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 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.
Highlights
From the $Laboratoryof Genetics, Howard Hughes Medical Institute, Johns Hopkins University Schoolof Medicine, Baltimore, Maryland 21205 and the TDepartment of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NewJersey 07103
The cDNA hybridizes to a 1.85-kilobase mRNA in samples enzyme from cultured human fibroblasts and LHN cells is from human cell lines and predicts a 319-amino acid, inhibited by proline but not by NADP+; theconverse is true
Phenotypic complementation occurred despite the relatively low level of P5C reductase activity measured in the transformants (Table11).Either wild type S. cereuisiae have far more P5C reductase activity than required for growth on a medium lacking proline or the enzymatic activity measured i n vitro does not accurately reflect that in uiuo
Summary
P5C, pyrroline 5-carboxylate; bp, base pair(s); LHN, lymphoblastoid, SDS, sodium dodecyl sulfate; elected to utilize a functional cloning strategy, namely complementationin a Saccharomyces cereuisiae mutantstrain lacking the reductase. Mutant stroafinSs. cereukiue lacking P5C reductase activityhave been well characterized [12] and are auxotrophic for proline,providing a selection system for complementation. The human homolog for the cellcycle control gene (cdc2) [14], a CCAATbinding transcription factor [15], and three multifunctional genes involved in de mvo purine synthesis [13,16]have been cloned. We describethe.isolation and characterization of human P5C reductase cDNA by complementation in S. cereuisiae and mapping of the human structuralgene
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