We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations. These procedures were used to examine the subcellular distribution ofchitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast. Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters. One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-P-glucan synthetase activity. The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase. This fraction cosedimented with the main peak of 1,3-13-glucan synthetase. A third, minor population of chitin synthetase particles was also detected. Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of cbitin synthetase whose location in the cell has yet to be unequivocally determined.Because of the importance of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in cell wall formation and morphogenesis in fungi, considerable attention has been devoted to the characterization of this enzyme and the cell biological mechanisms by which it functions. Conflicting claims have been made about the cellular localization of chitin synthetase: some studies emphasize the localization in chitosomes (1-4); others maintain it is mainly, if not exclusively, a plasma membrane-bound enzyme (5-7). Saccharomyces cerevisiae (2, 5) has been a salient subject in this controversy.Our recent experience with improved methods for separating membranous organelles by density gradient sedimentation (8) was applied to examine the intracellular distribution of chitin synthetase in cell-free homogenates of S. cerevisiae. We used exponentially growing, walled cells rather than protoplasts (4,5) since the former represent a normal morphogenetic state of this organism and thus offer a greater potential for revealing the in vivo distribution of chitin synthetase. METHODS Cultivation and Cell Disruption. S. cerevisiae (ATCC 26109) was grown in 1200 ml offilter-sterilized medium (0.7% Difco yeast nitrogen base; 2% glucose) (9) in a shaker bath at 14 E l-.

The biocontrol agent Trichoderma harzianum IMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular β-1,3-glucanases upon induction with laminarin, a soluble β-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible β-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-β-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.

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The existence of more than one chitin synthetase in fungal cells poses the question of whether these enzymes have similar or different localization. The subcellular distribution of chitin synthetases 1 and 2 (Chsl and Chs2) was determined in cell-free extracts of Saccharomyces cerevisiae fractionated by sucrose density gradient sedimentation. Chsl was examined in two strains : ATCC 26109, a wild-type strain, and D3C (MATE ura3-52). Chs2 was investigated in a strain (D3B) freed of Chsl by gene disruption (MATa his4 ura3-52 chsl::URA3). A prolonged, strong centrifugation (20 h a t 265000 g) was necessary to cleanly resolve two major populations of chitin synthetase particles : chitosomes (a population of microvesicles of low buoyant density, d = 1-15 g ml-I) and plasma membrane (a population of vesicles of high buoyant density, d = 1.21 g ml-I). Chsl and Chs2 were both present in chitosomes and plasma membrane, but the relative distribution of each chitin synthetase in these two membranous populations varied. Chs2 was much less abundant than Chsl and required Co2+ rather than Mg2+ as a cofactor. A salient finding was the high sensitivity of chitosomal Chs2 to high centrifugal forces. The subcellular distribution of 1,3-P-glucan synthetase was the same in the three strains studied, i.e. unaffected by the presence or absence of Chsl. Culture conditions affected the profiles of chitin and glucan synthetases : the relative abundance of Chsl in chitosomes or plasma membrane was quite different in cells grown on two different media but the buoyant density was not affected; in contrast, there was shift in the buoyant density of the two peaks of 1,3-P-glucan synthetase. We concluded that the subcellular localization of Chsl and Chs2 remains the same despite genetic and other differences in the properties of these enzymes. We confirmed that 1,3-P-glucan synthetase and chitin synthetase exhibit a partially different subcellular distribution -an indication that these two enzymes are mobilized through different secretory pathways.

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