THE YEAST NUCLEOPORIN NSP1 BINDS NUCLEAR-LOCALIZATION SEQUENCES IN-VITRO

Facilitated transport of proteins into the nucleus requires nuclear lo calization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear tr ansport apparatus is essential for these targeting reactions. We now r eport that the yeast nucleoporin Nsp1 binds specifically nuclear local ization sequences in vitro. This nucleoporin recognizes several NLSs t hat are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Ga14. Nsp1 is or ganized into three domains, and we have located NLS binding sites to t he N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we ob tained 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 bin ding 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-antige n, 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.