Figure 1.

Purification of human Rab GTPases used in this reconstitution study. A, schematic representation of the endosomal Rabs (Rab4a, Rab5a, Rab7a, Rab9a, Rab11a, and Rab14), non-endosomal Rabs (Rab1a and Rab3a), and HRas GTPase in humans showing their amino acid residues, domains (Ras-superfamily GTPase domains and hypervariable regions), and intracellular locations, which include early endosome (EE), recycling endosome (RE), plasma membrane (PM), late endosome (LE), lysosome (Ly), ER, Golgi, and secretory vesicle (SV). B, Coomassie Blue-stained gels of purified recombinant proteins of the C-terminally His₁₂-tagged endosomal Rab, non-endosomal Rab, and HRas GTPases and the untagged form of Rab11a used in this study. C, intrinsic GTP hydrolysis activities of purified Rab proteins. Purified Rab-His₁₂, HRas-His₁₂, and untagged Rab11a proteins (2 μm) were incubated at 30 °C for 1 h in RB150 containing MgCl₂ (6 mm), DTT (1 mm), and GTP (1 mm) or GTPγS (1 mm) for the control followed by an assay of the released free phosphate molecules using a malachite green-based reagent.