BicD interacts with Rab6 but not Rab7.

Source was adult female ovary of transgenic fly line; bait produced from tagged transgenic construct; prey produced from endogenous gene.

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from embryo cDNA expression library).

62 distinct clones of BicD recovered as Rab6-binding partners.

Two-hybrid system: yeast LexA-BD/GAL4-AD

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type ovary extract.

BicD binds to Rab6 but not Rab1.

Preloading Rab6 with the non-hydrolyzable GTP analog GTP-γ-S improved binding to BicD.

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

Two-hybrid system: yeast GAL4-BD/GAL4-AD

Proteins were purified, mixed and complex size was resolved by size exclusion chromatography.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

BicD and Rab6 form a heterotetramer with 2:2 stoichiometry.

Interaction was tested for purified recombinant proteins.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

BicD and Rab6 form a heterotetramer with 2:2 stoichiometry.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

Purified recombinant proteins were cross-linked, trypsin digested and peptides were analyzed by mass spectrometry.

A BicD-Rab6 cross-linked peptide was recovered with linkage between BicD K730 and Rab6 K12 residues.

Purified recombinant proteins were tested for interaction.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey produced as a recombinant fusion protein in bacterial system.

BicD binds to GTP-bound Rab6, not GDP-bound Rab6.

There is overlap of egl and Rab6 binding sites in BicD.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from S2 cell extract (endogenous gene).

Specificity S-score=11.86.

Constitutively active (GTP-locked) form of Rab was used for pull down. Parallel pull downs performed for several Rabs proteins in this study. Yield was measured using spectral counts. A specificity score (S score) was calculated, where greater weight was given to interactions seen with fewer baits.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from S2 cell detergent-free extract (endogenous gene).

Specificity S-score=25.83.

Constitutively active (GTP-locked) form of Rab was used for pull down. Parallel pull downs performed for several Rabs proteins in this study. Yield was measured using spectral counts. A specificity score (S score) was calculated, where greater weight was given to interactions seen with fewer baits.