The PROJ gene of Saccharomyces cerevisiae encodes the 428-amino-acid protein y-glutamyl kinase (ATP:L-glutamate 5-phosphotransferase, EC 2.7.2.11), which catalyzes the first step in proline biosynthesis. Amino acid sequence comparison revealed significant homology between the yeast and Escherichia coli -y-glutamyl kinases throughout their lengths. Four close matches to the consensus sequence for GCN4 protein binding and one close match to the RAP1 protein-binding site were found in the PROJ upstream region. The response of the PROJ gene to changes in the growth medium was analyzed by measurement of steady-state mRNA levels and of 13-galactosidase activity encoded by a PROJ-lacZ gene fusion. PROJ expression was not repressed by exogenous proline and was not induced by the presence of glutamate in the growth medium. Although expression of the PROJ gene did not change in response to histidine starvation, both steady-state PROJ mRNA levels and 13-galactosidase activities were elevated in a gcdJ strain and reduced in a gcn4 strain.In addition, a pro) bradytrophic strain became completely auxotrophic for proline in a gcn4 strain background. These results indicate that PROJ is regulated by the general amino acid control system.The yeast Saccharomyces cerevisiae synthesizes proline from glutamate via the intermediates y-glutamyl phosphate, -y-glutamyl semialdehyde, and A1-pyrroline-5-carboxylic acid (P5C) (12,13,49,59). The same pathway is found in members of the family Enterobactenaceae (for a review, see reference 38) and mammals (56, 60). The three enzymes involved in this pathway are -y-glutamyl kinase (the product of the PROI gene), -y-glutamyl phosphate reductase (the product of the PRO2 gene), and P5C reductase (the product of the PRO3 gene). The gene-enzyme relationships were demonstrated by interspecies complementation of proB, proA, and proC mutants of Escherichia coli and Salmonella typhimurium by the yeast PRO], PRO2, and PRO3 genes, respectively (59), and of a yeast prol mutant by the E. coli proB gene (49). There is substantial deduced amino acid sequence similarity between the E. coli (23) and S. cerevisiae (this work) -y-glutamyl kinases, between the E. coli (23) and S. cerevisiae (10) -y-glutamyl phosphate reductases, and among the P5C reductases of bacteria (24, 30, 52), S. cerevisiae (13), plants (22), and mammals (27).Proline-requiring S. cerevisiae strains were isolated long after auxotrophs for almost all of the other amino acids had been obtained (12). This was because of their peculiar property of being unable to grow in the standard rich medium in common use in yeast laboratories. Repression and inactivation of both the general amino acid and the proline-specific permeases in media rich in nitrogen sources (20) is believed to be responsible for this inability of proline auxotrophs to grow.