Nucleotide exchange factor Rab3GEP requires DENN and non-DENN elements for activation and targeting of Rab27a.

Paolo Sanzà,Richard D Evans,Alistair N Hume,Ana C Figueiredo,Lluis Montoliu,Marta Cantero,Shyamal Patel,Aymelt Itzen,Miguel C Seabra,Elena V Sviderskaya,Deborah A Briggs

doi:10.1242/jcs.212035

Abstract

ABSTRACTRab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3GKO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3GKO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.

Highlights

Rab proteins are a family (>60 in humans) of small GTPases that regulate vesicle trafficking in eukaryotic cells (Stenmark, 2009; Hutagalung and Novick, 2011)
Based on this we suggest that Rab3GEP differs from other differentially expressed in normal and neoplastic cells (DENN)-containing RabGEFs in the mechanism by which it activates Rabs, and that other factors work alongside Rab3GEP to activate and target Rab27a
Melanosome dispersion is partially disrupted in melan-R3GKO melanocytes To investigate Rab3GEP function in Rab27a-dependent organelle transport we generated Rab3GEP-deficient immortal melanocyte lines, melan-R3GKO1, melan-R3GKO2 and melan-R3GKO3 (Lavado et al, 2005; Tanaka et al, 2001)

Summary

Introduction

Rab proteins are a family (>60 in humans) of small GTPases that regulate vesicle trafficking in eukaryotic cells (Stenmark, 2009; Hutagalung and Novick, 2011). Rab3GEP promotes melanosome dispersion by acting as a Rab27a GEF and melanosome targeting factor (Figueiredo et al, 2008; Tarafder et al, 2011).

Results

Conclusion