Comprehensive Screening for Novel Rab-Binding Proteins by GST Pull-Down Assay Using 60 Different Mammalian Rabs‡

Large scale screening for novel rab effectors reveals unexpected broad Rab binding specificity.

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this s...

SnapShot: Mammalian Rab Proteins in Endocytic Trafficking

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin-binding domain-tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled ve...

TBC proteins: GAPs for mammalian small GTPase Rab?

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to funct...

In vitro pull-down assay without expression constructs.

The in vitro pull-down assay is a well-established method to confirm direct binding in protein-protein interactions that was inferred from other interaction assays, such as two-hybrid analysis (1). The assay is usually carried out using glutathione-S-transferasetagged or His-tagged fusion proteins as the pull-down drivers and in vitro-translated 35S-labeled proteins as probes to detect interact...