Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.
Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.
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DOI：
10.1371/journal.pone.0022417
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Minamino T
中科院分区：
文献类型：
--
作者：
Hara N;Namba K;Minamino T
为了组装细菌鞭毛，大多数鞭毛蛋白通过由质子动力（PMF）驱动的鞭毛III型蛋白输出装置穿过细胞质膜被运输到鞭毛的远端。FlhA是输出装置中不可或缺的膜蛋白，与三种可溶性蛋白FliH、FliI和FliJ一起沿着于输出过程的早期阶段，但能量偶联机制仍未知。在这里，我们进行了定点诱变的8个高度保守的带电残基在假定的flhA的跨膜螺旋和跨膜螺旋。只有Asp-208是必需的酸性残基。大多数FlhA取代是耐受的，但在ΔfliH-fliI突变背景下导致功能丧失，即使在缺乏FliH和FliI的情况下增加鞭毛蛋白输出概率的第二位点flhB（P28 T）突变也是如此。FliH和FliI的添加允许D45 A、R85 A、R94 K和R270 A突变蛋白即使在flhB（P28 T）突变存在下也起作用。FlhA（K203 W）突变的抑制子分析显示FlhA和FliR之间的相互作用。综上所述，我们认为Asp-208直接参与PMF驱动的蛋白质输出，FlhA与FlhB、FliH、FliI和FliR的协同相互作用驱动输出底物的易位。
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.
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DOI：
10.1073/pnas.92.26.12046
发表时间：
1995-12-19
影响因子：
11.1
作者：
Choe, S;Stevens, CF;Sullivan, JM
通讯作者：
Sullivan, JM
影响因子：
3.6
作者：
Fan, F;Ohnishi, K;Macnab, RM
通讯作者：
Macnab, RM
影响因子：
3.2
作者：
Kihara, M;Minamino, T;Macnab, RM
通讯作者：
Macnab, RM
影响因子：
3.2
作者：
Karlinsey, JE;Hughes, KT
通讯作者：
Hughes, KT
影响因子：
3.2
作者：
Minamino, T;González-Pedrajo, B;Macnab, RM
通讯作者：
Macnab, RM