SUBCELLULAR-LOCALIZATION, ABUNDANCE AND STABILITY OF CHITIN SYNTHETASE-1 AND SYNTHETASE-2 FROM SACCHAROMYCES-CEREVISIAE

The existence of more than one chitin synthetase in fungal cells poses the question of whether these enzymes have similar or different local ization. The subcellular distribution of chitin synthetases 1 and 2 (C hs1 and Chs2) was determined in cell-free extracts of Saccharomyces ce revisiae fractionated by sucrose density gradient sedimentation. Chs1 was examined in two strains: ATCC 26109, a wild-type strain, and D3C ( MAT alpha ura3-52). Chs2 was investigated in a strain (D3B) freed of C hs1 by gene disruption (MAta his4 ura3-52 chs1::URA3). A prolonged, st rong centrifugation (20 h at 265000 g) was necessary to cleanly resolv e two major populations of chitin synthetase particles: chitosomes (a population of microvesicles of low buoyant density, d = 1.15 g ml(-1)) and plasma membrane (a population of vesicles of high buoyant density , d = 1.21 g ml(-1)). Chs1 and Chs2 were both present in chitosomes an d plasma membrane, but the relative distribution of each chitin synthe tase in these two membranous populations varied. Chs1 was much less ab undant than Chs1 and required Co2+ rather than Mg2+ as a cofactor. A s alient finding was the high sensitivity of chitosomal Chs2 to high cen trifugal forces. The subcellular distribution of 1.3-beta-glucan synth etase was the same in the three strains studied, i.e. unaffected by th e presence or absence of Chs1. Culture conditions affected the profile s of chitin and glucan synthetases: the relative abundance of Chs1 in chitosomes or plasma membrane was quite different in cells grown on tw o different media but the buoyant density was not affected; in contras t, there was shift in the buoyant density of the two peaks of 1,3-beta -glucan synthetase. We concluded that the subcellular localization of Chs1 and Chs2 remains the same despite genetic and other differences i n the properties of these enzymes. We confirmed that 1,3-beta-glucan s ynthetase and chitin synthetase exhibit a partially different subcellu lar distribution - an indication that these two enzymes are mobilized through different secretory pathways.