Identification of a putative effector protein for rab11 that participates in transferrin recycling

Abstract

We have identified and cloned the cDNA for a 912-aa protein, rab11BP, that interacts with the GTP-containing active form of rab11, a GTP-binding protein that plays a critical role in receptor recycling. Although rab11BP is primarily cytosolic, a significant fraction colocalizes with rab11 in endosomal membranes of both the sorting and recycling subcompartments. In vitro binding of rab11 to native rab11BP requires partial denaturation of the latter to expose an internal binding site located between residues 334 and 504 that is apparently masked by the C-terminal portion of the protein, which includes six repeats known as WD40 domains. Within the cell, rab11BP must undergo a conformational change in which the rab11-binding site becomes exposed, because when coexpressed with rab11 in transfected cells the two proteins formed abundant complexes in association with membranes. Furthermore, although overexpression of rab11BP did not affect transferrin recycling, overexpression of a truncated form of the protein, rab11BP(1-504), that includes the rab11-binding site but lacks the WD40 domains inhibited recycling as strongly as does a dominant negative 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 on recycling. We propose that rab11BP is an effector for rab11, whose association with this GTP-binding protein is dependent on the action of another membrane-associated factor that promotes the unmasking of the rab11-binding site in rab11BP.

Figure 1

Identification and cDNA cloning of a 130-kDa protein (rab11BP) that specifically interacts with rab11-GTP in a filter-overlay assay. (A) Lanes a–c, presence of rab11BP in MDCK (a), 293T (b), and 3T3 (c) cells. Cell lysates (100 μg of protein) were analyzed by the filter-overlay assay using [α-³²P]GTP-rab11 as a probe. Lanes d–f, though primarily cytosolic (f), a fraction of rab11BP is membrane-associated (e). A total MDCK cell lysate (TH, lane d) and aliquots of sedimentable (P, lane e) and soluble subfractions (S, lane f) were analyzed by the blotting assay for the presence of rab11BP. Lanes g and h, only the active GTP-containing form of rab11 binds to rab11BP. Filter-overlay assays with MDCK cell lysates and [α-³²P]GTP-rab11 as probe were carried out in the presence of a 30-fold excess of rab11 charged with nonradioactive GTP (g) or GDP (h). (B) Specificity of the rab11–rab11BP interaction. When used as probes charged with [α-³²P]GTP, rab 3a (b), rab5 (c), and a rab11 effector domain mutant (rab11T43A) (d) fail to bind to rab11BP. Aliquots (100 μg protein) of a rat brain cytosolic subfraction obtained by ammonium sulfate precipitation (25–45% saturation) were analyzed by the blotting assay with the indicated [α-³²P]GTP-charged probes. Lanes e–h, when used as competitors in the blotting assay with the brain cytosolic subfraction, rab8 and the dominant negative rab11 mutant (rab11S25N) do not diminish binding of [α-³²P]GTP-rab11 to rab11BP. The various competitors were incubated for charging with cold GTP and used in the blotting assay at a 30-fold excess over the concentration of the labeled [³²P]GTP-rab11 probe. (C) The cloned cDNA encodes a protein that comigrates with rab11BP and binds rab11-GTP. Lane a, an immunoprecipitate obtained from a partially purified brain cytosolic extract with an antibody to a peptide (residues 88–103) within the amino acid sequence derived from the cloned cDNA was used for the filter-overlay assay with [α-³²P]GTP-rab11 as a probe. The protein in the immunoprecipitate that bound the probe (a) had the same electrophoretic mobility as rab11BP (d) in the original bovine brain protein fraction. Lanes b and c, aliquots of lysates of 293T cells transfected with the pcDNA vector alone (b) or the vector containing the cloned cDNA (c) were analyzed by the filter-overlay assay. The amount of extract analyzed in lane b, to reveal the endogenous human rab11BP, was 30 times greater than that from the transfected cells used in lane c.

Figure 2

Structure of Rab11BP. (A) Amino acid sequence of bovine rab11BP derived from the cDNA. The sequences of peptides originally identified by microsequencing in the purified protein are underlined. A proline-rich region extends from residues 210 to 256. The protein has a calculated molecular mass of 101,290 Da and a pI of 5.18. The nucleotide sequence that encodes the bovine rab11BP has been submitted to GenBank with accession no. AF117897. (B) Rab11BP contains six WD40 repeats in the region extending from residues 508 to 813. (a) The six repeats, whose positions are indicated by the numbers within the parentheses at the right of each sequence, are shown in an alignment to maximize their agreement (shaded residues) with the loose consensus sequence for WD40 repeats defined by Neer et al. (30), which is presented in b. (C) Schematic representation of the structure of rab11BP. In addition to the WD40 repeats and the proline-rich region, the figure indicates the location of the rab11-binding domain, as defined by the experiments in Fig. 3.

Figure 3

The rab11-binding region in rab11BP is located between residues 334 and 504. (A) HA-tagged intact rab11BP (lane a, 1–912) or the specific HA-tagged deleted forms of the protein indicated above each lane (b–d) were expressed in transfected 293T cells, and the cell lysates were analyzed by immunoblotting with the anti-HA antibody (Left, a–d) or, for [³²P]GTP-rab11 binding, by the overlay assay (Right, a′–d′). The white asterisks indicate degradation products of rab11BP(1–912). (B) Purified fusion proteins containing the Escherichia coli maltose-binding protein linked to rab11BP segments extending from residues 334 to 504 (a and a′) or from 504 to 912 (b and b′) were fractionated by SDS gel electrophoresis and transferred to nitrocellulose. The Poinceau red-stained protein pattern (Left) and [³²P]GTP-rab11 binding pattern (Right) are shown.

Figure 4

Colocalization of rab11 and rab11BP in subcellular fractions enriched in endosomes. A rat liver homogenate was fractionated (20) to obtain three sedimentable fractions (P1–2, P3, P4) enriched in nuclei and mitochondria (P1–2), smaller particulate components of the cytoplasm (P3 and P4), and a final supernatant (S). The sedimentable fractions were subfractionated by flotation in discontinuous sucrose gradients to obtain from each four subfractions (A, B, C, and D) and a pellet. Aliquots (100 μg protein) of each fraction and subfraction were analyzed by Western blotting with antibodies specific for rab11 or rab11BP, or for the marker proteins indicated in each chart. Bound antibodies were detected with ¹²⁵I-labeled protein A and quantified by PhosphorImager analysis. The bars represent the specific activities for each protein in the various fractions and subfractions indicated on the abscissa at the bottom of the figure.

Figure 5

Immunofluorescence colocalization of rab11 and membrane-associated rab11BP in endosomes and the pericentriolar recycling compartment. Permanent transformants of Chinese hamster ovary cells that express the human transferrin receptor (TRVb-1 cells) were transfected with a plasmid-encoding HA-tagged rab11BP. The cells grown on coverslips were permeabilized with 0.05% saponin in PBS for 4 min to release soluble cytosolic components and processed for double-label immunofluorescence with primary mouse mAbs to the HA epitope (A) and rabbit antibodies to rab11 (B), followed by Texas red-conjugated donkey anti-mouse antibody and fluorescein-conjugated donkey anti-rabbit antibody, respectively. The arrows and arrowheads point to the pericentriolar compartment and to peripheral-sorting endosomes in a transfected cell, respectively. Note that a trio of untransfected cells in B shows intense staining of the pericentriolar compartment with anti-rab11 antibodies.

Figure 6

In vivo association of rab11 and rab11BP in cotransfected cells. Cultures of 293T cells were transfected with (a) a pcDNA3-based expression plasmid encoding HA-Rab11BP alone or together with plasmids encoding (b) WT Rab11, (c) Rab11Q70L (GTPase-deficient mutant), (d) Rab11T43A (effector domain mutant), (e) Rab11S25N (GDP-bound mutant), or (f) Rab11ΔC (a deletion mutant that lacks the last 6 amino acids and therefore cannot be prenylated). Forty hours posttransfection, complexes containing wild-type or rab11 mutant proteins were immunoprecipitated from detergent-solubilized cell lysates using immobilized polyclonal antibody to rab11. (A) Recoveries of HA-rab11BP in the immunoprecipitates analyzed by immunoblotting with a monoclonal anti-HA antibody. (B) Recoveries of rab11 or its mutants in the same immunoprecipitates analyzed with rabbit anti-rab11 antibodies. (C) Comparable levels of HA-Rab11BP are expressed in all the transfected cells. Equal aliquots of the cell lysates (2% of the total) were analyzed by immunoblotting with the anti-HA antibody.

Figure 7

Inhibition of transferrin recycling by truncated rab11BP(1–504) and its relief by the nonprenylated rab11ΔC. Rates of transferrin recycling were assessed as described previously (16) in cultures of TRVb cells, a mutant line of Chinese hamster ovary cells lacking a functional endogenous transferrin receptor, that were cotransfected with a plasmid encoding the human transferrin receptor (A–D) and plasmids encoding the intact rab11BP (A), or its truncated variants, rab11BP (1–336) (B) and rab11BP (1–504) (C and D). A also shows the recycling kinetics (▵) for cells that express the receptor together with the rab11 dominant negative mutant (rab11S25N). In D, the cells express—in addition to the transferrin receptor and the truncated rab11BP(1–504)—the nonprenylatable rab11ΔC. In A–D, the recycling kinetics also are shown (○) for control cells that were cotransfected with the plasmid encoding the transferrin receptor and the pCDNA3 vector lacking an insert.