Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface.
Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome. Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways. This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP). REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear. We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs. Here, we tested this hypothesis. We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs. We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay. We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions. These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP.
Pereira-Leal
José B
JB
Cell and Molecular Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, UK.
Strom
Molly
M
Godfrey
Richard F
RF
Seabra
Miguel C
MC
D048868:Adaptor Proteins, Signal Transducing; D019883:Alkyl and Aryl Transferases; D020816:Amino Acid Motifs; D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C081787:CHM protein, human; D011993:Recombinant Fusion Proteins; D019883:Alkyl and Aryl Transferases; D020691:rab GTP-Binding Proteins