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: Several proteins bound with high specificity to Rab18 (Figure 6A). These include subunits of the Dsl1 complex (or NRZ in mammals), which is localized to ER membranes and tethers and then fuses vesicles returning from the Golgi (Civril et al., 2010; Wainman et al., 2012). These subunits (Zw10, Rod, Zwilch, RINT1, and syntaxin-18) were only recovered from the detergent lysate, consistent with syntaxin-18 being a SNARE with a transmembrane domain. The only subunits not detected were two further SNAREs, which are below the ∼45 kDa minimum of this data set. Antisera against two NRZ subunits confirmed the interaction with Rab18 and showed it to be GTP specific (Figure 6B). In S2 cells, red fluorescent protein (RFP)-Rab18 colocalized with ER and early Golgi, and with GFP-tagged ZW10 (Figures S6A and S6B). These results suggest that Rab18 may assist the tethering of COPI-coated vesicles to the ER. In mammalian cells, Rab18 localizes to the ER and Golgi, but when overexpressed, it accumulates on lipid droplets (Ozeki et al., 2005). When we overexpressed Rab18 in mammalian cells, the endogenous NRZ subunits shifted from a diffuse distribution to being clustered around lipid droplets (Figures 6C and S6C). In addition, when human Rab18 was used for affinity chromatography of human cell lysates, subunits of NRZ were among the most abundant proteins showing GTP-specific binding (Figure S6D). The physiological relevance of overexpressed Rab18 being on lipid droplets is unclear, but these data at least indicate that its interaction with NRZ is relevant to mammalian cells and would explain why overexpression of Rab18 induces an association between lipid droplets and ER (Ozeki et al., 2005).
Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.