Purification and Identification of Novel Rab Effectors Using Affinity Chromatography

• DOI: 10.1006/meth.2000.0953
• Publication Date: 2002-09-18T19:46:30Z
• Authors: Savvas Christofor...; Marino Zerial
• Keywords: Brain Chemistry; 0303 health sciences; rab5 GTP-Binding Proteins/*metabolism; Membrane Proteins/isolation & purification; Vesicular Transport Proteins; Membrane Proteins; Nerve Tissue Proteins; Nerve Tissue Proteins/*isolation & purification; Ligands; Chromatography, Affinity; 03 medical and health sciences; Chromatography, Affinity/*methods; Guanine Nucleotide Exchange Factors/isolation & purification; Animals; Guanine Nucleotide Exchange Factors; Cattle; *Vesicular Transport Proteins; Protein Binding; rab5 GTP-Binding Proteins

Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effectors is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effectors of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effectors 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-dependent biological functions.