Proline biosynthesis in Saccharomyces cerevisiae: molecular analysis of the PRO1 gene, which encodes gamma-glutamyl kinase

Abstract

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.

Transcriptional Profiling Shows that Gcn4p Is a Master Regulator of Gene Expression during Amino Acid Starvation in Yeast

“…Surprisingly, PRO1 was repressed by 3AT (Fig. 6A, Pro), consistent with a previous report that PRO1 is not induced by histidine starvation (59). As expected (36), we found that nine of the arginine biosynthetic genes (ARG1, -2, -3, -4, -5, 6, -7, and -8; CPA1; and CPA2) are Gcn4p targets and that most were strongly induced by 3AT (Fig.…”

l -Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding γ-Glutamyl Kinase

“…In plants, the first steps of L-proline biosynthesis from L-glutamate are catalyzed by P5C synthetase (an EC number has not been not assigned), a bifunctional enzyme with ␥-GK and ␥-GPR activities (15,44); the ␥-GK activity of the P5C synthetase was also found to be sensitive to L-proline. In contrast, the PRO1 gene of S. cerevisiae was regulated by the general amino acid control system, which increased the expression of many amino acid biosynthetic genes in cells subjected to amino acid starvation conditions (20). However, feedback inhibition of ␥-GK activity remains unknown.…”

“…A fragment of the PRO1 gene including the probable TATA box sequences (20) and the tripartite terminator (20) was prepared by PCR performed with genomic DNA from strains MB329-17C and FH515 and oligonucleotide primers 5Ј-ACC CAA GCT TTG GTC AGT GGC ACA G-3Ј and 5Ј-ACC CGA GCT CGA AGG ATT TTA ACG GAT CAC-3Ј (the underlined sequences are the positions of a HindIII site and a SacI site, respectively). The unique 1.8-kb band amplified from genomic DNA of MB329-17C and FH515 was digested with HindIII and SacI and then ligated to the large fragment of pRS415 digested with HindIII and SacI to construct pRS-WTPRO1 and pRS-D154NPRO1, respectively.…”

Isolation, characterization, and chromosomal location of a gene encoding the Δ1‐pyrroline‐5‐carboxylate synthetase in Arabidopsis thaliana

“…Interestingly in yeast, ? '-glutamyl kinase [31] and ~'-glutamyl phosphate reductase ( [32]; M.C. Brandriss, personal communication) functions are carried by two different proteins.…”