Ballas N, Citovsky V
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10723-8. doi: 10.1073/pnas.94.20.10723.
T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5' end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPalpha, specifically bound VirD2 in vivo and in vitro. VirD2-AtKAPalpha interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPalpha was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin alpha family. Indeed, AtKAPalpha efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPalpha specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.
T-DNA核输入是农杆菌对植物细胞进行遗传转化的核心事件。据推测，T-DNA运输中间体是一个与两种细菌蛋白VirD2和VirE2相关联的单链DNA分子，这两种蛋白很可能介导了运输过程。在VirE2协同包裹被运输的单链DNA时，VirD2共价连接于其5'端。为了更好地理解VirD2的作用机制，鉴定了一种VirD2的细胞受体，并从拟南芥中克隆了其编码基因。所鉴定的蛋白命名为AtKAPα，它在体内和体外均能特异性结合VirD2。VirD2与AtKAPα的相互作用绝对依赖于VirD2羧基末端的双元核定位信号序列。AtKAPα推导的氨基酸序列与属于核转运蛋白α家族的酵母和动物核定位信号结合蛋白同源。实际上，AtKAPα能有效拯救一个核输入缺陷的酵母突变体。此外，AtKAPα特异性介导VirD2转运至透化酵母细胞的细胞核中。
