Muramoto K, Macnab R M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Mol Microbiol. 1998 Sep;29(5):1191-202. doi: 10.1046/j.1365-2958.1998.00998.x.
MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.
MotA和MotB是细胞质膜蛋白，它们构成了鼠伤寒沙门氏菌及许多其他细菌鞭毛马达的力产生单元。已知这两种蛋白中的许多错义突变会导致马达旋转缓慢（慢速运动表型）或根本不旋转（非运动或麻痹表型）。然而，MotA细胞质区域和MotB周质区域中的大片序列未能产生这类突变。在本研究中，我们研究了这些表型沉默区域中一系列10个氨基酸缺失的影响。就MotA而言，我们发现只有C末端的5个氨基酸是完全可缺失的；相邻的10个氨基酸是部分可缺失的。在MotA的细胞质环区域，缺失会使蛋白质不稳定。对于MotB，我们发现周质区域的两个大片段是可缺失的：单个缺失的结果表明，第一个片段由唯一跨膜区段和肽聚糖结合基序之间的6个缺失组成，而第二个片段由C末端的4个缺失组成。我们还发现MotB N末端细胞质区域的缺失会损害运动性，但不会使其完全丧失。通过组合可缺失的缺失片段对MotB进行了进一步研究。仍然保留一定程度功能的MotB最极端版本在跨膜区段之后紧接着的周质区域总共缺少99个氨基酸。这些结果表明，MotA细胞质环区域中的缺失区域对于稳定性至关重要；它们可能直接参与扭矩产生，也可能不参与。MotA C末端细胞质区域的一部分对于扭矩产生不是必需的。MotB可分为三个区域：细胞质中约30个氨基酸的N末端区域、一个跨膜区段以及周质中约260个氨基酸，包括一个肽聚糖结合基序。在周质区域，我们认为MotB中两个可缺失区段中的第一个可能构成MotB跨膜区段与其与肽聚糖层附着点之间连接子的一部分，并且该连接子序列的大部分长度或特定序列并不关键。C末端约40个残基也不重要。
