Structural features of the GTP-binding defective Rab5 mutants required for their inhibitory activity on endocytosis.
Rab5 is a Ras-like small GTPase that regulates early events of endocytosis. Previous work indicates that two GTP-binding defective Rab5 mutants (Rab5:S34N and Rab5:N133I) are dominant inhibitors of endocytosis. In this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two Rab5 mutants. Second-site mutations were introduced into Rab5:S34N and Rab5:N133I, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector. Endocytic activity of the cells was monitored by following the uptake of a fluid-phase endocytic marker (horseradish peroxidase). The effects of the Rab5 mutants on endosome fusion in vitro were also examined. Truncation of the C-terminal isoprenylation motif CCSN abolished the inhibitory activity of both Rab5:S34N and Rab5:N133I. The same held true when the secondary mutation was a substitution mutation (F57S) in the effector domain. Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:N133I. The final mutation (R81A) was created immediately downstream of the second GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on the structural model of Ras. This mutation greatly decreased the isoprenylation of Rab5:N133I and its inhibitory activity on endocytosis. It is believed that Rab5 function requires protein-protein interactions with Rab5-specific regulators and effectors. Some of these interactions are disrupted by Rab5:S34N and Rab5:N133I. By analogy to Ras, both Rab5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor. This interaction requires the effector domain Phe57 residue and C-terminal isoprenylation of Rab5.
Li
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Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Barbieri
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Colombo
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Stahl
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007668:Kidney; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012845:Sindbis Virus; D014162:Transfection; D020696:rab5 GTP-Binding Proteins
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins