Enenkel C, Schülke N, Blobel G
Laboratory of Cell Biology, Rockefeller University, Howard Hughes Medical Institute, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12986-91. doi: 10.1073/pnas.93.23.12986.
Using truncated forms of recombinant yeast karyopherins alpha and beta in in vitro binding assays, we mapped the regions of karyopherin alpha that bind to karyopherin beta and the regions of karyopherin beta that interact with karyopherin alpha and with Ran-GTP. Karyopherin alpha's binding region for karyopherin beta was localized to its N-terminal domain, which contains several clusters of basic residues, whereas karyopherin beta's binding region for karyopherin alpha was localized to an internal region containing two clusters of acidic residues. Karyopherin beta's binding region for Ran-GTP overlaps with that for karyopherin alpha and comprises at least one of the two acidic clusters required for karyopherin alpha binding in addition to further downstream determinants not required for karyopherin alpha binding. Overexpression in yeast of fragments containing either karyopherin beta's binding region for alpha and Ran-GTP or karyopherin alpha's binding region for beta resulted in sequestration of most of the cytosolic karyopherin alpha or karyopherin beta, respectively, in complexes containing the truncated proteins. As these binding region-containing fragments lack other domains required for function of the corresponding protein, the overexpression of either fragment also inhibited in vivo nuclear import of a model reporter protein as well as cell growth.
在体外结合试验中，我们使用重组酵母核转运蛋白α和β的截短形式，绘制了核转运蛋白α与核转运蛋白β结合的区域，以及核转运蛋白β与核转运蛋白α和Ran-GTP相互作用的区域。核转运蛋白α与核转运蛋白β的结合区域定位于其N端结构域，该结构域包含几个碱性残基簇，而核转运蛋白β与核转运蛋白α的结合区域定位于一个内部区域，该区域包含两个酸性残基簇。核转运蛋白β与Ran-GTP的结合区域与核转运蛋白α的结合区域重叠，并且除了核转运蛋白α结合不需要的更远端决定簇之外，还包含核转运蛋白α结合所需的两个酸性簇中的至少一个。在酵母中过表达包含核转运蛋白β与α和Ran-GTP的结合区域或核转运蛋白α与β的结合区域的片段，分别导致大部分胞质核转运蛋白α或核转运蛋白β隔离在含有截短蛋白的复合物中。由于这些含结合区域的片段缺乏相应蛋白质功能所需的其他结构域，任一片段的过表达也抑制了模型报告蛋白的体内核输入以及细胞生长。
