EXPRESSION IN YEAST OF BINDING REGIONS OF KARYOPHERIN-ALPHA AND KARYOPHERIN-BETA INHIBITS NUCLEAR IMPORT AND CELL-GROWTH

Using truncated forms of recombinant yeast karyopherins alpha and beta in in vitro binding assays, se mapped the regions of karyopherin alph a that hind to karyopherin beta and the regions of karyopherin beta th at 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 ka ryopherin beta's binding region for karyopherin alpha was localized to an internal region containing two clusters of acidic residues, Karyop herin beta's binding region for Ran-GTP overlaps with that for karyoph erin alpha and comprises at least one of the two acidic clusters requi red for karyopherin alpha binding in addition to further downstream de terminants not required for karyopherin alpha binding, Overexpression in yeast of fragments containing either karyopherin beta's binding reg ion for alpha and Ran GTP or karyopherin alpha's binding region for be ta resulted in sequestration of most of the cytosolic karyopherin alph a or karyopherin beta, respectively, in complexes containing the trunc ated proteins. As these binding region-containing fragments lack other domains required for function of the corresponding protein, the overe xpression of either fragment also inhibited in vivo nuclear import of a model reporter protein as well as cell growth.