Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Binding Sites
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Cell Line
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Cell Nucleus / metabolism
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Dimerization
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Dose-Response Relationship, Drug
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Fungal Proteins / chemistry
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Fungal Proteins / metabolism
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GTP-Binding Proteins*
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Glutathione Transferase / metabolism
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Guanosine Diphosphate / metabolism
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Guanosine Triphosphate / metabolism
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HeLa Cells
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Humans
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Immunoblotting
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Leucine / chemistry
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Microscopy, Fluorescence
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Models, Genetic
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Molecular Sequence Data
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Monomeric GTP-Binding Proteins / chemistry*
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Monomeric GTP-Binding Proteins / genetics*
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Monomeric GTP-Binding Proteins / metabolism
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Nucleotides / metabolism
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Precipitin Tests
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Protein Binding
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins*
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Sequence Homology, Amino Acid
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Time Factors
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Transfection
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Two-Hybrid System Techniques
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beta-Galactosidase / metabolism
Substances
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Fungal Proteins
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GTR2 protein, S cerevisiae
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Nucleotides
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RRAGC protein, human
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RRAGD protein, human
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Recombinant Fusion Proteins
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Guanosine Diphosphate
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Guanosine Triphosphate
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Glutathione Transferase
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beta-Galactosidase
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GTP-Binding Proteins
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RRAGA protein, human
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RRAGB protein, human
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Monomeric GTP-Binding Proteins
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Leucine
Associated data
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GENBANK/AF272035
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GENBANK/AF272036