The affinity of TIP47 for Rab9 bound to GDP was lower ( em K /em d = 160 nM; open circles)

The affinity of TIP47 for Rab9 bound to GDP was lower ( em K /em d = 160 nM; open circles). ; Barbero as described previously (Diaz and Pfeffer, 1998 ). Recombinant Rab9CLLL was purified as described previously (Shapiro at 4C. Equal amounts of the clarified cell lysates (30 g/lane) were resolved by SDS-PAGE and immunoblotted for EGFR and Rab9 or TIP47. EGFR was quantified using ImageJ software and plotted against time to obtain the rate of receptor down-regulation. TIP47/Rab9 Binding Studies Rab9 protein (100 nM) was allowed to bind radiolabeled GDP or GTPS to equilibrium for 4 h at 37C in the presence of TIP47 protein, added at the concentrations indicated (0C800 nM; Shapiro em et al /em ., 1993 ). The percent of Rab9 protein that can actively exchange nucleotide is increased upon the addition of recombinant TIP47 protein (Hanna em et al /em ., 2002 ). The stabilizing effect that TIP47 has for Rab9 nucleotide exchange Ivacaftor benzenesulfonate is specific to TIP47 and is not seen with control proteins such as BSA or GFP (Hanna em et al Ivacaftor benzenesulfonate /em ., 2002 ). Thus, increasing Ivacaftor benzenesulfonate TIP47 concentrations in the nucleotide exchange assay are reflected by increasing amounts of radiolabeled nucleotide binding to Rab9. For ease of comparison, the data for the Rab9 binding to GDP and guanosine 5- em O /em -(3-thio)triphosphate (GTPS) have been normalized and are Rabbit Polyclonal to CEACAM21 plotted as fraction Rab9 bound to TIP47. Both nucleotide binding and TIP47 stabilization are complete at 4 h (Hanna em et al /em ., 2002 ). Crude Membrane Fractionation Control and TIP47 siRNA-transfected cells grown in 35-mm dishes were washed three times with PBS and once with 10 mM HEPES, pH 7.4, followed by a 15-min incubation at 4C in 10 mM HEPES, pH 7.4, supplemented with protease inhibitors. Cells were harvested by scraping in homogenization buffer (20 mM HEPES, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol plus protease inhibitors) and were homogenized with five passes through a 22-gauge needle. A postnuclear supernatant (PNS) was obtained by centrifugation of the homogenate at 3000 rpm at 4C for 5 min. The PNS was further centrifuged at 98,000 rpm for 15 min at 4C, and the supernatant (cytosolic fraction) was removed and the pellet (membrane fraction) was resuspended in a volume of homogenization buffer equal to that of the supernatant. Equal volumes of cytosol and membrane fractions were then subject to SDS-PAGE and immunblotting to determine the concentrations of membrane-bound and cytosolic TIP47 and Rab9. RESULTS We have shown previously that cells expressing a GDP-preferring Rab9 mutant protein (Rab9 S21N) display defects in MPR transport from endosomes to the Golgi complex (Riederer em et al /em ., 1994 ). To investigate further the importance of Rab9 for the function and morphology of late endosomes, we used siRNA to deplete Rab9 from Ivacaftor benzenesulfonate cultured cells. Immunoblot analysis showed that Rab9 protein was decreased 90% upon siRNA treatment (Figure 1A). The depletion seemed to be specific for Rab9 as the steady-state level of the late endosomal Rab7 protein was unchanged (Figure 1A). Loss of Rab9 did not alter the steady-state level of TIP47. This may not be surprising because the majority of TIP47 is cytosolic and that pool of TIP47 does not contain bound Rab9 (Diaz and Pfeffer, 1998 ). Similarly, the level of the late endosome/lysosomal marker LAMP1 increased only slightly upon Rab9 depletion (58%, Figure 1A; see below). However, loss of Rab9 led to a more significant increase in CI-MPR protein levels (Figure 1A). Open in a separate window Figure Ivacaftor benzenesulfonate 1. Rab9 depletion destabilizes CI-MPRs and induces their expression. (A) Immunoblot analysis of HeLa cell lysates (equal protein amounts) treated for 72 h with luciferase control siRNA (-) or Rab9 siRNA (+) by using the indicated antibodies. (B) HeLa cells, transfected with Rab9 or control siRNA, were pulse labeled with [35S]methionine/cysteine and chased.