Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae.
Pyrroline-5-carboxylate reductase (EC 1.5.1.2) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline. We cloned a human pyrroline-5-carboxylate reductase cDNA by complementation of proline auxotrophy in a Saccharomyces cerevisiae mutant strain, DT1100. Using a HepG2 cDNA library in a yeast expression vector, we screened 10(5) transformants, two of which gained proline prototrophy. The plasmids in both contained similar 1.8-kilobase inserts, which when reintroduced into strain DT1100, conferred proline prototrophy. The pyrroline-5-carboxylate reductase activity in these prototrophs was 1-3% that of wild type yeast, in contrast to the activity in strain DT1100 which was undetectable. The 1810-base pair pyrroline-5-carboxylate reductase cDNA hybridizes to a 1.85-kilobase mRNA in samples from human cell lines and predicts a 319-amino acid, 33.4-kDa protein. The derived amino acid sequence is 32% identical with that of S. cerevisiae. By genomic DNA hybridization analysis, the human reductase appears to be encoded by a single copy gene which maps to chromosome 17.
Dougherty
K M
KM
Laboratory of Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Brandriss
M C
MC
Valle
D
D
D000595:Amino Acid Sequence; D001345:Autoradiography; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011762:Pyrroline Carboxylate Reductases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D012333:RNA, Messenger; D004247:DNA; D011762:Pyrroline Carboxylate Reductases