Abstract
To detect specific partners of the small Golgi-localized GTPase rab1b we generated rab1b mutants and used them as bait proteins in yeast two-hybrid screens. We isolated several specifically interacting clones. Two of them encode large protein fragments highly homologous to rat GM130 and to human Golgin95. The full-length human GM130 cDNA was cloned and its interaction with rab1b was characterized in detail by yeast two-hybrid and in vitro binding assays. Here we report for the first time that the rab1b protein interacts specifically with GM130 in a GTP-dependent manner and therefore needs the hypervariable regions of the N- and C-termini. We mapped the rab1b binding site of GM130 and provide evidence that it is different to the previously described p115 and Grasp65 binding sites of the GM130 protein.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Autoantigens
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Binding Sites
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Carrier Proteins / metabolism
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Cell Line
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Cloning, Molecular
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DNA, Complementary / metabolism
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Gene Library
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Golgi Apparatus / metabolism*
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Golgi Matrix Proteins
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HeLa Cells
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Humans
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Microscopy, Fluorescence
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Models, Genetic
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Molecular Sequence Data
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Mutation
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Plasmids / metabolism
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Protein Binding
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Protein Structure, Tertiary
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Rats
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Transfection
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Transformation, Genetic
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Two-Hybrid System Techniques
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Vesicular Transport Proteins*
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beta-Galactosidase / metabolism
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rab GTP-Binding Proteins / chemistry
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rab GTP-Binding Proteins / metabolism*
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rab1 GTP-Binding Proteins*
Substances
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Autoantigens
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Carrier Proteins
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DNA, Complementary
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GORASP1 protein, human
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Golgi Matrix Proteins
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Golgin subfamily A member 2
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Gorasp1 protein, rat
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Membrane Proteins
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Recombinant Proteins
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Vesicular Transport Proteins
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vesicular transport factor p115
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beta-Galactosidase
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RAB1B protein, rat
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rab GTP-Binding Proteins
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rab1 GTP-Binding Proteins