Gene-enzyme relationships in the proline biosynthetic pathway of Saccharomyces cerevisiae.
The PRO1, PRO2, and PRO3 genes were isolated by functional complementation of pro1, pro2, and pro3 (proline-requiring) strains of Saccharomyces cerevisiae. Independent clones with overlapping inserts were isolated from S. cerevisiae genomic libraries in YEp24 (2 microns) and YCp50 (CEN) plasmids. The identity of each gene was determined by gene disruption, and Southern hybridization and genetic analyses confirmed that the bona fide genes had been cloned. Plasmids containing each gene were introduced into known bacterial proline auxotrophs, and the ability to restore proline prototrophy was assessed. Interspecies complementation demonstrated that the S. cerevisiae PRO1 gene encoded gamma-glutamyl kinase, PRO2 encoded gamma-glutamyl phosphate reductase, and PRO3 encoded delta 1-pyrroline-5-carboxylate reductase. The presence of the PRO3 gene on a high-copy-number plasmid in S. cerevisiae caused a 20-fold overproduction of delta 1-pyrroline-5-carboxylate reductase. The PRO2 gene mapped on chromosome XV tightly linked to cdc66, and the PRO3 gene was located on the right arm of chromosome V between HIS1 and the centromere.
Tomenchok
D M
DM
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103.
Brandriss
M C
MC
D000445:Aldehyde Oxidoreductases / Q000235:genetics; D055598:Chemical Phenomena; D002621:Chemistry; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010770:Phosphotransferases / Q000235:genetics; D017851:Phosphotransferases (Carboxyl Group Acceptor); D010957:Plasmids; D011392:Proline / Q000096:biosynthesis* / Q000235:genetics; D011762:Pyrroline Carboxylate Reductases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012486:Salmonella typhimurium / Q000235:genetics; D014170:Transformation, Genetic; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase
D000445:Aldehyde Oxidoreductases; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D010770:Phosphotransferases; D017851:Phosphotransferases (Carboxyl Group Acceptor); D011392:Proline; D011762:Pyrroline Carboxylate Reductases; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase; C020650:glutamate 5-kinase