Differential Trafficking and Timed Localization of Two Chitin Synthase Proteins, Chs2p and Chs3p

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 pulsechase 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. Temperaturesensitive mutations that block either the late secretory pathway (secl-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. T HE polysaccharide chitin is deposited into the cell wall of Saccharomyces cerevisiae in a spatially and temporally regulated manner (for review see Cabib et al., 1982; Bulawa, 1993; Cid et al., 1995). In contrast with the major cell wall polysaccharides, glucans and mannans, which are found over the entire cell surface, chitin is specifically localized. A chitin ring forms on the cell surface in late G1, before bud emergence. This annulus predicts the site of bud emergence and persists at the junction between the mother and the growing bud. At the end of mitosis, centripetal synthesis within the chitin ring results in the formation of a disk of chitin called the primary division septum. This event initiates the process of cell separation. Secondary septa, comprised of glucans and mannans, are subsequently laid down on each side of the primary division septum. Finally, cell separation is effected by the action of an endochitinase, which partially hydrolyzes the Address all correspondence to Randy W. Schekman, Department of Molecular and Cell Biology, Howard Hughes Institute, University of California, Berkeley, Berkeley, CA 04720. Tel.: (510) 642-5686. Fax (510) 6427846. chitin septum, resulting in the physical separation of the mother and the daughter cell. The localized deposition of chitin during specific points in the cell cycle has been of interest as a model for the development of cell surface asymmetry. Three chitin synthase (CS) 1 activities (CSI, II, and III) have been identified in S. cerevisiae (Bulawa, 1993; Cabib et al., 1982; Orlean, 1987; Sburlati and Cabib, 1986). All three enzymes are membrane associated and catalyze the synthesis of chitin chains from UDP-N-acetylglucosamine. Chslp is required for CSI activity (Bulawa et al., 1986). CSII activity is encoded by the CHS2 gene (Silverman et al., 1988). CSIII activity requires the function of CHS3 (recently renamed from CAL1/CSD2/DITIO1/KTI2; Cid et al., 1995), which is the presumed catalytic subunit, as well as CHS4 (CAL2/CSD4) and CHS5 (CAL3) (Bulawa, 1992, 1. Abbreviat ions used in this paper:. CS, chitin synthase; CWP, cell wall protein; DAPI, 4',6-diamidino-2-phenylindole; GST, glutathione-S-transferase; HA, hemagglutinin; nt, nucleotide; ORF, open reading frame; PGK, phosphoglycerate kinase; PIC, protease inhibitor cocktail: TEA, triethanolamine. © The Rockefeller University Press, 0021-9525/96/11/597/14 $2.00 The Journal of Cell Biology, Volume 135, Number 3, November 1996 597~10 597 on M ay 8, 2017 D ow nladed fom Published November 1, 1996