title: GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.
abstract: We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.
authors.affiliation: Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
authors.forename: E J
authors.initials: EJ
authors.lastname: Tisdale
authors.forename: J R
authors.initials: JR
authors.lastname: Bourne
authors.forename: R
authors.initials: R
authors.lastname: Khosravi-Far
authors.forename: C J
authors.initials: CJ
authors.lastname: Der
authors.forename: W E
authors.initials: WE
authors.lastname: Balch
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014162:Transfection; D014759:Viral Envelope Proteins / Q000378:metabolism*; D020761:rab1 GTP-Binding Proteins; D020693:rab2 GTP-Binding Protein
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; C061569:RAB1B protein, rat; D020761:rab1 GTP-Binding Proteins; D020693:rab2 GTP-Binding Protein