Multiple factors contribute to inefficient prenylation of Rab27a in Rab prenylation diseases.
Post-translational geranylgeranylation of Rab GT-Pases is essential for their membrane association and function as regulators of intracellular vesicular transport. The reaction is catalyzed by Rab geranylgeranyltransferase (RGGT) and is assisted by the Rab escort proteins (REP), which form stable complexes with newly synthesized GDP-bound Rabs. Two genetic diseases involve the Rab geranylgeranylation machinery: choroideremia, an X-linked retinal degeneration resulting from loss-of-function mutations in REP1, and gunmetal, a mouse model of Hermansky-Pudlak syndrome resulting from mutations in the alpha-subunit of RGGT. A small subset of Rab proteins is selectively under-prenylated in both diseases, most notably Rab27a. Here we analyze why Rab27a is selectively affected in diseases of Rab geranylgeranylation. Semi-quantitative immunoblotting suggests that mass action, i.e. the amount of Rab27a relative to other Rabs, is unlikely to be a factor as the expression level of Rab27a is similar to other Rabs not affected in these diseases. In vitro binding assays and fluorescence resonance energy transfer detected by fluorescence lifetime imaging microscopy in intact cells demonstrate that Rab27a binds equally well to both REP1 and REP2, suggesting differential affinity of Rab27a for REP isoforms is not an important factor. However, steady-state kinetic analysis of the geranylgeranylation reaction indicates that REP2-Rab27a has lower affinity for RGGT compared with REP1-Rab27a. Furthermore, we show that Rab27a has relatively low GTPase activity, presumably decreasing the affinity of the REP interaction in vivo. We suggest that the restricted phenotypes observed in these diseases result from multiple contributing factors.
Larijani
Banafshé
B
Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London SW7 2AZ, U.K.
Hume
Alistair N
AN
Tarafder
Abul K
AK
Seabra
Miguel C
MC
D048868:Adaptor Proteins, Signal Transducing; D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D015794:Choroideremia / Q000209:etiology / Q000378:metabolism; D004285:Dogs; D015785:Eye Diseases, Hereditary / Q000209:etiology* / Q000378:metabolism; D022861:Hermanski-Pudlak Syndrome / Q000209:etiology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017368:Protein Prenylation / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; C081787:CHM protein, human; C085460:CHML protein, human; D002352:Carrier Proteins; C513166:Chm protein, rat; C486217:Chml protein, mouse; D011993:Recombinant Fusion Proteins; D000075745:rab27 GTP-Binding Proteins; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; C066694:RAB27A protein, human; C512962:Rab27a protein, mouse; C512961:Rab27a protein, rat; D020691:rab GTP-Binding Proteins