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© The Rockefeller University Press, 0021-9525/1997//797 $5.00
The Journal of Cell Biology, Volume 137, Number 4, , 1997 797-811

Article

A Nuclear Export Signal in Kap95p Is Required for Both Recycling the Import Factor and Interaction with the Nucleoporin GLFG Repeat Regions of Nup116p and Nup100p



M. Kathryn Iovine and Susan R. Wente

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A–tagged Nup116p or protein A–tagged Nup100p complexes. The protein A–tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.

1. Abbreviations used in this paper: DAPI, 4',6-diamidino-2-phenylindole; 5-FOA, 5-fluoroorotic acid; GST, glutathione-S-transferase; HSA, human serum albumin; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; PKI, protein kinase inhibitor.

M.K. Iovine performed this work as a predoctoral trainee supported by National Institutes of Health (NIH) training grant 2T32GM07067-21. This work was supported by a grant from the NIH, GM51219-02, and a Junior Faculty Research Award from American Chemical Society to S.R. Wente.

Please address all correspondence to Susan R. Wente, Department of Cell Biology and Physiology, Box 8228, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110. Tel.: (314) 362-2713. Fax: (314) 362-7463. e-mail: swente{at}cell.bio.wustl.edu


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