Novel G proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B.
Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p of Saccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [(3)H]GTP and [(3)H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotide-bound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.
Sekiguchi
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. sekigu@molbiol.med.kyushu-u.ac.jp
Hirose
E
E
Nakashima
N
N
Ii
M
M
Nishimoto
T
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007930:Leucine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D005656:Fungal Proteins; C121263:GTR2 protein, S cerevisiae; D009711:Nucleotides; C430722:RRAGC protein, human; C430723:RRAGD protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins; C484342:RRAGA protein, human; C484345:RRAGB protein, human; D020559:Monomeric GTP-Binding Proteins; D007930:Leucine