Kuge O, Saito K, Kojima M, Akamatsu Y, Nishijima M
Department of Biochemistry and Cell Biology, National Institute of Health, Tokyo, Japan.
Biochem J. 1996 Oct 1;319 ( Pt 1)(Pt 1):33-8. doi: 10.1042/bj3190033.
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.
我们分离出了中国仓鼠卵巢（CHO）pssC基因的全长cDNA克隆，该基因编码线粒体磷脂酰丝氨酸脱羧酶。此cDNA克隆能够使CHO-K1细胞中的磷脂酰丝氨酸脱羧酶活性提高到11倍。根据cDNA序列预测，pssC基因产物由409个氨基酸残基组成。在与体外转录偶联的体外翻译系统中，该cDNA克隆指导形成一种表观分子量为46 kDa的蛋白质。在CHO-K1细胞中，用针对重组pssC蛋白产生的抗体进行蛋白质印迹分析表明，该cDNA克隆导致产生两种主要的肽，表观分子量分别为38 kDa和34 kDa。当用[35S]甲硫氨酸对转染了该cDNA克隆的CHO-K1细胞进行短期标记时，用该抗体免疫沉淀的蛋白质缺乏放射性的38 kDa和34 kDa肽，但含有两种表观分子量分别为46 kDa和42 kDa的放射性肽。根据cDNA序列预测的pssC基因产物在其C末端附近有一个独特的Leu-Gly-Ser-Thr序列，该序列是大肠杆菌磷脂酰丝氨酸脱羧酶的加工位点。一个突变的pssC cDNA克隆，其中保守序列中的Ser378被Ala取代，导致46 kDa、42 kDa和38 kDa肽的过量产生，但不产生34 kDa肽。与野生型克隆相比，该突变克隆不能提高磷脂酰丝氨酸脱羧酶活性。这些结果表明，Leu-Gly-Ser-Thr序列处的加工对于活性酶的形成至关重要。因此，pssC基因产物通过多个翻译后加工步骤转化为成熟的磷脂酰丝氨酸脱羧酶。
