Vlasuk G P, Inouye S, Ito H, Itakura K, Inouye M
J Biol Chem. 1983 Jun 10;258(11):7141-8.
We have examined the importance of the positively charged NH2 terminus of the major outer membrane lipoprotein precursor, prolipoprotein, in the early steps of secretion in Escherichia coli. For this purpose, we have generated three mutants using oligonucleotide-directed mutagenesis in which the charge at the NH2-terminal region was changed from +2 to +1, 0, and -2. The results indicate that the synthesis of prolipoprotein is facilitated by the presence of a positively charged NH2 terminus. In addition, the translocation of prolipoprotein across the cytoplasmic membrane does not absolutely require any basic amino acids at its NH2 terminus. However, the presence of a net negatively charged NH2 terminus causes an initial cytoplasmic accumulation of prolipoprotein which is slowly, post-translationally translocated across the cytoplasmic membrane at a rate which is dependent on the number of positive charges present in this region. The analysis of these mutants clearly demonstrates the importance of the NH2 terminus of the lipoprotein signal peptide in initiating the secretion of this protein in E. coli.
我们研究了主要外膜脂蛋白前体——前脂蛋白带正电荷的NH2末端在大肠杆菌分泌早期步骤中的重要性。为此，我们利用寡核苷酸定向诱变产生了三个突变体，其中NH2末端区域的电荷从+2变为+1、0和-2。结果表明，带正电荷的NH2末端有助于前脂蛋白的合成。此外，前脂蛋白跨细胞质膜的转运在其NH2末端并不绝对需要任何碱性氨基酸。然而，净带负电荷的NH2末端的存在会导致前脂蛋白最初在细胞质中积累，该积累的前脂蛋白在翻译后会以依赖于该区域正电荷数量的速率缓慢地跨细胞质膜转运。对这些突变体的分析清楚地证明了脂蛋白信号肽的NH2末端在启动大肠杆菌中该蛋白分泌方面的重要性。
