A protein required for nuclear-protein import, interacts ath GTP-Gsplp, the Saccharomyces Ran homologue Mogip, directly cerevisiae

We previously isolated 25 temperaturesensitive gspl alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors— PDE2, NTF2, and a gene designated MOG1—all of which rescued a growth defect of these gspl strains. The gspl suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gspl but not PDE2. Moglp, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gspl alleles with similar efficiency, and both suppressed gspl even with a single gene dose. Consistent with the high efficiency of gspl suppression, Mogip directly bound to GTP, but not to GDP-Gsplp. The disruption of MOG1 made yeast temperature-sensitive for growth. Amogl, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Moglp, which was localized in the nucleus, is a Gsplp-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gspl and rnal-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway. Gsplp is the Saccharomyces cerevisiae homologue of the Ran GTPase, which is essential for nucleocytoplasmic exchange of macromolecules (1-9). Indeed, all temperature-sensitive (ts)gspl alleles have a defect in nuclear-protein import (10, 11). However, mutants of Rcclp, which is the guanine nucleotide-exchanging factor (GEF) of Ran (12), show a pleiotropic phenotype, such as cell cycle-specific arrest and defects in mRNA export and mRNA splicing (13-18). Furthermore, rnal-1, a ts mutant of yeast Ran GTPase activator (RanGAP; ref. 19) was initially identified as a mutant defective in RNA processing (20) and was subsequently found to have defects in mRNA metabolism similar to prp20, a ts mutant of the S. cerevisiae RCC1 homologue (21-23) and in nuclear-protein import (24). It is a matter of debate how Ran controls all of the biological defects observed. To clarify the pathways downstream of Gsplp, we previously isolated a series of ts mutants of GSPI (11). In this report, we isolated multicopy suppressor genes NTF2, PDE2, and MOGI from S. cerevisiae that conferred the temperature resistance (ts+) phenotype to these gspl strains. The suppression of gspl with these genes occurred even in the absence of another S. cerevisiae GSP1-like gene, GSP2, that encodes a protein 97% identical to Gsplp and is expressed in a nonglucose medium (25). Ntf2p has been reported to rescue gspl-1 and gspl-2 (10). It is a highly conserved protein essential for nuclear-protein import (26, 27) and specifically binds to GDP-Gsplp but not to GTP-Gsp 1 (28, 29). The role of Ntf2p in nuclear-protein import, however, is still unknown. Pde2p, which is a high-affinity phosphodiesterase (30), has been reported to suppress real-1 (31). In contrast, Mogip is encoded by the S. cerevisiae ORF YJR074W. Both NTF2 and MOGI genes suppressedgspl with a similar efficiency. Consistent with this finding, Moglp was found to have a functional interaction with Ntf2p. Thus, Mog1p, which was found to be required for nuclear-protein import and to bind to GTP-Gsp1p, may help us analyze the function of Ntf2p in the nuclear protein-import process. MATERIALS AND METHODS Strains and Media. All of the S. cerevisiae strains used in this study are shown in Table 1. The media used for S. cerevisiae and bacterial strains were described previously (11). The strain PSY962-479 was derived from the PSY962 strain (a gift from P. Silver, Dana-Farber Cancer Institute; ref. 10) by plasmid shuffling using 5-f luoroorotic acid (5FOA) as described (11). Determination of the Critical Temperature. The multicopy vector, YEplac195, was introduced into all of the gspl strains. Ura transformants isolated at 26°C were incubated at 26°C, 30°C, 33°C, 34°C, 35°C, 36°C, or 37°C on a synthetic medium (ura-) plate. Thus, the lowest temperature above which the gspl strain did not papillate with expression of the multicopy plasmids was determined for each gspl allele. Isolation of Multicopy Suppressors. The genomic library (RB236) constructed by using the vector YEp24 (32) was introduced into cultures of gspl strains. Transfected cells (4.5 x 105) were incubated in synthetic medium (ura-) for 1 day at 26°C and then for 3-6 days at the nonpermissive temperature for each gspl allele. As described (33), plasmids were isolated from transformants (ts+ Lira+) of gspl cells which grew at the nonpermissive temperature in synthetic medium (ura-). Subsequently, DNA inserts of resulting plasmids were sequenced by using as the primers 5 '-GTCCTGCTCGCTTCGCTACT-3' and 5'-GCGATATAGGCGCCAGCAAC-3'. The nucleotide sequences of both the 5' and 3' ends of the DNA insert were used to search for ORFs of S. cerevisiae genomic sequence by using the Munich Information Center for Protein Sequences (MIPS) (http:// www.mips.biochem.mpg.del). Construction of Plasmids. All of the plasmids used in this study are listed in Table 1. For pGEX-MOG1, 1 kb of DNA-fragment containing MOGI was amplified from p195M0G1 by PCR. Resultant DNA fragments were partially digested and inserted into the NcoI/HindIII site of pGEX-KG, resulting in pGEXMOG1. For pHIS3MOG1, 0.97 kb of the 5' noncoding region of MOGI was amplified by using PCR and was inserted into the Xbal/BamH1 site of pBluescript II SK(+), resulting in pSKMOG1XB. On the other hand, 1.03 kb of the 3' noncoding Abbreviations: ts, temperature-sensitive; GEF, gaunine nucleotide exchange factor; RanGAP, RAN GTPase activator; NLS, nuclear localization signal; GST, glutathione S-transferase, DAPI, 4',6diamidino-2-phenylindole. Table 1. Yeast strains and plasmids used in this study Relevant markers Genotype/comment Source