Rab3GEP Is the Non-redundant Guanine Nucleotide Exchange Factor for Rab27a in Melanocytes

Ana C Figueiredo,Christina Wasmeier,Abul K Tarafder,José S Ramalho,Rudi A Baron,Miguel C Seabra

doi:10.1074/jbc.m804134200

Ana C Figueiredo, Christina Wasmeier + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m804134200

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Abstract

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.

Highlights

Regulation of Rab function is determined by the ability to cycle between a GDP-bound inactive form and a GTP-bound active form [6]
Rab27a controls organelle transport in melanocytes and cytotoxic T lymphocytes [4, 16] as well as regulated exocytosis in secretory cells such as pancreatic ␤ cells [17]
We identify Rab3GEP, previously isolated as a guanine nucleotide exchange factors (GEFs) for Rab3 [14], as the non-redundant GEF required for the activation of Rab27a in melanocytes

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Mouse wild-type melanocytes (melan-ink4a) and Rab27a-deficient ashen melanocytes (melan-ash2) [20] were maintained in RPMI 1640 supplemented with 10% fetal. To generate pFastBacHT-B-Rab3GEP, the 5Ј 4.3-kb coding sequence of rat Rab3GEP was amplified by PCR using flanking primers containing SalI and Psp1406I restriction sites and pCMV-Rab3GEP4.3 Transfection—Melanocytes were seeded into wells or onto glass coverslips at ϳ30% confluency the day before and were transfected with siRNA oligonucleotides and Oligofectamine (Invitrogen) according to the manufacturer’s protocol. HEK-293 cells were transfected with siRNA oligonucleotides as described for melanocytes, followed by transfection with plasmid DNA and FuGENE6 (Roche Applied Science) according to the manufacturer’s instructions. S. frugiperda Virus Production—pFastBacHT-B-Rab3GEP and pFastBacHT-B-GRAB were used to transform DH10Bac Escherichia coli (Invitrogen) for transposition into bacmid according to the manufacturer’s protocol. Beads were washed three times in lysis buffer, resuspended in SDS-PAGE sample buffer, and boiled for 5 min. In cells treated with Mlph-specific siRNA (Fig. 1E)

Melanosome clustering was induced by depleting cells of

DISCUSSION