Pi J, Dogovski C, Pittard A J
Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3052, Australia.
J Bacteriol. 1998 Nov;180(21):5515-9. doi: 10.1128/JB.180.21.5515-5519.1998.
The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650-2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [35S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.
PheP蛋白是大肠杆菌的一种高亲和力苯丙氨酸特异性通透酶。此前曾提出一种基于遗传分析的拓扑模型，该分析涉及构建与碱性磷酸酶的蛋白融合体，其中PheP有12个跨膜区段，N端和C端均位于细胞质中（J. Pi和A. J. Pittard，《细菌学杂志》178:2650 - 2655，1996）。定点诱变已被用于研究PheP蛋白16个脯氨酸残基中每个残基的功能重要性。仅在三个位置P54、P341和P442处将丙氨酸替换，导致活性丧失50%或更多。P341处的替换产生的影响最为显著。然而，这些转运活性的变化均与突变蛋白插入膜的任何缺陷无关，如[35S]甲硫氨酸标记和使用抗PheP血清的免疫沉淀所示。讨论了这三个脯氨酸各自可能的作用。在IX跨段及其紧邻的环内的不同位点插入单个丙氨酸残基也对转运活性有重大影响，表明该蛋白这一区域高度有序的结构起重要作用。
