POLYMORPHISM OF THE YEAST PYRUVATE-CARBOXYLASE-2 GENE AND PROTEIN - EFFECTS ON PROTEIN BIOTINYLATION

In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carbo xylase (Pyc) encoded by separate genes designated PYC1 and PYC2. We re port the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S. cerevisiae. Comparison with t he previously reported sequence [Stucka, Dequin, Salmon and Gancedo (1 991) Mol. Gen. Genet. 229, 307-315] revealed significant differences w ithin the open reading frame. The most notable difference was near the 3' end, where we found a single base deletion reducing the open readi ng frame by 15 bases. We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-aci d biotin-domain peptide. In addition, we investigated the effects of t he two changes in the Pyc2 biotin domain (K1155R substitution and Q117 8P/five-amino-acid extension) on the extent of biotinylation in vivo b y Escherichia coli biotin ligase, and compared the biotinylation of pe ptides containing these changes with that of two different-length Pyc1 biotin domain peptides. The K1155R substitution had very little effec t on biotinylation, but the five-amino-acid C-terminal extension to Py c2 and the N-terminal extension to Pyc1 both improved biotinylation in vivo.