MOLECULAR ANALYSIS OF CHS3P PARTICIPATION IN CHITIN SYNTHASE-III ACTIVITY

Chitin is a minor but essential component of the cell wall of Saccharo myces cerevisiae, with functions in septum formation in the vegetative life cycle and also in conjugation and spore cell-wall synthesis in t he sexual cycle. Of the three chitin synthases present in yeast, chiti n synthase III (CSIII) is responsible for the synthesis of most of the chitin found in the cell, including a chitin ring at early budding, c hitin interspersed in the cell wall, and chitin laid down during the s exual cycle. We have tagged Chs3p, the putative catalytic subunit of C SIII, with the immunoreactive epitope of influenza virus hemagglutinin to follow expression of the protein. Little correlation was found bet ween the levels of transcription and translation of Chs3p and in vivo function, supporting our previous conclusion that regulation of CSIII occurs at the posttranslational level. To identify possible regions of the protein involved in catalysis or regulation, mutations were gener ated in the QRRRW 'signature sequence' of chitin synthases. Arginine r esidue mutations in Chs3p, and in Chs1p and Chs2p, resulted in a loss of both function in vivo and enzymatic activity. Mutations in a serine residue adjacent to glutamine in Chs3p caused loss of function in viv o with a moderate decrease in CSIII activity, suggesting a regulatory role for the serine residue in chitin biosynthesis. Several truncation s in the unique hydrophilic carboxy-terminal region of Chs3p identifie d a sequence of about 25 amino acids that is required for both functio n and in vitro activity. Since this region is not present in Chs1 or C hs2,, it may be involved in the specific regulation of CSIII.