Purification and identification of novel Rab effectors using affinity chromatography

Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as we ll as organelle dynamics Rab proteins interact with effector molecules in t he GTP-bound active state. The identification of Rab effecters is, therefor e, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to b iochemically purify and identify effecters of the small GTPase Rab5. The me thod, which is based on an affinity chromatography procedure, results in th e large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional cha racterization. In the case of Rab5, the procedure allows a comprehensive an alysis of the downstream effecters and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-de pendent biological functions. (C) 2000 Academic Press.