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
Related in: MedlinePlus
Mentions: Drosophila melanogaster has 27 Rabs, of which 23 have at least one mammalian ortholog, with these mammalian orthologs representing 50 of the 66 human Rabs (Figures S1A and S1B available online). All 23 Drosophila Rabs that have a mammalian ortholog were expressed as fusions to glutathione S-transferase (GST) with the Q→L mutation that is known in several Rabs to stabilize the GTP-bound form (Hyvola et al., 2006; Li and Stahl, 1993). They were coupled to glutathione Sepharose for affinity chromatography of lysates of Drosophila S2 cells, a widely used cell line thought to be from a macrophage-like lineage (Figure 1A). S2 cells were lysed in the detergent CHAPS, and the clarified lysate was applied to each GST-Rab column. After washing, proteins were eluted and separated on SDS gels. The gel lanes above the GST-Rab (∼45–50 kDa) were cut into sections and digested with trypsin, and peptides were sequenced by tandem mass spectrometry (Table S1).
Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.