Ziemienowicz A, Merkle T, Schoumacher F, Hohn B, Rossi L
Friedrich Miecher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland.
Plant Cell. 2001 Feb;13(2):369-83. doi: 10.1105/tpc.13.2.369.
To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.
为了研究T-DNA的核输入机制，对由毒性蛋白VirD2和VirE2以及单链DNA（ssDNA）组成的复合物进行了体外导入植物细胞核的测试。这些复合物的导入快速且高效，并且可以被一种竞争者——与牛血清白蛋白（BSA）偶联的核定位信号（NLS）所抑制。对于短ssDNA的导入，VirD2就足够了，而长ssDNA的导入还需要VirE2。一个缺失其C端NLS的VirD2突变体无法介导T-DNA复合物导入细胞核。尽管游离的VirE2分子可以导入细胞核，但一旦与ssDNA结合就不能导入了，这意味着当与DNA复合时，VirE2的NLS没有暴露，因此不起作用。RecA，另一种ssDNA结合蛋白，在T-DNA的核输入中可以替代VirE2，但在T-DNA转移到植物细胞的早期事件中则不能。我们提出，VirD2将T-DNA复合物导向核孔，而这两种蛋白介导其通过核孔。因此，通过与ssDNA结合，VirE2可能会塑造T-DNA复合物，使其被接受转运到细胞核中。
