Analysis and Preparation of Stable Complexes between Rab GTPases, Rab Escort Protein, and Rab Gerany

Rab proteins are small Ras-like GTPases that regulate vesicular trafficking events in the cell. More than 50 Rabs have been described in mammalian cells (1 ), each with a specific subcellular localization reflecting the functional specificity of Rabs to specific trafficking steps (2 –5 ). Rabs contain two cysteine residues at or near the carboxyl terminus, arranged in a variety of motifs. Both cysteine residues are modified by the attachment of geranylgeranyl groups via thioether bonds, in a reaction catalyzed by Rab geranylgeranyl transferase (also known as GGTase type II, RGGT) (6 ). This enzyme is a tightly bound heterodimer, composed of a 60-kDa α-subunit and a 38-kDa β-subunit, both related to the α- and β-subunits of the other known protein prenyltransferases, farnesyl transferase and caax geranylgeranyl transferase (also known as GGTase type I).