Cloning of a gene (PSD1) encoding phosphatidylserine decarboxylase from Saccharomyces cerevisiae by complementation of an Escherichia coli mutant.
A gene (PSD1) encoding a phosphatidylserine decarboxylase of Saccharomyces cerevisiae was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Expression of the cDNA clone in EH150 corrected growth, phospholipid, and phosphatidylserine decarboxylase activity defects. Expression of the genomic clone in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. A 1500-base pair open reading frame encodes a 56,558-Da protein with a potential mitochondrial targeting sequence. Upstream regulatory elements found in other enzymes of the phospholipid biosynthetic pathway are present in PSD1. The derived amino acid sequence shows 44 and 35% identity with the phosphatidylserine decarboxylases from Chinese hamster ovary cells and E. coli, respectively. Near the carboxyl terminus is an LGST sequence which, in E. coli, is the site of proteolytic cleavage of the proenzyme into the alpha and beta subunits and formation of the pyruvate prosthetic group (Dowhan, W., and Li, Q.-X. (1992) Methods Enzymol. 209, 348-359). Disruption of the PSD1 gene in a haploid strain of yeast resulted in loss of detectable decarboxylase activity but little alteration of the growth properties or phospholipid composition. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.
Clancey
C J
CJ
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Chang
S C
SC
Dowhan
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010743:Phospholipids / Q000378:metabolism; D011003:Ploidies; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D024101:Mitochondrial Proteins; D010743:Phospholipids; D002262:Carboxy-Lyases; C577156:Psd1 protein, S cerevisiae; C022353:phosphatidylserine decarboxylase