Bosch D, Voorhout W, Tommassen J
Department of Molecular Cell Biology, State University Utrecht, The Netherlands.
J Biol Chem. 1988 Jul 15;263(20):9952-7.
To identify export and sorting information in outer membrane protein PhoE of Escherichia coli K-12, a set of deletions was created, resulting in the removal of N-terminal amino acids of the mature protein. Pulse-chase experiments revealed that some mutant proteins were slowly or not at all processed, but there was not correlation between processing rate and the extent of the deletions. The unprocessed precursors were accessible to trypsin in the periplasm showing that processing by leader peptidase rather than translocation is affected by these deletions. The results show that no specific sequences in the N-terminal part of the mature PhoE protein are required for translocation through the inner membrane. The capability of the processed mutant proteins to assemble into the outer membrane was correlated to the exten of the deletions. Thus, mutants which lack up to amino acid residue 14 are normally incorporated into the outer membrane. Larger deletions which removed the first postulated membrane-spanning fragment of the protein affected the efficiency of assembly: in addition to trimers of the protein in the outer membrane, also monomers were detected in the periplasm. If the deletions extended C-terminally to residue 48, only monomeric forms of the proteins were found in the periplasm.
为了鉴定大肠杆菌K-12外膜蛋白PhoE中的输出和分选信息，构建了一系列缺失突变体，导致成熟蛋白的N端氨基酸被去除。脉冲追踪实验表明，一些突变蛋白加工缓慢或根本未被加工，但加工速率与缺失程度之间没有相关性。周质中的胰蛋白酶可作用于未加工的前体，这表明这些缺失影响的是前导肽酶的加工而非转运。结果表明，成熟PhoE蛋白的N端部分不存在特定序列也可通过内膜进行转运。加工后的突变蛋白组装到外膜的能力与缺失程度相关。因此，缺失至氨基酸残基14的突变体通常可整合到外膜中。更大的缺失去除了该蛋白第一个假定的跨膜片段，影响了组装效率：除了外膜中的蛋白三聚体，周质中还检测到了单体。如果缺失延伸至C端的残基48，则在周质中仅发现蛋白的单体形式。
