The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro.
Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions. We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro. This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Ga14. Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 x 10(7) M-1 and 5 x 10(7) M-1. An association constant of 5 x 10(7) M-1 was determined for NLS binding to the repetitive domain of Nsp1. We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo. The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.
Barth
W
W
Department of Physiology, McGill University, Montreal, QC, Canada.
Stochaj
U
U
D001665:Binding Sites; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013539:Simian virus 40
D002135:Calcium-Binding Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; C062748:NSP1 protein, S cerevisiae; C064550:NUP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins