For each Rab column, 5E8 S2 cells were harvested by centrifugation for lysis.
Each of the 23 D. melanogaster Rabs that have a mammalian ortholog were expressed as recombinant GST fusions (in E. coli) bearing the Q>L mutation that is known to stabilize the GTP-bound form (constitutively active). GST-Rab fusions were coupled to glutathione sepharose and incubated with clarified S2 cell lysates (10-20mg/ml protein extract) that were prepared in the absence of any detergent, in the presence of non-hydrolysable GTP analogue GppNHp. Columns were washed and bound proteins were eluted using high salt and GDP. Proteins were concentrated by chloroform/methanol protein precipitation, separated by SDS-PAGE, and trypsin digested for subsequent identification by mass spectrometry.
Purified proteins were identified by liquid chromatography-mass spectrometry. Peptide searches were against FlyBase Dmel 5.9.
Peptide searches were against FlyBase Dmel 5.9, and protein identifications were accepted if they could be established at >95.0% probablility and contained at least two identified peptides. The predicted false discovery rate (FDR) was less than 1%. Spectral counts were used to estimate protein abundance. An "S-score" was calculated for each interaction, which gives greater weight to interactions seen with fewer baits.
Lysis of cells in the absence of detergent prevented solubilization of common contaminants for a cleaner dataset, but some known Rab-binding proteins were not recovered. The use of detergent during S2 cell lysis in the Rab_Effectors_1 dataset solubilized more proteins, which allowed for the recovery of more known Rab interacting proteins, but also increased the number of non-specifing binding proteins isolated across the parallel affinity purifications. Poor results (due to low expression or few specific effectors) were obtained for Rab3, Rab8, Rab27, Rab32 and Rab40.
