The acidic C-terminal domain of rna1p is required for the binding of Ran.GTP and for RanGAP activity.
The small GTP binding protein Ran is an essential component of the nuclear protein import machinery whose GTPase cycle is regulated by the nuclear guanosine nucleotide exchange factor RCC1 and by the cytosolic GTPase activating protein RanGAP. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae the RanGAP activity is encoded by the RNA1 genes which are essential for cell viability and nucleocytoplasmic transport in vivo. Although of limited sequence identity the two yeast 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 acidic amino acid residues. By analyzing the RanGAP activity of a series of recombinantly expressed rna1p mutant derivatives, we show that the highly 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 rna1p.Ran complex formation indicating that the loss of GAP activity in the C-terminally truncated rna1p mutants results from an impaired interaction with Ran. The predominant species stabilized through the covalent cross-link is a rna1p.Ran heterodimer whose formation requires the GTP-bound conformation of Ran. As the acidic C-terminal domain of rna1p is required for establishing the interaction with Ran, the leucine-rich repeats domain in rna1p is potentially available for additional protein interactions perhaps required for directing a fraction of rna1p to the nuclear pore.
Haberland
J
J
Institute for Medical Biochemistry, University of Münster, Von-Esmarch-Strasse 56, D-48149 Münster, Federal Republic of Germany.
Becker
J
J
Gerke
V
V
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D020931:ran GTP-Binding Protein
D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; C060997:RNA1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C499298:rna1 protein, S pombe; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein