THE ACIDIC C-TERMINAL DOMAIN OF RNA1P IS REQUIRED FOR THE BINDING OF RAN-CENTER-DOT-GTP AND FOR RANGAP ACTIVITY

The small GTP binding protein Ran is an essential component of the nuc lear protein import machinery whose GTPase cycle is regulated by the n uclear guanosine nucleotide exchange factor RCC1 and by the cytosolic GTPase activating protein RanGAP. In the yeasts Schizosaccharomyces po mbe and Saccharomyces cerevisiae the RanGAP activity is encoded by the RNA1 genes which are essential for cell viability and nucleocytoplasm ic transport in vivo. Although of limited sequence identity the two ye ast proteins show a conserved structural organization characterized by an N-terminal domain of eight leucine-rich repeats, motifs implicated in protein-protein interactions, and a C-terminal domain rich in acid ic amino acid residues. By analyzing the RanGAP activity of a series o f recombinantly expressed rna1p mutant derivatives, we show that the h ighly acidic sequence in the C-terminal domain of both yeast proteins is indispensable for activating Ran-mediated GTP hydrolysis. Chemical cross-linking reveals that the same sequence in rna1p is required for rnalp.Ran complex formation indicating that the loss of GAP activity i n the C-terminally truncated rna1p mutants results from an impaired in teraction with Ran. The predominant species stabilized through the cov alent cross-link is a rna1p.Ran heterodimer whose formation requires t he GTP-bound conformation of Ran. As the acidic C-terminal domain of r na1p is required for establishing the interaction with Ran, the leucin e-rich repeats domain in rna1p is potentially available for additional protein interactions perhaps required for directing a fraction of rna 1p to the nuclear pore.