Chitinase is required for cell separation during growth of Saccharomyces cerevisiae.

M.J Kuranda,P.W Robbins

doi:10.1016/s0021-9258(18)55057-2

Abstract

The Saccharomyces cerevisiae chitinase described by Correa et al. (Correa, J. U., Elango, N., Polacheck, I., and Cabib, E. (1982) J. Biol. Chem. 257, 1392-1397) has been cloned and sequenced. Analysis of the derived amino acid sequence suggests that the protein contains four domains: a signal sequence, a catalytic domain, a serine/threonine-rich region, and a carboxyl-terminal domain with high binding affinity for chitin. Most of the enzyme produced by cells is secreted into the growth medium and is extensively glycosylated with a series of short O-linked mannose oligosaccharides ranging in size from Man2 to Man5. Chitinase O-mannosylation was further examined in the temperature-sensitive secretion mutants sec18, sec7, and sec6. Oligosaccharides isolated from chitinase accumulating in cells at the nonpermissive temperature revealed Man1 and Man2 associated with the sec18 mutant. sec6 and sec7 accumulated Man2-Man5 with a higher proportion of Man5 relative to the secreted protein. A significant amount of chitinase is also found associated with the cell wall through binding of COOH-terminal domain to chitin. Disruption of the gene for the enzyme leads to a defect in cell separation but does not substantially alter the level of cellular chitin.

Highlights

From the Center for Cancer Research and the Departmentof Biology, Massachusetts Institute of Techrwbgy, Cambridge, Massachusetts 02139 al
Five clones containing insertswere transformed into MKY1315.Four contained invertase activity and produced a protein product which reacted with both antiinvertase and antichitinase antibodies on Western blots. One of these was chosen for further analysis and designated reagents used in polymerase chain reactions were included in the pCT33
Chitin binding domain (Fig. 4).The linearized plasmid was used as a Isolation of CTSl-&-The CTSl-disrupted strain MKY1315 was template for in uitro amplification by the polymerase chain reaction transformed with a YCp50 yeast genomic library prepared from strain which resulted in replication of vector sequences as well as deletion DBY918 (11)

Summary

MATERIALS AND METHODS

Purification of chitinase on a smasllcale for rapid analysis by SDS precise segment of the yeast invertase coding sequences with NotI gels was accomplished by placing 15 ml of YPD medium containing and XhoI cohesive ends was produced by in vitro DNA amplification. Five clones containing insertswere transformed into MKY1315.Four contained invertase activity and produced a protein product which reacted with both antiinvertase and antichitinase antibodies on Western blots One of these was chosen for further analysis and designated reagents used in polymerase chain reactions were included in the pCT33. Chitin binding domain (Fig. 4).The linearized plasmid was used as a Isolation of CTSl-&-The CTSl-disrupted strain MKY1315 was template for in uitro amplification by the polymerase chain reaction transformed with a YCp50 yeast genomic library prepared from strain which resulted in replication of vector sequences as well as deletion DBY918 (11). Transformation of E. coli and restriction analysis of plasmids obtained from transformants indi-

RESULTS

A TOTTKTLTSSTKTKSSLGTTTTESTLNSVAITSMKTTLSS

Findings

DISCUSSION