Fikes J D, Bassford P J
J Bacteriol. 1987 Jun;169(6):2352-9. doi: 10.1128/jb.169.6.2352-2359.1987.
The Escherichia coli maltose-binding protein (MBP) R2 signal peptide is a truncated version of the wild-type structure that still facilitates very efficient export of MBP to the periplasm. Among single amino acid substitutions in the R2 signal peptide resulting in an export-defective precursor MBP (pMBP) were two that replaced residues in the consensus Ala-X-Ala sequence (residues -3 to -1) that immediately precedes the cleavage site. It was suggested that the functional hydrophobic core and signal peptidase recognition sequence of this signal peptide substantially overlap and that these two alterations affect both pMBP translocation and processing. In this study, the export of pMBP by the mutants, designated CC15 and CC17, with these two alterations was investigated further. The pMBP of mutant CC17 has an Arg substituted for Leu at the -2 position. It was found that CC17 cells exported only a very small amount of MBP, but that which was exported appeared to be correctly processed. This result was consistent with other studies that have concluded that virtually any amino acid can occupy the -2 position. For mutant CC15, which exhibits a fully Mal+ phenotype, an Asp is substituted for the Ala at the -3 position. CC15 cells were found to export large quantities of unprocessed, soluble pMBP to the periplasm, although such export was achieved in a relatively slow, posttranslational manner. This result was also consistent with other studies that suggested that charged residues are normally excluded from the -3 position of the cleavage site. Using in vitro oligonucleotide-directed mutagenesis, we constructed a new signal sequence mutant in which Asp was substituted for Arg at the -3 position of an otherwise wild-type MBP signal peptide. This alteration had no apparent effect on pMBP translocation across the cytoplasmic membrane, but processing by signal peptidase was inhibited. This pMBP species with its full-length hydrophobic core remained anchored to the membrane, where it could still participate in maltose uptake. The implications of these results for models of protein export are discussed.
大肠杆菌麦芽糖结合蛋白（MBP）的R2信号肽是野生型结构的截短版本，它仍然能够非常有效地将MBP输出到周质空间。在导致输出缺陷型前体MBP（pMBP）的R2信号肽的单氨基酸替换中，有两个替换发生在紧接切割位点之前的共有Ala-X-Ala序列（-3至-1位残基）中的残基上。有人提出，该信号肽的功能性疏水核心和信号肽酶识别序列基本重叠，并且这两个改变会影响pMBP的转运和加工。在本研究中，进一步研究了具有这两个改变的突变体（命名为CC15和CC17）对pMBP的输出情况。突变体CC17的pMBP在-2位的Leu被Arg取代。发现CC17细胞仅输出极少量的MBP，但输出的MBP似乎被正确加工。这一结果与其他研究一致，这些研究得出结论，实际上任何氨基酸都可以占据-2位。对于表现出完全Mal+表型的突变体CC15，其-3位的Ala被Asp取代。发现CC15细胞将大量未加工的可溶性pMBP输出到周质空间，尽管这种输出是以相对缓慢的翻译后方式实现的。这一结果也与其他研究一致，这些研究表明带电荷的残基通常被排除在切割位点的-3位之外。利用体外寡核苷酸定向诱变，我们构建了一个新的信号序列突变体，其中在野生型MBP信号肽的-3位将Arg替换为Asp。这种改变对pMBP跨细胞质膜的转运没有明显影响，但信号肽酶的加工受到抑制。这种具有全长疏水核心的pMBP物种仍锚定在膜上，在那里它仍然可以参与麦芽糖的摄取。讨论了这些结果对蛋白质输出模型的意义。
