Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae

K. M. Dougherty, M. C. Brandriss, D. Valle

Research output: Contribution to journalArticlepeer-review

Abstract

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 105 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.

Original languageEnglish (US)
Pages (from-to)871-875
Number of pages5
JournalJournal of Biological Chemistry
Volume267
Issue number2
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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