Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey produced and labeled by in vitro translation.

The Rab5-Rbsn-5 interaction is stronger in the presence of GTP than GDP.

Bait expressed and purified from bacteria.

Prey expressed and purified from bacteria.

Rbsn-5 binds the GTP-bound form of Rab5, but not GDP- or GTP-bound forms of Rab2, Rab4 or Rab11.

GST pull down.

Rab proteins were loaded with either GDP or the non-hydrolyzable GTP analog GTPγS

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey produced and labeled by in vitro translation.

Rbsn-5 binds Rab5 but not Rab4, Rab7 or Rab11. This specificity is conserved in human orthologs, and the fly and human orthologs appear to be interchangeable in these binding assays. No direct interaction was observed between Rbsn-5 and GST-stan.

Binding was tested in the presence of GDP or a non-hydrolyzable GTP analog (GTPγS).

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

Specificity S-score=13.78.

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=28.77.

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.