Expression in yeast of an Escherichia coli gene encoding a phospholipid biosynthetic enzyme.
Cardiolipin (CL) is a structurally unique phospholipid having important functional roles in both prokaryotic and eukaryotic cells. The genes encoding CL biosynthetic enzymes have been identified and extensively studied in Escherichia coli, and manipulation of CL biosynthesis in this organism has elucidated a great deal about CL function in prokaryotes. In contrast, little is known about CL biosynthesis or its regulation in eukaryotic cells. We sought to determine whether we could utilize E. coli genes to manipulate expression of CL biosynthetic enzymes and CL content in yeast. The E. coli pgsA gene encodes phosphatidylglycerophosphate synthase (PGPS), catalyzing the first step in the CL biosynthetic pathway. We constructed plasmids with pgsA under the control of the yeast CUP1 promoter. Extracts of Saccharomyces cerevisiae cells transformed with this plasmid contained high levels of E. coli PGPS activity. However, when compared to cells transformed with a control plasmid, pgsA-transformed cells did not exhibit differences in phospholipid composition. The most likely explanation is that the in vitro activity of the E. coli pgsA product is not indicative of its activity in vivo, due to mislocalization of the enzyme and/or inaccessibility of the enzyme to the substrates. To our knowledge, this is the first demonstration of expression of a bacterial phospholipid biosynthetic enzyme in yeast.
Kelly
B L
BL
Cellular and Molecular Biology Program, University of Michigan, Ann Arbor 48109-0606.
Greenberg
M L
ML
D001426:Bacterial Proteins / Q000235:genetics*; D002308:Cardiolipins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015966:Gene Expression Regulation, Fungal; D005798:Genes, Bacterial; D005818:Genetic Engineering; D008565:Membrane Proteins; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics*
D001426:Bacterial Proteins; D002308:Cardiolipins; D008565:Membrane Proteins; D012333:RNA, Messenger; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase