Abstract
Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.
Highlights
I1 is measured in wild type strains without preincuba- chitinsynthase(chitinsynthase I) is nonessential: cells in tion with trypsin, the condition under which highest which CHSl has been disrupted with the URA3 gene bud and contain normal levels of chitin from the chsl ::URA3 strain
Chitin synthaseI is not required for a-mating pheromone-induced chitin synthesis iMn ATa cells, yet trypsin.This activity-chitinsynthase 11-can be distinguished clearly in uitro from trypsin-activated chitin synthase I in the wild type strain
Since chitinsynthase I1 chitin synthaseI1 is responsible for chitin synthesisin vivo, whereas nonessential chitin synthase I, detectawas the major, if, chitin syntheticactivity measurable in vitro without prior treatment of extracts with trypsin, it is ble in vitroonly after trypsin treatmentm, ay not nor- proposed that chitinsynthase I1 is responsible for chitin mally be active in vivo
Summary
Disruption of the yeast CHSl gene, which encodes chitin synthesis, Polyoxin D, the primary septum is not trypsin-activable chitin synthasIe, yielded strains that formed, and lysis can occur at the junction between mother apparently lacked chitin synthase activtyin vitro, yet and daughtercells [2]. Identification of Two Chitin Synthases in the Wild Type C H S l Strain-As expected, pretreatment of the particulate fraction from the CHSl culture with trypsin caused a 3-fold increase in a chitin synthaseactivity that was further stimulated when digitonin (0.2% w/v) was present during assay (Fig. 1B). It is this activity-chitin synthase I-that is encoded by the C H S l gene; it is absent from chsl ::URA3. Chitin synthases I and I1 are V,, of 0.55 nmol of GlcNAc incorporated/min/mg of protein, clearly distinct in their responses to trypsin treatment, chiwtihnereas thaotf chitin synthaseI,measured in trypsin-treated, CSII from chsl: :URA3
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