Dang T A, Christie P J
Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, 77030, USA.
J Bacteriol. 1997 Jan;179(2):453-62. doi: 10.1128/jb.179.2.453-462.1997.
The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid. VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and 'phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2). Conversely, VirB4::beta-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low beta-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high beta-galactosidase activities. Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K treatment of A. tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of approximately 35 and approximately 45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains.
根癌土壤杆菌的VirB4 ATP酶是T复合体转运装置的一个假定组成部分，它独立于Ti质粒的其他产物与细胞质膜结合。VirB4难以从野生型菌株A348或从IncP复制子表达virB4的无Ti质粒菌株的膜中提取出来。为了评估VirB4的膜拓扑结构，采用嵌套缺失法在virB4和“phoA”之间产生高频随机融合，“phoA”编码一种去除了信号序列的周质活性碱性磷酸酶（AP）。在大肠杆菌和根癌土壤杆菌中表现出AP活性的VirB4::PhoA杂合蛋白的连接位点定位于两个区域，即58至84位残基之间（区域1）和450至514位残基之间（区域2）。相反，连接位点定位于区域1和2的VirB4::β-半乳糖苷酶杂合蛋白表现出低β-半乳糖苷酶活性，而连接位点在其他位置的杂合蛋白表现出高β-半乳糖苷酶活性。具有酶活性的VirB5::PhoA杂合蛋白的连接位点分布在整个蛋白质长度上。用蛋白酶K处理根癌土壤杆菌原生质球导致87 kDa的VirB4蛋白消失，并同时出现两种免疫反应性条带，大小约为35 kDa和45 kDa。综合来看，我们的数据支持一个模型，即VirB4在拓扑结构上是一种具有两个周质结构域的整合细胞质膜蛋白。
