Rusch S L, Chen H, Izard J W, Kendall D A
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269.
J Cell Biochem. 1994 Jun;55(2):209-17. doi: 10.1002/jcb.240550208.
In order to titrate the dependence of individual steps in protein transport on signal peptide hydrophobicity, we have examined a series of mutants which involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide. The core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine. Analysis of this series identified a midpoint for rapid precursor processing between alanine to leucine ratios of 6:4 and 5:5 [Doud et al. (1993): Biochemistry 32:1251-1256]. Examination of precursors that are processed more slowly indicates a lower limit of signal peptide hydrophobicity that permits membrane association and translocation. Analysis of precursors that are processed rapidly defines an intermediate range of hydrophobicity that is optimum; above this level precursors become insensitive to transport inhibitors such as sodium azide and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in parallel with substantial inhibition of beta-lactamase processing. Our data indicate that there is a surprisingly narrow range of signal peptide hydrophobicity which both supports transport of the protein to which it is attached and which does not have such a high affinity for the transport pathway that it disrupts the appropriate balance of other secreted proteins.
为了测定蛋白质转运中各个步骤对信号肽疏水性的依赖性，我们研究了一系列突变体，这些突变体涉及大肠杆菌碱性磷酸酶信号肽疏水核心片段的替换。核心区域中丙氨酸与亮氨酸残基的比例从10:0到0:10不等。因此，用疏水性更强的残基亮氨酸滴定无功能的含多聚丙氨酸信号肽。对该系列的分析确定了丙氨酸与亮氨酸比例为6:4和5:5之间快速前体加工的中点[杜德等人（1993年）：《生物化学》32:1251 - 1256]。对加工较慢的前体的研究表明，允许膜结合和转运的信号肽疏水性下限。对快速加工的前体的分析确定了一个最佳的疏水性中间范围；高于此水平，前体对诸如叠氮化钠和羰基氰3 - 氯苯腙（CCCP）等转运抑制剂变得不敏感，同时β - 内酰胺酶加工受到显著抑制。我们的数据表明，信号肽疏水性的范围惊人地狭窄，它既能支持与其相连蛋白质的转运，又不会对转运途径具有如此高的亲和力以至于破坏其他分泌蛋白的适当平衡。
