Chen H, Kim J, Kendall D A
Department of Molecular and Cell Biology, The University of Connecticut, Storrs 06269, USA.
J Bacteriol. 1996 Dec;178(23):6658-64. doi: 10.1128/jb.178.23.6658-6664.1996.
We have developed a system for examining the relative affinity of two different signal peptides for the protein secretion pathway in Escherichia coli. This system involves the expression of a modified alkaline phosphatase which possesses two signal peptides arranged in tandem. When both signal peptides have the wild-type sequence, cleavage after the first and cleavage after the second occur with nearly equal frequency. In both cases the remainder of the protein is transported to the periplasm. Thus both signal peptides effectively compete with each other for entrance to the secretion pathway. When the hydrophobicity of the second signal peptide is altered by small increments, we find that the more hydrophobic signal peptide is preferentially utilized. Thus, a more hydrophobic signal peptide can outcompete even an efficient wild-type signal sequence. The crossover point, for utilization of the second to the first signal peptide, is marked and occurs over a very small change in hydrophobicity. Our results suggest that the small differences in the hydrophobicity of wild-type signal peptides may have critical consequences: preproteins with the more hydrophobic signals could dominate one pathway, leaving those with only slightly less hydrophobic signals to require additional factors such as chaperonins, SecB, and other binding proteins.
我们开发了一种系统，用于检测两种不同信号肽对大肠杆菌蛋白质分泌途径的相对亲和力。该系统涉及表达一种修饰的碱性磷酸酶，其具有两个串联排列的信号肽。当两个信号肽都具有野生型序列时，第一个信号肽之后和第二个信号肽之后的切割频率几乎相等。在这两种情况下，蛋白质的其余部分都被转运到周质中。因此，两个信号肽有效地相互竞争进入分泌途径。当第二个信号肽的疏水性以小幅度增加而改变时，我们发现疏水性更强的信号肽被优先利用。因此，疏水性更强的信号肽甚至可以胜过高效的野生型信号序列。第二个信号肽相对于第一个信号肽的利用交叉点很明显，并且发生在疏水性的非常小的变化范围内。我们的结果表明，野生型信号肽疏水性的微小差异可能产生关键影响：具有更强疏水性信号的前体蛋白可能主导一条途径，而那些疏水性略弱的前体蛋白则需要额外的因子，如伴侣蛋白、SecB和其他结合蛋白。
