EMBO J. with -COP binding but stimulates the release of slowly sedimenting vesicles comprising Rab2, -COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15C. These data suggest that the Rab2 protein plays a role in the low-temperatureCsensitive step that regulates membrane circulation from VTCs to the Golgi complex and back to the ER. Intro Protein transport through the secretory pathway is definitely mediated Rabbit polyclonal to IQCE by carrier vesicles and tubular elements that selectively deliver cargo between two membrane-bounded compartments. Despite this continuous movement of membrane parts, intracellular organelles preserve their identity. This truth shows that there is a mechanism that ensures temporal and spatial specificity of vesicular traffic. In recent years, a large number of polypeptides have been characterized that participate in this complex trafficking network and include members of the Rab family (Nuoffer and Balch, 1994 ; Pfeffer, 1994 ). The Rab proteins are small, monomeric GTPases that interconvert between a GDP- and GTP-bound form that catalyzes a cycle of membrane association and launch to the cytosol. When these proteins are membrane connected, they show a unique subcellular location that suggests that Rab proteins regulate distinct Garenoxacin Mesylate hydrate transport events. However, the molecular part of Rab proteins in membrane traffic is Garenoxacin Mesylate hydrate not clearly understood. Our desire for characterizing the early events of the secretory pathway offers led us to study the Rab2 protein. Rab2 immunolocalizes to pre-Golgi intermediates that consist of clusters of small vesicles and tubules (termed vesicular-tubular clusters or VTCs) (Chavrier for 10 min at 4C, and the cell pellet was resuspended in 50 mM Tris, pH 7.4, 1 mM dithiothreitol (DTT), 0.1 mM PMSF, 0.1 mM benzamidine, 1 mM EDTA, and 1% Triton X-100 and then homogenized by 20 passes having a Dounce cells grinder. Lysozyme Garenoxacin Mesylate hydrate (400 g/ml), DNase I (40 g/ml), and MgCl2 (25 mM) were added to the homogenate, which was allowed to break down for 30 min at 4C and then centrifuged at 22,000 for 30 min at 4C. The supernatant was applied to a 70-ml Garenoxacin Mesylate hydrate column comprising Q Sepharose Fast Circulation (Pharmacia, Piscataway, NJ) equilibrated with buffer A (50 mM Tris, pH 7.4, 10 mM MgCl2, and 1.0 mM EDTA), washed with two bed quantities of buffer A, and then eluted having a linear NaCl gradient (0C400 mM) in buffer A. Three-milliliter fractions were collected, Garenoxacin Mesylate hydrate and an aliquot of each portion was separated by SDS-PAGE and immunoblotted having a Rab2 polyclonal antibody. Rab2- or Rab2 Q65LCenriched fractions were pooled, concentrated, applied to a 200-ml column comprising Sephacryl S-100 (Pharmacia), and eluted with buffer A. Fractions comprising Rab2 or Rab2 Q65L protein were recognized by SDS-PAGE and immunoblotting and then pooled and concentrated. To assess the purity of the pooled Rab2 and Rab2 Q65L fractions, we subjected an aliquot of each protein to SDS-PAGE and Coomassie blue staining and then scanned the gel on a densitometer (Molecular Dynamics, Sunnyvale, CA). The recombinant proteins were found to be 90% genuine. For use in the assays, the purified proteins were first prenylated in an in vitro reaction. Briefly, the isoprenylation reaction was performed in a total volume of 50 l that contained 0.5 g of recombinant Rab2 or Rab2 mutant, 10 g of geranylgeranyl pyrophosphate (Sigma), 1 mM DTT, 25 l of rat liver cytosol, 10 mM MgCl2, 1 mM ATP, 5 mM creatine phosphate, and 0.2 U of rabbit muscle creatine kinase. The reaction was incubated for 1 h at 37C and then desalted through a 10-ml column of Sephadex G-25 (Pharmacia) to remove incompatible reagents that may inhibit the in vitro assays. Mock prenylation reactions lacking recombinant Rab2 were performed as explained above. The portion comprising prenylated Rab2 and the equivalent portion of the mock prenylation reaction were collected and concentrated, and the protein concentration was determined by Micro BCA Protein Assay Reagent (Axiovert fluorescence microscope.
