Characterization of the molecular defects in Rab27a, caused by RAB27A missense mutations found in patients with Griscelli syndrome.
Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.
Bahadoran
Philippe
P
INSERM U385, Biologie et Physiopathologie de la Peau, Faculté de Médecine, Avenue de Valombrose, 06107, Nice cedex 2, France.
Busca
Roser
R
Chiaverini
Christine
C
Westbroek
Wendy
W
Lambert
Jo
J
Bille
Karine
K
Valony
Gäelle
G
Fukuda
Mitsunori
M
Naeyaert
Jean-Marie
JM
Ortonne
Jean-Paul
JP
Ballotti
Robert
R
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000235:genetics*; D051379:Mice; D020125:Mutation, Missense; D018995:Myosin Heavy Chains / Q000378:metabolism; D024701:Myosin Type V / Q000378:metabolism; D009422:Nervous System Diseases / Q000235:genetics*; D011485:Protein Binding; D013577:Syndrome; D014407:Tumor Cells, Cultured; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C492410:MLPH protein, human; C435724:Mlph protein, mouse; C513082:Myo5a protein, mouse; D000075745:rab27 GTP-Binding Proteins; C077511:MYO5A protein, human; D006160:Guanosine Triphosphate; D024701:Myosin Type V; C066694:RAB27A protein, human; C512962:Rab27a protein, mouse; D018995:Myosin Heavy Chains; D020691:rab GTP-Binding Proteins