YEAST CARBAMOYL-PHOSPHATE-SYNTHETASE-ASPARTATE-TRANSCARBAMYLASE MULTIDOMAIN PROTEIN IS PHOSPHORYLATED INVITRO BY CAMP-DEPENDENT PROTEIN-KINASE

The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl‐phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease‐B‐deficient URA2‐transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP‐dependent protein kinase. After electrophoresis under denaturing conditions, a single 240‐kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg‐Arg‐Phe‐Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase‐like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor. Copyright © 1990, Wiley Blackwell. All rights reserved