Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells
Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells
复制标题
DOI：
10.1042/bj3190033
复制
发表时间：
1996-10-01
影响因子：
4.1
通讯作者：
Nishijima, M
中科院分区：
文献类型：
--
作者：
Kuge, O;Saito, K;Nishijima, M
我们分离到了一个编码线粒体磷脂酰丝氨酸脱羧酶的中国仓鼠卵巢（CHO） pssC基因的全长cDNA克隆。cDNA克隆能够将CHO-KI细胞的磷脂酰丝氨酸脱羧酶活性提高到2倍。cDNA序列预测的pssC基因产物由409个氨基酸残基组成。在体外翻译与体外转录耦合的系统中，cDNA克隆指导表观分子质量为46 kDa的蛋白质的形成。在CHO-K1细胞中，cDNA克隆导致两种主要肽的产生，其明显分子质量为38和34 kDa，通过Western blotting检测针对重组pssC蛋白的抗体。当转染cDNA克隆的CHO-K1细胞用[S-35]蛋氨酸短时间标记时，用抗体免疫沉淀的蛋白缺乏放射性38和34 kDa肽，而含有两种明显分子质量为46和42 kDa的放射性肽。cDNA序列预测的pssC基因产物在其c端附近有一个独特的Leu-Gly-Ser-Thr序列，该序列被称为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，其中保守序列的Ser（378）被Ala取代，导致过量产生46、42和38 kDa肽，但不产生34 kDa肽。与野生型克隆相比，该突变克隆不能增加磷脂酰丝氨酸脱羧酶的活性。这些结果表明，在Leu-Gly-Ser-Thr序列上的加工对活性酶的形成至关重要。因此，pssC基因产物经过多步翻译后加工转化为成熟的磷脂酰丝氨酸脱羧酶。
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 Ii-fold in CHO-KI 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 [S-35]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 Ser(378) 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.