Spudich G M, Fernandez D, Zhou X R, Christie P J
Department of Microbiology and Molecular Genetics, The University of Texas Health Sciences Center, Houston 77030, USA.
Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7512-7. doi: 10.1073/pnas.93.15.7512.
The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus. Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue. Three types of beta-mercaptoethanol-dissociable complexes were visualized with VirB7 and/or a VirB7::PhoA41 fusion protein: (i) a 9-kDa complex corresponding in size to a VirB7 homodimer, (ii) a 54-kDa complex corresponding in size to a VirB7/VirB7::PhoA41 mixed dimer, and (iii) a 102-kDa complex corresponding to a VirB7::PhoA41 homodimer. A VirB7C24S mutant protein was immunologically undetectable, whereas the corresponding VirB7C24S::PhoA41 derivative accumulated to detectable levels but failed to form dissociable homodimers or mixed dimers with wild-type VirB7. We further report that VirB7-dependent stabilization of VirB9 is correlated with the ability of these two proteins to dimerize via formation of a disulfide bridge between reactive Cys-24 and Cys-262 residues, respectively. Two types of dissociable complexes were visualized: (i) a 36-kDa complex corresponding in size to a VirB7/VirB9 heterodimer and (ii) an 84-kDa complex corresponding in size to a VirB7/VirB9::PhoA293 heterodimer. A VirB9C262S mutant protein was immunologically undetectable, whereas the corresponding VirB9C262S::PhoA293 derivative accumulated to detectable levels but failed to form dissociable heterodimers with wild-type VirB7. Taken together, these results support a model in which the formation of disulfide cross-linked VirB7 dimers represent critical early steps in the biogenesis of the T-complex transport apparatus.
根癌土壤杆菌的VirB7脂蛋白在假定的T复合体转运装置的生物合成过程中有助于VirB蛋白的稳定。在此，我们报告VirB7自身的稳定与其通过反应性半胱氨酸-24残基形成二硫键交联同型二聚体的能力相关。用VirB7和/或VirB7::PhoA41融合蛋白观察到三种类型的β-巯基乙醇可解离复合物：（i）大小对应于VirB7同型二聚体的9 kDa复合物，（ii）大小对应于VirB7/VirB7::PhoA41混合二聚体的54 kDa复合物，以及（iii）对应于VirB7::PhoA41同型二聚体的102 kDa复合物。VirB7C24S突变蛋白在免疫上无法检测到，而相应的VirB7C24S::PhoA41衍生物积累到可检测水平，但未能与野生型VirB7形成可解离的同型二聚体或混合二聚体。我们进一步报告，VirB7依赖的VirB9稳定与这两种蛋白通过分别在反应性半胱氨酸-24和半胱氨酸-262残基之间形成二硫键而二聚化的能力相关。观察到两种类型的可解离复合物：（i）大小对应于VirB7/VirB9异二聚体的36 kDa复合物，以及（ii）大小对应于VirB7/VirB9::PhoA293异二聚体的84 kDa复合物。VirB9C262S突变蛋白在免疫上无法检测到，而相应的VirB9C262S::PhoA293衍生物积累到可检测水平，但未能与野生型VirB7形成可解离的异二聚体。综上所述，这些结果支持了一个模型，其中二硫键交联的VirB7二聚体的形成代表了T复合体转运装置生物合成中的关键早期步骤。
