Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly
Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly
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DOI：
10.1046/j.1365-2958.1999.01372.x
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发表时间：
1999-05-01
影响因子：
3.6
通讯作者：
Hughes, C
中科院分区：
文献类型：
--
作者：
Fraser, GM;Bennett, JCQ;Hughes, C
在游动肠杆菌的鞭毛组装过程中，鞭毛轴蛋白被认为是以部分未折叠的单体形式通过25-30埃鞭毛中心通道输出到细胞表面结构中。如何防止胞浆中的过早折叠和寡聚？我们以前已经使用超级鞭毛变形杆菌和一个能动但不成群的flgN转座子突变体表明，表面上胞质16.5 kDa的鞭毛蛋白flgN有助于有效的鞭毛细丝组装。在这里，我们进一步研究FlgN是否作为轴蛋白的伴侣，预测它含有典型的III型出口伴侣蛋白的C端两亲性螺旋。将来自鼠伤寒沙门氏菌的可溶性放射性标记FlgN与野生型鼠伤寒沙门氏菌的硝酸纤维素裂解物和一个非鞭毛的1类flhDC突变体孵育，结果表明FlgN与鞭毛蛋白结合。对野生型和flhDC、flgI、flgK、flgL、flc或fld鞭毛突变体的培养上清液进行相同亲和印迹分析表明，flgN与鞭毛挂钩相关蛋白(HAPS)FlgK和FlgL结合。通过在大肠杆菌中人工表达单个HAPS的印迹证实了这一点。对原始奇异P.mirabilis flgN突变体分泌到培养液中的轴蛋白的分析表明，FlgK和FlgL的输出明显减少，伴随而来的是鞭毛直接远端成分未聚合鞭毛(Flc)的释放增加。这些数据表明FlgN作为FlgK和FlgL的出口伴侣发挥作用。平行实验表明，Flit是一种类似的小(14 KDa)的潜在螺旋鞭毛蛋白，专门与鞭毛丝帽蛋白Flid(Happ)结合，表明它也可能是一种出口伴侣。鞭毛轴蛋白的末端都含有两亲性螺旋。在每种情况下，去除HAP C-末端螺旋结构域都取消了与FlgN和Flit的结合，并且由每个HAP C-末端组成的多肽与FlgN和Flit特异性结合。我们认为FlgN和Flit是底物特异性的鞭毛伴侣蛋白，通过在出口前与其螺旋结构域结合来防止HAP的寡聚。
During flagellum assembly by motile enterobacteria, flagellar axial proteins destined for polymerization into the cell surface structure are thought to be exported through the 25-30 Angstrom flagellum central channel as partially unfolded monomers. How are premature folding and oligomerization in the cytosol prevented? We have shown previously using hyperflagellated Proteus mirabilis and a motile but nonswarming flgN transposon mutant that the apparently cytosolic 16.5 kDa flagellar protein FlgN facilitates efficient flagellum filament assembly. Here, we investigate further whether FlgN, predicted to contain a C-terminal amphipathic helix typical of type III export chaperones, acts as a chaperone for axial proteins. Incubation of soluble radiolabelled FlgN from Salmonella typhimurium with nitrocellulose-immobilized cell lysates of wild-type S. typhimurium and a non-flagellate class 1 flhDC mutant indicated that FlgN binds to flagellar proteins. Identical affinity blot analysis of culture supernatants from the wild-type and flhDC, flgI, flgK, flgL, fliC or fliD flagellar mutants showed that FlgN binds to the flagellar hook-associated proteins (HAPs) FlgK and FlgL. This was confirmed by blotting artificially expressed individual HAPs in Escherichia coli. Analysis of axial proteins secreted into the culture medium by the original P. mirabilis flgN mutant demonstrated that export of FlgK and FlgL was specifically reduced, with concomitant increased release of unpolymerized flagellin (FliC), the immediately distal component of the flagellum. These data suggest that FlgN functions as an export chaperone for FlgK and FlgL. Parallel experiments showed that FliT, a similarly small (14 kDa), potentially helical flagellar protein, binds specifically to the flagellar filament cap protein, FliD (HAPP), indicating that it too might be an export chaperone. Flagellar axial proteins all contain amphipathic helices at their termini. Removal of the HAP C-terminal helical domains abolished binding by FlgN and FliT in each case, and polypeptides comprising each of the HAP C-termini were specifically bound by FlgN and FliT. We suggest that FlgN and FliT are substrate-specific flagellar chaperones that prevent oligomerization of the HAPs by binding to their helical domains before export.