Figure 7. RhoBTB3 is a Rab9-regulated ATPase that binds TIP47.
(A). ATP hydrolysis was determined at the times indicated using 100nM active His-RhoBTB3 full length protein or GST-Rho domain, in the absence or presence of either Rab9 (7.6μM active protein) or Rab5 (8.25μM active protein) and 50μM ATP. Right inset, SDS-PAGE analysis of proteins used. (B). Immunoblot analysis of blue native gel electrophoresis of K562 Golgi/endosome-enriched membranes or K562 cytosol (10μg each). Native protein marker migration is shown at left (kD). Left panel, RhoBTB3; right panel, TIP47. X-link indicates samples that were reacted with DTSSP crosslinker. (C). Rab9 and ATP stimulate release of TIP47 from the RhoBTB3 complex. Top panel, representative immunoblot; below, quantitation of duplicate experiments, with standard deviation.