THE SMALL GTPASE GSP1P BINDS TO THE REPEAT DOMAIN OF THE NUCLEOPORIN NSP1P

The small GTPase Gsp1p of Saccharomyces cerevisiae and its homologue R an play essential roles in several nuclear processes, such as cell-cyc le progression, nuclear organization and nucleocytoplasmic traffic of RNA and proteins. Gsp1p/Ran is an abundant nuclear protein that intera cts with different cytoplasmic and nuclear factors. Several of the pre viously identified Ran-binding proteins located at the nuclear-pore co mplex carry a specific Ran-binding domain. So far, direct interactions between the GTPase and other proteins have been mostly characterized in higher eukaryotes. Here we report that the yeast protein Gsp1p can directly bind to the nucleoporin Nsp1p in vitro. Nsp1p does not contai n a Ran-binding domain and therefore represents a distinct type of nuc leoporin that associates with Gsp1p. We demonstrate that the middle do main of Nsp1p is sufficient to mediate this interaction. Importantly, we show that a conserved cluster of positively charged amino acid resi dues of Gsp1p located at positions 142-144 is essential for the bindin g reaction. Thus we have identified Nsp1p as a new candidate protein l ocated at the nuclear pore complex of the yeast S. cerevisiae that int eracts directly with Gsp1p. We further demonstrate that both Gsp1p and Nsp1p are components of larger protein complexes in vivo, supporting the idea that the association between both proteins takes place in gro wing cells.