The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: Putative components for flagellar assembly
The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: Putative components for flagellar assembly
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DOI：
10.1128/jb.179.19.6092-6099.1997
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发表时间：
1997-10-01
影响因子：
3.2
通讯作者：
Macnab, RM
中科院分区：
文献类型：
--
作者：
Ohnishi, K;Fan, F;Macnab, RM
鼠伤寒沙门氏菌的纤毛基因Flio、Flip、FliQ和FliR在FliLMNOPQR操纵子内是连续的。它们是鞭毛形成所必需的，但不编码任何已知的结构或调节成分，它们可能参与鞭毛蛋白的输出，这是通过III型输出途径进行的。这些基因已经被克隆和测序。这些序列预测的蛋白质的相对分子质量分别为13,068，26,755，9,592和28,933 Da。所有四种基因产物都经过实验鉴定；与它们的高疏水残基含量一致，它们与膜组分分离。从N-端氨基酸序列分析，我们得出结论，Flio是在FLIN之后立即开始的，而不是在之前提出的下游位点。FliP以25 kDa和23 kDa两种形式存在，对23 kDa形式的N末端氨基酸分析表明，它经历了信号肽的切割-这是原核细胞质膜蛋白罕见的过程。裂解位点的定点突变导致加工受损，这减少了但不能消除翻转突变体的互补。在群体平板试验中，编码成熟蛋白的克隆片段也可以补充翻转突变体，但这一作用更差。最后，当MOTA(一种不经历信号肽切割的细胞质膜蛋白)的第一个跨膜跨膜与成熟的FliP融合时，融合蛋白的补充作用非常弱。更高水平的突变蛋白的合成极大地改善了功能，我们得出结论，对于将翻转插入膜中，切割在动力学上是重要的，但不是绝对必需的。
The fIagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components, They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form, N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide-a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays, A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function, We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.