Figure 2.

Endosomal Rab GTPases directly initiate membrane tethering by themselves in a chemically defined reconstitution system. A, schematic representation of liposome turbidity assays for testing Rab-mediated liposome tethering (shown in B–G). B–G, endosomal Rab-His₁₂ proteins (each at 0.5–4 μm), Rab4a-His₁₂ (B), Rab5a-His₁₂ (C), Rab7a-His₁₂ (D), Rab9a-His₁₂ (E), Rab11a-His₁₂ (F), and Rab14-His₁₂ (G), were incubated with synthetic liposomes bearing physiological mimic lipid composition (400 nm diameter, 0.5 mm lipids) in RB150 containing MgCl₂ (5 mm) and DTT (1 mm) at room temperature for 300 s. During incubation, turbidity changes in the Rab-liposome–mixed reactions were monitored by measuring the absorbance at 400 nm. The protein-to-lipid molar ratios used for these turbidity reactions were from 1:1000 to 1:125 as indicated.