Jain R G, Rusch S L, Kendall D A
Department of Molecular & Cell Biology, University of Connecticut, Storrs 06269.
J Biol Chem. 1994 Jun 10;269(23):16305-10.
As a first step toward understanding the topology of the signal peptide with respect to the membrane during the protein export process, we have examined the constraints on the length of the cleavage region needed to achieve signal peptidase recognition and cleavage. Using the signal peptide of Escherichia coli alkaline phosphatase, a series of cleavage region mutants has been constructed. Variations in length were brought about by replacing the wild type cleavage region of the signal peptide with polymers of increasingly more residues. In each case, alanine residues are used exclusively in the -1 and -3 positions to provide only one viable cleavage site. Glutamine residues are used in all other positions in order to vary the length from 3 to 13 total residues. Analysis of these mutants revealed that cleavage regions ranging from 3 to 9 residues are completely and efficiently processed. The extent of processing drops substantially thereafter, with no processing observed for signal peptides with 13-residue long cleavage regions. A second mutant with a 13-residue long cleavage region was designed and analyzed to ensure that the lack of processing reflected a cleavage problem and not a translocation defect. The results are consistent with the notion that the signal peptidase active site is in close proximity to the periplasmic surface of the inner membrane and that interaction of the cleavage region with the signal peptidase probably depends on, and is constrained by, other interactions involving the signal peptide.
作为理解蛋白质输出过程中信号肽相对于膜的拓扑结构的第一步，我们研究了实现信号肽酶识别和切割所需的切割区域长度的限制因素。利用大肠杆菌碱性磷酸酶的信号肽，构建了一系列切割区域突变体。通过用越来越多残基的聚合物取代信号肽的野生型切割区域来实现长度变化。在每种情况下，仅在 -1 和 -3 位置使用丙氨酸残基以提供唯一可行的切割位点。在所有其他位置使用谷氨酰胺残基，以使总残基长度从 3 变化到 13。对这些突变体的分析表明，3 至 9 个残基的切割区域能够完全且有效地被加工。此后加工程度大幅下降，对于具有 13 个残基长切割区域的信号肽未观察到加工现象。设计并分析了第二个具有 13 个残基长切割区域的突变体，以确保缺乏加工反映的是切割问题而非转运缺陷。结果与以下观点一致：信号肽酶活性位点紧邻内膜的周质表面，并且切割区域与信号肽酶的相互作用可能取决于并受涉及信号肽的其他相互作用的限制。
