Iino T, Takahashi M, Sako T
J Biol Chem. 1987 May 25;262(15):7412-7.
Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis. When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative. A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing. Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy. Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region. From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process.
通过体外寡核苷酸定向诱变产生了在信号肽氨基末端带电荷区段内具有氨基酸取代的葡萄球菌激酶突变体。当在大肠杆菌细胞中分析基因产物的加工过程时，随着净电荷变得更负，突变型葡萄球菌激酶前体的加工速率降低。氨基末端区段需要净正电荷而非特定氨基酸残基以实现高效加工。具有净负电荷的葡萄球菌激酶前体在细胞质中积累，通过亚细胞分级分离和免疫电子显微镜确定其倾向于结合到细胞质膜上。尽管在疏水区段具有氨基酸取代的突变体和野生型葡萄球菌激酶对其他正常分泌蛋白的加工有干扰作用，但当它们在氨基末端区域也含有改变电荷的取代时，这种作用就消失了。从这些结果我们得出结论，葡萄球菌激酶信号肽氨基末端区段上的正电荷是进入蛋白质输出过程所必需的。
