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Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells.

ABSTRACT: 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.

SUBMITTER: Kuge O

PROVIDER: S-EPMC1217732 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Project description:Externalization of PtdSer (phosphatidylserine) is an important event in signalling removal of apoptotic cells. In contrast with previous work [Yu, Byers, Ridgway, McMaster and Cook (2000) Biochim. Biophys. Acta 1487, 296-308] with U937 cells showing that specific stimulation of PtdSer biosynthesis during apoptosis was caspase dependent, PtdSer biosynthesis in CHO (Chinese-hamster ovary)-K1 increased 2.5-fold during UV-induced apoptosis but was not reversed by a caspase inhibitor, Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone). Also, in CHO-K1 cells, stimulation of synthesis was less specific for PtdSer as similar levels of stimulation were observed for sphingomyelin biosynthesis. Involvement of PtdSer synthase isoforms was tested in CHO-K1 cells overexpressing PSS I (PtdSer synthase I) and PSS II. Both types of transformed cells showed resistance to UV-induced apoptosis based on the decreased levels of caspase 3 activation and morphology changes; externalization of PtdSer was reduced with UV treatment even though expression of endogenous scramblase increased slightly. Serine-labelling experiments showed that PSS I- or PSS II-expressing cells had higher basal levels of PtdSer biosynthesis compared with vector control cells. When cells were exposed to UV light to induce apoptosis, PtdSer biosynthesis was further stimulated 1.5- and 2-fold in PSS I- and PSS II-expressing cells respectively compared with UV-treated vector cells. Caspase activation was not required, as Z-VAD-FMK did not change PtdSer synthesis. Although enhanced PtdSer synthesis was supposed to facilitate apoptosis, cells overexpressing PSS I and II were actually resistant to UV-induced apoptosis. Whereas enhanced PtdSer synthesis was associated with apoptosis, potential anti-apoptotic effects were observed when excess activity of these synthetic enzymes was present. This suggests a tightly regulated role for PtdSer synthesis and/or an important dependence on compartmentation of PSS enzymes in association with scramblase facilitated enrichment of this phospholipid at the cell surface.

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Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells.

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