Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells.
We have isolated a full-length cDNA clone of the Chinese hamster ovary (CHO) pssC gene, which encodes mitochondrial phosphatidylserine decarboxylase. The cDNA clone is capable of increasing phosphatidylserine decarboxylase activity to 11-fold in CHO-K1 cells. The pssC gene product predicted from the cDNA sequence is composed of 409 amino acid residues. In an in vitro translation system coupled with in vitro transcription, the cDNA clone directs the formation of a protein with an apparent molecular mass of 46 kDa. In CHO-K1 cells, the cDNA clone leads to the production of two major peptides with apparent molecular masses of 38 and 34 kDa, as determined by Western blotting with an antibody raised against a recombinant pssC protein. When CHO-K1 cells transfected with the cDNA clone are labelled with [35S]methionine for a short period, proteins immunoprecipitated with the antibody lack radioactive 38 and 34 kDa peptides, but contain two radioactive peptides with apparent molecular masses of 46 and 42 kDa instead. The pssC gene product predicted from the cDNA sequence has, near its C-terminus, a unique Leu-Gly-Ser-Thr sequence which is known as a processing site for Escherichia coli phosphatidylserine decarboxylase. A mutant pssC cDNA clone, in which Ser378 in the conserved sequence is replaced by Ala, leads to overproduction of 46, 42 and 38 kDa peptides, but not a 34 kDa peptide. This mutant clone is incapable of increasing phosphatidylserine decarboxylase activity, in contrast to the wild-type clone. These results indicate that the processing at the Leu-Gly-Ser-Thr sequence is essential for formation of the active enzyme. Thus, the pssC gene product is converted into mature phosphatidylserine decarboxylase through multiple steps of post-translational processing.
Kuge
O
O
Department of Biochemistry and Cell Biology, National Institute of Health, Tokyo, Japan.
Saito
K
K
Kojima
M
M
Akamatsu
Y
Y
Nishijima
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D005260:Female; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000737:chemistry; D013347:Subcellular Fractions / Q000201:enzymology
D018076:DNA, Complementary; D012313:RNA; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase