrin adaptor protein complex 1 (AP-1) restores Chs3p transport to the plasma membrane. Similarly, in AP-1 deficient cells, the resident TGN/early endosome syntaxin, Tlg1p, is missorted. We propose that clathrin and AP-1 act to recycle Chs3p and Tlg1p from the early endosome to the TGN.  相似文献   

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