An aliquot (150 g) of the pooled final fractions was fractionated by SDS/PAGE (10%), and, after transfer to nitrocellulose, the major band at 130 kDa, which was positive in the filter-overlay assay, was excised for proteolytic fragmentation and peptide microsequencing (22, 23). conformational switch in which the rab11-binding site becomes exposed, because when coexpressed with rab11 in transfected cells the two proteins created abundant complexes in association with membranes. Furthermore, although overexpression of rab11BP did not impact transferrin recycling, overexpression of a truncated form of the protein, rab11BP(1C504), that includes the rab11-binding site but lacks the WD40 domains inhibited recycling as strongly as does a dominant unfavorable rab11 mutant protein that does not bind GTP. Strikingly, the inhibition caused by the truncated rab11BP was prevented completely when the cells also expressed a C-terminally deleted, nonprenylatable form of rab11 that, by itself, has no effect Icariin on recycling. We propose that rab11BP is an effector for rab11, whose association with this GTP-binding protein is dependent around the action of another membrane-associated factor that promotes the unmasking of the rab11-binding site in rab11BP. Small GTP-binding proteins of the rab family have characteristic subcellular distributions, and specific rab proteins have been implicated in the control of vesicular transport actions between different subcellular compartments (observe ref. 1). The precise mechanism of action of rab proteins has not yet been elucidated, although there is usually Tap1 evidence that this active GTP-containing form of some rab proteins serves to stabilize SNARE complexes required for vesicle fusion (2C4). Several proteins have been recognized (5C12), in most cases by using a yeast two-hybrid interaction screen, that are capable of binding to the active forms of rab proteins and may serve as their effectors in mediating specific transport steps. In general, these proteins do not show sequence similarity to each other and, in fact, may have diverse functional roles ranging from stabilizing the active form of the rab (9, 13, 14) to mediating interactions with the cytoskeleton (7). At least five rab proteins (rabs 4, 5, 7, 9, and 11) have been shown to participate in endocytosis and/or transport of endocytosed material or receptors between endosomal compartments or between endosomes and the plasma membrane or the trans-Golgi network (TGN; observe ref. 1). Rab11, in particular, has been shown to play a key role in plasma membrane receptor recycling, because expression of a form of this protein that cannot be activated by GTP binding inhibited transport of endocytosed transferrin receptors from sorting endosomes to a pericentriolar recycling compartment, from where receptors normally are returned to the Icariin cell surface (15, 16). In addition to its presence in sorting endosomes and the recycling compartment, rab11 has been localized to TGN membranes and post-Golgi secretory vesicles (17, 18) and is highly concentrated in the apical tubulovesicular system of parietal cells in the gastric epithelium (19). To further our understanding of the mechanism of action of rab11, we sought to identify proteins that interact with its active GTP-binding form and, therefore, may serve as the downstream effectors that execute its function. Using a blotting assay, we have recognized a rab11-binding protein, rab11BP, that interacts with the GTP form of rab11 only if the latter contains an intact effector domain name. Moreover, we present evidence that rab11BP, together with rab11, participates in the regulation of transferrin receptor recycling. MATERIALS AND METHODS DNA Cloning and Construction of Expression Plasmids Encoding Wild-Type or Mutant rab11 or rab11BP. Wild-type and mutant human rab11 cDNAs (16) were cloned into the bacterial expression vector pET-11d (Novagen), and the bacterially expressed rab11 proteins were purified from lysates by ammonium sulfate precipitation, followed by fast protein liquid chromatography on a Superdex HR-75 gel-filtration matrix (Pharmacia). The full-length rab11BP cDNA coding region and various subfragments of it were cloned into pcDNA3 (Invitrogen), downstream of a hemagglutinin epitope tag, or into pMAL-C2 (New England Biolabs) downstream of sequences encoding the maltose-binding protein moiety, used to purify the hybrid proteins made in bacteria on an amylose-containing resin. Transfection of cultured cells was carried out as explained (16). Cell Fractionation. Confluent monolayers of cultured cells (MDCK, 293T, Icariin or 3T3) were harvested by scraping in a buffer made up of 25 mM Hepes/150 mM NaCl/5 mM MgCl2/3 mM DTT/1 g/ml leupeptin/5 g/ml Trasylol. Cells were broken by sonication, and sedimentable membrane (P100) and cytosolic supernatant fractions (S100) were obtained by centrifugation (60 min at 100,000 (20), and the distribution of various markers was determined by immunoblotting. Filter-Blotting Assay and Purification of rab11BP. Gel-electrophoresis/filter-binding overlay assays, including the competition experiments with rab proteins charged with nonradioactive nucleotides, were performed essentially as explained previously (21). Rab11BP was purified from a high-speed supernatant (40,000 rpm, 2 hr in a Beckman Ti60 rotor) prepared from a bovine brain homogenate (1.5 kg in 2 vol of 50 mM Mes, pH 7.0/0.5 mM MgCl2/3 mM NaN3/1 mM EGTA/2 mM EDTA/1.