A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2 Delta gtrld gtr2 Delta was lethal, while a double mutant: gtrld gtr2 Delta survived well, indicating that Yrb2p protected cells from the killing effect of gtrld gtr2 Delta. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtrlp-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtrlp-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p. (c) 2005 Elsevier Inc. All rights reserved.