Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase.
Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase.
复制标题
根癌农杆菌 VirB11 ATP 酶的膜和蛋白质相互作用决定因素的表征。
DOI：
10.1128/jb.179.3.583-591.1997
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发表时间：
1997
影响因子：
3.2
通讯作者：
Christie,PJ
中科院分区：
文献类型：
--
作者：
Rashkova,S;Spudich,GM;Christie,PJ
VirB11 ATPase是负责引导核蛋白颗粒(T复合体)通过根癌农杆菌被膜输出到敏感植物细胞的运输机制的一个假定组成部分。分离和膜处理研究表明，大约30%的VirB11以可溶性蛋白的形式存在，而其余的蛋白只被野生型菌株A348和表达IncP复制子的无钛质粒菌株的细胞膜中的尿素部分溶解。VirB11中影响蛋白质功能的突变位于氨基端(Q6L、P13L和E25G)附近，恰好位于Walker A核苷酸结合位点(F154H；L155M)的上游，并位于Walker A基序(P170L、K175Q和Delta GKT174-176)内。K175Q和Delta GKT174-176突变蛋白几乎完全与细胞膜结合，这表明与核苷酸结合相关的活性可能调节VirB11与细胞膜的亲和力。在二倍体分析中，VirB11F154H；L155M等位基因对野生型VirB11易位显性，这提供了至少一种形式的VirB11具有同源或异源多聚体功能的有力证据。在二倍体分析中，缺失该基因前半部分的等位基因VirB11 Delta1-156是反显性的，表明VirB11的C端半部分含有蛋白质相互作用结构域。VirB11 Delta1-156和VirB11 Delta158-343的产物都与根癌农杆菌膜紧密结合，提示VirB11的两个半部分都含有膜相互作用决定因素。
The VirB11 ATPase is a putative component of the transport machinery responsible for directing the export of nucleoprotein particles (T complexes) across the Agrobacterium tumefaciens envelope to susceptible plant cells. Fractionation and membrane treatment studies showed that approximately 30% of VirB11 partitioned as soluble protein, whereas the remaining protein was only partially solubilized with urea from cytoplasmic membranes of wild-type strain A348 as well as a Ti-plasmidless strain expressing virB11 from an IncP replicon. Mutations in virB11 affecting protein function were mapped near the amino terminus (Q6L, P13L, and E25G), just upstream of a region encoding a Walker A nucleotide-binding site (F154H;L155M), and within the Walker A motif (P170L, K175Q, and delta GKT174-176). The K175Q and delta GKT174-176 mutant proteins partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. The virB11F154H;L155M allele was transdominant over wild-type virB11 in a merodiploid assay, providing strong evidence that at least one form of VirB11 functions as a homo- or heteromultimer. An allele with a deletion of the first half of the gene, virB11 delta1-156, was transdominant in a merodiploid assay, indicating that the C-terminal half of VirB11 contains a protein interaction domain. Products of both virB11 delta1-156 and virB11 delta158-343, which synthesizes the N-terminal half of VirB11, associated tightly with the A. tumefaciens membrane, suggesting that both halves of VirB11 contain membrane interaction determinants.