Toward a comprehensive map of the effectors of rab GTPases.
Bottom Line: However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear.For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors.In addition, we found numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function.
Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.Show MeSH
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Mentions: Rab4 is widely conserved in evolution but has been lost in various lineages including budding yeasts. It is localized to endosomes and is proposed to play a role in recycling back to the surface. The best characterized Rab4 effector is RUFY1 (or Rabip4), one of three closely related paralogs (Cormont et al., 2001). CG31064, the Drosophila ortholog of these proteins, showed a strong and highly specific interaction with Rab4 under both lysis conditions (which we confirmed to be GTP-specific using a GFP-tagged CG31064), indicating that the Rab4-GTP was functional (Figures 4A and 4B). In addition to CG31064, there were several other proteins that were specific to Rab4 that have not previously been reported to be effectors. Most striking were Vps51, Vps52, and Vps53, three subunits of the GARP tethering complex that is found on the Golgi and involved in retrograde traffic from endosomes (Bonifacino and Hierro, 2011). These were found with or without detergent, and we were able to confirm that GARP subunit binding is GTP specific (Figure 4B). We also examined a GARP subunit (Vps53) in mammalian cells and found that it could be relocated to endosomes by overexpression of Rab4 (Figure 4C).
Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.