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Mol Biol Cell. 2011 May 15; 22(10): 1699–1715.
PMCID: PMC3093322
A Highlights from MBoC Selection

Clathrin-dependent mechanisms modulate the subcellular distribution of class C Vps/HOPS tether subunits in polarized and nonpolarized cells

Keith E. Mostov, Monitoring Editor
University of California, San Francisco

Abstract

Coats define the composition of carriers budding from organelles. In addition, coats interact with membrane tethers required for vesicular fusion. The yeast AP-3 (Adaptor Protein Complex 3) coat and the class C Vps/HOPS (HOmotypic fusion and Protein Sorting) tether follow this model as their interaction occurs at the carrier fusion step. Here we show that mammalian Vps class C/HOPS subunits and clathrin interact and that acute perturbation of clathrin function disrupts the endosomal distribution of Vps class C/HOPS tethers in HEK293T and polarized neuronal cells. Vps class C/HOPS subunits and clathrin exist in complex with either AP-3 or hepatocyte growth factor receptor substrate (Hrs). Moreover, Vps class C/HOPS proteins cofractionate with clathrin-coated vesicles, which are devoid of Hrs. Expression of FK506 binding protein (FKBP)–clathrin light chain chimeras, to inhibit clathrin membrane association dynamics, increased Vps class C/HOPS subunit content in rab5 endosomal compartments. Additionally, Vps class C/HOPS subunits were concentrated at tips of neuronal processes, and their delivery was impaired by expression of FKBP–clathrin chimeras and AP20187 incubation. These data support a model in which Vps class C/HOPS subunits incorporate into clathrin-coated endosomal domains and carriers in mammalian cells. We propose that vesicular (AP-3) and nonvesicular (Hrs) clathrin mechanisms segregate class C Vps/HOPS tethers to organelles and domains of mammalian cells bearing complex architectures.

INTRODUCTION

Membranous organelles maintain their steady-state structural and functional identity despite the continuous flow of protein and lipids through them. Flow is dictated by departing carriers budding off organelles and incoming vesicles fusing with their targets. Thus fidelity in vesicle budding and fusion is central to generating and maintaining organelle identity. Coats and adaptors specify vesicle­budding events by selecting cargoes loaded into vesicle carriers. In contrast, Soluble NSF Attachment Protein REceptors (SNAREs), rabs, and tethers determine vesicle fusion fidelity (Bonifacino and Glick, 2004 blue right-pointing triangle). Vesicular and nonvesicular mechanisms regulate flow of macromolecules between endosomes in mammals. Flow between compartments along the endocytic pathway occurs by vesicle-mediated mechanisms (Stoorvogel et al., 1996 blue right-pointing triangle; Peden et al., 2004 blue right-pointing triangle), and the processes of tubule-mediated transfer of cargoes (Delevoye et al., 2009 blue right-pointing triangle), kiss-and-run (Bright et al., 2005 blue right-pointing triangle), and endosome maturation (Stoorvogel et al., 1991 blue right-pointing triangle; Rink et al., 2005 blue right-pointing triangle; Poteryaev et al., 2010 blue right-pointing triangle). Here we show that the coat clathrin interacts with endosomal tethers involved in endosome maturation and fusion (class C Vps/HOPS subunits) by means of vesicular (clathrin and the clathrin adaptor AP-3 [Adaptor Protein Complex 3]) and nonvesicular mechanisms (clathrin and hepatocyte growth factor receptor substrate, Hrs).

Mammalian endosomes possess clathrin domains in the form of budding profiles that contain AP-1/AP-3 clathrin adaptors (Stoorvogel et al., 1996 blue right-pointing triangle; Peden et al., 2004 blue right-pointing triangle; Theos et al., 2005 blue right-pointing triangle) or as flat lattices defined by the presence of Hrs (Raiborg et al., 2002 blue right-pointing triangle, 2006 blue right-pointing triangle). Of these coats, buds containing AP-3 and clathrin mature into vesicles that transport their contents between early endosomes and late endosomal/lysosomal compartments (Di Pietro and Dell’Angelica, 2005 blue right-pointing triangle; Dell’Angelica, 2009 blue right-pointing triangle). In contrast, Saccharomyces cerevisiae generates AP-3 vesicles at the Golgi complex destined for the lysosomal equivalent, the vacuole (Odorizzi et al., 1998 blue right-pointing triangle). This process is independent of clathrin in yeast (Seeger and Payne, 1992 blue right-pointing triangle; Anand et al., 2009 blue right-pointing triangle). Mutation of the human clathrin adaptor AP-3 causes Hermansky Pudlak Syndrome (HPS), in which lysosome-related organelles such as melanosomes, dense-platelet granules, and lamellar bodies are defective. HPS patients exhibit pigment dilution, bleeding diathesis, and pulmonary fibrosis, and this pathology is directly attributable to defects in vesicular biogenesis (Di Pietro and Dell’Angelica, 2005 blue right-pointing triangle; Dell’Angelica, 2009 blue right-pointing triangle). HPS phenotypes are triggered in mice when any one of 15 genes is defective. These 15 gene products assemble into protein complexes: AP-3, BLOC-1 to BLOC-3 (biogenesis of lysosome-related organelles complex), and tethering complexes constituted by class C Vps subunits (Li et al., 2004 blue right-pointing triangle). Vps33a mutant mice possess a phenotype similar to other HPS mutants, which might be explained by interactions of Vps33a with other HPS complexes (Suzuki et al., 2003 blue right-pointing triangle). Whether these genetic similarities underlie associations of Vps33a with HPS complexes, such as AP-3, into common complexes is presently unknown. This hypothesis is supported, however, by genetic and biochemical interactions between yeast class C Vps proteins and AP-3 (Anand et al., 2009 blue right-pointing triangle; Angers and Merz, 2009 blue right-pointing triangle; Salazar et al., 2009 blue right-pointing triangle).

The mammalian class C Vps protein Vps33a/b assembles into a core complex of class C Vps proteins including Vps11, Vps16, and Vps18. This core associates with Vps39 and Vps41 to form the HOmotypic fusion and Protein Sorting (HOPS) complex (Kim et al., 2001 blue right-pointing triangle; Nickerson et al., 2009 blue right-pointing triangle; Zhu et al., 2009 blue right-pointing triangle). The class C Vps core complex could also associate with Vps8/KIAA0804 and Vps3/TGFBRAP1 to constitute a hypothetical mammalian CORVET complex, a molecular species so far only documented in yeast (Peplowska et al., 2007 blue right-pointing triangle; Markgraf et al., 2009 blue right-pointing triangle). Class C core subunits and accessory HOPS subunits are critical for fusion events at the vacuole, a lysosome equivalent in the yeast S. cerevisiae. In this organism, HOPS orchestrates interactions with the GTPase ypt7, a rab7 ortholog, SNARES, and, recently, AP-3 to achieve selective membrane fusion (Ostrowicz et al., 2008 blue right-pointing triangle; Angers and Merz, 2009 blue right-pointing triangle; Nickerson et al., 2009 blue right-pointing triangle; Wickner, 2010 blue right-pointing triangle). Association of AP-3 and HOPS occurs at the vesicle fusion step with the vacuole when incoming Golgi-derived AP-3–coated vesicles reach the vacuole, the compartment where HOPS complexes reside in yeast (Angers and Merz, 2009 blue right-pointing triangle).

Here we show that mammalian Vps class C/HOPS subunits coimmunoprecipitated and/or colocalized with the endosomal coats clathrin, AP-3, and Hrs. Isolated clathrin-coated vesicles had Vps class C/HOPS subunits on them, yet they were depleted of Hrs. We also found that acute perturbation of clathrin dynamics on and off membranes using FKBP–clathrin light chain (CLC) chimeras (Moskowitz et al., 2003 blue right-pointing triangle; Deborde et al., 2008 blue right-pointing triangle) altered the distribution of class C Vps/HOPS proteins in two different types of mammalian cells. HEK293T cells with reduced clathrin function had elevated levels of Vps class C proteins in rab5-positive endosomes, suggesting that clathrin dynamics at vesicular AP-3– or nonvesicular Hrs-positive endosomal domains regulate Vps class C protein content on early endosomes. We hypothesized that coats in endosomal tether-coat complexes could contribute to polarized distribution of tethers, such as the HOPS complex. In support of this idea, differentiated neuroendocrine cells accumulated Vps class C/HOPS subunits at the growing tip of processes, where they were concentrated with clathrin. Targeting of Vps class C/HOPS subunits from cell bodies to neurites was sensitive to clathrin function perturbation. Our data suggest that vertebrate cells differ from yeast in that the vertebrates engage in novel interactions between class C Vps–containing tethers and coats, perhaps for specialized demands such as polarized targeting in neuroendocrine and epithelial cells.

RESULTS

Subunits of the Vps class C/HOPS tethering complexes associate with clathrin–AP-3 adaptor subunits

We initially explored molecular interactions between tethering complexes and coats taking advantage of protein complexes affected in HPS. Mouse mutations on genes encoding the class C-Vps protein Vps33a or AP-3 subunits trigger a phenotype that recapitulates HPS (Suzuki et al., 2003 blue right-pointing triangle; Li et al., 2004 blue right-pointing triangle), suggesting that AP-3 and complexes containing Vps33a act on a common pathway. Consistent with this interpretation, subunits of the tethering HOPS complex interact with AP-3 in both S. cerevisiae (Angers and Merz, 2009 blue right-pointing triangle) and mammalian cells (Salazar et al., 2005 blue right-pointing triangle, 2009 blue right-pointing triangle). Despite this similarity, in mammalian cells, but not yeast, AP-3 shows interactions with clathrin (Seeger and Payne, 1992 blue right-pointing triangle; Dell’Angelica et al., 1998 blue right-pointing triangle; Anand et al., 2009 blue right-pointing triangle). This observation suggests that Vps class C/HOPS tethers interact with coats differently in vertebrates as compared with yeast.

We tested whether all four class C Vps proteins, class C Vps33a-b isoforms, and the two HOPS-specific subunits associated with AP-3 and clathrin complexes isolated from HEK293T cells. As described in Materials and Methods, we used in vivo cross-linking with dithiobissuccinimidyl propionate (DSP) coupled to immunoaffinity chromatography with monoclonal antibodies (mAbs) against the AP-3 δ subunit or clathrin chains. DSP treatment decreases immunoreactivity of AP-3 δ subunit detected with a mAb against δ adaptin (see Materials and Methods). Vps class C and HOPS subunits present in AP-3 or clathrin immune complexes were detected with antibodies either against endogenously expressed Vps33b or tags engineered in recombinantly expressed subunits (Vps11, Vps16, Vps18, Vps33a, Vps33b, Vps39, and Vps41). We expressed tagged recombinant proteins because available class C Vps or HOPS subunit antibodies failed to detect endogenous proteins in multiple cell types, including HEK293T cells (our unpublished data).

We first determined whether phenotypic similarities between mice carrying mutations in either the class C Vps protein Vps33a or AP-3 subunits were predictors of a biochemical interaction between Vps33a and AP-3. Recombinantly expressed Vps33a selectively coprecipitated with AP-3 immunocomplexes (Figure 1A, compare lanes 3 and 5). This association was not restricted to Vps33a; similar results were obtained with tagged Vps33b (Figure 1A, compare lanes 4 and 6). The association of Vps33 isoforms with AP-3 could be due either to isolated Vps33 polypeptides interacting with AP-3 or Vps33 assembled into a Vps class C protein complex, such as HOPS. To discern between these hypotheses, we explored whether protein constituents of the HOPS complex were coisolated with AP-3 complexes together with endogenous Vps33b. Recombinantly expressed class C Vps proteins (Vps11, Vps16, and Vps18) or HOPS-specific subunits (Vps41 and Vps39) copurified with AP-3 and the endogenously expressed class C Vps protein, Vps33b. These associations were preferentially detected in the presence of DSP (Figure 1A, lanes 5 and 6; Figure 1B, lanes 8 and 10; Figure 1C, lane 10; Figure 1D, lanes 14, 16, and 18; Figure 1, E and F, lanes 10 and 12). The association specificity of AP-3 and these Vps subunits was probed using magnetic beads conjugated with anti–mouse immunoglobulin (Ig)G (beads alone, Figure 1B). Vps18-Myc and Vps33b coisolated with AP-3 when these beads contained mouse AP-3 δ antibodies, yet none of these proteins were detected with beads alone (Figure 1B, compare lanes 5 and 6 with 7–10). Second, we out-competed AP-3 δ binding to magnetic beads linked to mouse δ-antibodies with the δ peptide antigen (Salazar et al., 2009 blue right-pointing triangle). We monitored peptide competition by assessing the presence of AP-3 subunits (β3A or δ) and clathrin heavy chain (CHC; an AP-3 binding partner) in immunoisolated cross-linked AP-3 complexes (Dell’Angelica et al., 1998 blue right-pointing triangle; Salazar et al., 2009 blue right-pointing triangle). Pretreating beads displaying δ-antibodies with the δ-peptide abolished binding of AP-3 subunits, clathrin, and all Vps subunits to these beads (Figure 1A, compare lanes 3 and 4 with lanes 5 and 6; Figure 1, C, E, and F, compare lanes 6 and 8 with 10 and 12; Figure 1D, compare lanes 8, 10, and 12 with lanes 14, 16, and 18). Abolished binding indicates that class C Vps and HOPS subunits interact with the beads by binding to intact AP-3 and not via some nonspecific mechanism. As a control, we showed that AP-1 complexes were not cross-linked to Vps33b, Vps16-HA (hemagglutinin), or Vps41-Myc, showing that the association of class C Vps and HOPS subunits with AP-3 was selective (Figure 1G). AP-3-Vps subunit associations could result simply from abundantly expressed tagged proteins in endosomal compartments. To test this hypothesis, we asked whether the Vps33b-interacting protein Spe39 coisolates with AP-3 and clathrin complexes. We selected Spe39 because we previously demonstrated that Spe39 defines a subclass of Vps class C/HOPS complexes (Zhu et al., 2009 blue right-pointing triangle). In fact, Spe39 is present in a discrete pool of the total Vps33b and other class C protein as determined by quantitative immunomicroscopy (Zhu et al., 2009 blue right-pointing triangle). Importantly, endogenous Spe39 does not colocalize with AP-3 or CLCs (Supplemental Figure 1, A and B). Consistent with our immunomicroscopy, neither endogenous nor recombinant green fluorescent protein (GFP)-tagged Spe39 coprecipitated with AP-3 immunocomplexes (Supplemental Figure 1, C and D). These results argue against nonselective DSP cross-linking of recombinantly expressed proteins to AP-3.

FIGURE 1:
Association of class C Vps/HOPS subunits with the adaptor complex AP-3. HEK293T cells or cell lines expressing recombinant class C Vps/HOPS subunits (A–G) were left untreated (B–G, odd lanes) or treated (B–G, even lanes) with DSP ...

Others have proposed that the HOPS subunit Vps41 can associate with AP-3 as a clathrin-like coat (Rehling et al., 1999 blue right-pointing triangle; Darsow et al., 2001 blue right-pointing triangle). Consequently, we next looked for association of clathrin with Vps class C/HOPS in AP-3 cross-linked complexes. Cross-linked samples were treated with antibodies against CLC. Both Vps33 isoforms selectively coisolated with clathrin complexes isolated with CLC antibodies (Figure 2A, lane 3). We found that CLC consistently coimmunoprecipitated with CHC, the AP-3 δ subunit, endogenous Vps33b, and Vps41-Myc (Figure 2B, lane 7). Neither coats nor Vps proteins were detected with beads alone or beads coated with nonimmune IgG (Figure 2A, compare lanes 3 and 5 with lane 7). Immunoprecipitation of CLC after siRNA-mediated down-regulation of CHC not only showed lowered CHC levels, but also greatly diminished the abundance of the AP-3, Vps33b, and Vps41-Myc proteins (Figure 2B, compare lanes 7 and 8). We further assessed whether clathrin, AP-3, and Vps41-Myc form a complex together by performing sequential immunopurification of clathrin-containing AP-3 complexes from HEK293T cells stably expressing Vps41-Myc (Figure 2C). First, cross-linked AP-3 complexes were immunoaffinity purified with AP-3 δ antibodies in either the absence or presence of δ antigenic peptide as a control of binding selectivity to beads (Figure 2C, compare lanes 3 and 4 with lanes 5 and 6). Cross-linked AP-3 complexes bound to beads (Figure 2C, lane 6) were then eluted in native conditions from magnetic beads using the δ antigenic peptide. These eluted AP-3-cross-linked complexes were subjected to a second round of immunomagnetic isolation with either nonimmune mouse IgG (control) or a monoclonal anti-CLC. The composition of complexes bound to CLC antibody-coated beads was assessed by immunoblot. CLC antibodies allowed coimmunoprecipitation of the AP-3 δ subunit and Vps41-Myc (Figure 2C, compare lanes 7 and 8), indicating that at least a subset of AP-3, clathrin, and the HOPS subunit Vps41 exist in a multiprotein complex.

FIGURE 2:
CHC and CLC associate with class C Vps/HOPS subunits. (A) Extracts of DSP-treated HEK293T cells transfected with Vps33A or Vps33b were incubated with magnetic beads decorated with antibody directed against control IgG (lane 2) or CLC (lane 3). Immunocomplexes ...

The HOPS subunit Vps41 is known to directly interact with AP-3 through its δ subunit in yeast, whereas mammalian CHC interacts with AP-3 through its β3 subunit (Dell’Angelica et al., 1998 blue right-pointing triangle; Rehling et al., 1999 blue right-pointing triangle; Angers and Merz, 2009 blue right-pointing triangle). These interactions suggest that AP-3 could bridge between the HOPS complex and clathrin in mammalian cells. We examined this possibility by performing shRNA down-regulation of either AP-3 δ or CHC, cross-linking in vivo with DSP, and immunoprecipitating cell extracts with CLC or AP-3 δ antibodies (Figure 2, D and E). As compared with control cells, AP-3 δ subunit shRNA treatment caused a ~50% drop in the level of AP-3 δ polypeptide (49 ± 4.6%), whereas CHC shRNA eliminated nearly all CHC (95.5 ± 0.9% reduction, n = 4, Figure 2, D and E, compare lanes 1 and 2). Immunoaffinity purification with CLC antibodies from mock shRNA treated control cells recovered clathrin complexes that were cross-linked to AP-3 subunits, Vps33b, and Vps41-Myc (and Vps39; unpublished data) (Figure 2D, lane 5). Although CLC antibodies failed to isolate AP-3 subunits from AP-3 δ shRNA-treated cells, there were no discernible effects on the levels of Vps33b or Vps41-Myc present in CLC-isolated cross-linked complexes (Figure 2D, compare lanes 5 and 6). These results suggest that other tetrameric adaptors, such as AP-1, could mediate the association between Vps class C and HOPS subunits with clathrin. We did not detect, however, an association of Vps class C or HOPS subunits to cross-linked AP-1 complexes isolated from HEK293T cells (Figure 1G). Furthermore, cross-linked AP-3 complexes isolated from CHC-depleted cells, although devoid of clathrin, contained both endogenous Vps33b and recombinantly expressed Vps41-Myc (Figure 2E, compare lanes 8 and 10) or Vps39-GFP (unpublished data). In fact, cross-linked AP-3 complexes isolated from CHC shRNA–treated cells copurified with increased levels of Vps41-Myc and Vps33b. This effect may be attributable to an increased expression of Vps41-myc but not Vps33b in CHC-shRNA–treated cells (Figure 2E, compare lanes 1 and 2; Vps41-Myc 255 ± 18.5% of control shRNA, n = 2; Vps33b 138.5 ± 42%, n = 4). These data indicate that, despite the fact that clathrin–AP-3–HOPS subunits form a complex, clathrin is not required for the binding of Vps class C and HOPS subunits to AP-3 complexes, nor is AP-3 necessary for their binding to clathrin complexes. In addition to AP-3, other clathrin-interacting molecules present in endosomes may interact with Vps class C/HOPS subunits. One such mechanism is the Hrs-clathrin flat coat present in endosomes (Raiborg et al., 2002 blue right-pointing triangle, 2006 blue right-pointing triangle). Endogenous Hrs or recombinantly expressed Hrs-Myc coprecipitated endogenous CHC and Vps33b (Figure 2F, lanes 5–6 and 5′-6′). These interactions were selective as judged by the absence of Vps33b and CHC in GFP control immunoprecipitations as well as by the absence of transferrin receptor in all immunocomplexes (Figure 2F, lanes 3–4 and 3′-4′). Collectively, our results indicate that Vps class C/HOPS subunits copurify with at least two pools of clathrin present in early endosomes: clathrin–AP-3 and clathrin–Hrs complexes.

Vps class C and HOPS subunits are present in clathrin-coated organelles

Vps class C and HOPS subunits association with clathrin–AP-3 and clathrin–Hrs predicts that these Vps proteins should be present in domains of early endosomes as well as clathrin-coated vesicle carriers. The presence of Vps class C and HOPS subunits in early endosomes and clathrin-coated carriers would be consistent with the observation that AP-3, AP-3–clathrin budding profiles, clathrin–Hrs flat coats, Vps class C, and HOPS subunits are found in early endosomal compartments (Raiborg et al., 2002 blue right-pointing triangle, 2006 blue right-pointing triangle; Peden et al., 2004 blue right-pointing triangle; Richardson et al., 2004 blue right-pointing triangle; Theos et al., 2005 blue right-pointing triangle). We determined the subcellular distribution of Vps class C and HOPS subunits by immunofluorescence using quantitative Delta deconvolution microscopy. We used antibodies against coats (clathrin, AP-1 γ, and AP-3 δ) and either the early endosome marker, rab5, or the late endosomal markers rab7, rab7b, and LAMP-1. Our rationale for including the late endosomal marker rab7b is that its depletion selectively increases the protein levels of AP-3 (Progida et al., 2010 blue right-pointing triangle). We focused on endogenously expressed Vps33b and used Vps16-HA to further validate Vps33b results. In addition, we analyzed the subcellular localization of the HOPS subunits Vps39–GFP and Vps41–Myc in clathrin-positive compartments and endosomes. Endogenous Vps33b partially overlapped with puncta positive for AP-3 (Figure 3, B and C) and/or clathrin (Figure 3, E and F). Although partial, of these two coats, fluorescent signal overlap was more pronounced between Vps33b and clathrin than Vps33b with AP-3. One third of all Vps33b-positive pixels were positive for CHC (Figure 3, E, F, and J). Similar to Vps33b, Vps16-HA was preferentially found in puncta positive for CHC (Figure 3, G and J) or AP-3 δ (Figure 3H), where coats signals partially overlapped with class C Vps subunits. In contrast to CHC localization, Vps33b and Vps16-HA signals minimally overlapped with AP-1 γ (Figure 3, I and J).

FIGURE 3:
Vps class C/HOPS are present in AP-3, clathrin, Rab5, and Rab7b-compartments. HEK293T cells were fixed, and either triple (A–F) or double (G–I) labeled for indirect immunofluorescence with antibodies against the following antigens: Vps33b, ...

Vps33b was found in rab5 endosomes, in which we observed a partial overlap of fluorescent signals (Figure 3, A and J). In contrast, Vps33b was undetectable in late endosomes defined by rab7 and LAMP-1 staining (Figure 3J). Vps33b overlapped with rab7b (Figure 3, D and J), however, at levels similar to those of AP-3 (Figure 3J). Rab7b organelles were also positive for clathrin and the late endosomal SNARE vesicle-associated membrane protein 7 (VAMP7; Figure 3J). Another class C Vps protein, Vps16–HA, showed a similar pattern of endosomal localization compared with Vps33b (Figure 3J). Analysis of the distribution of the HOPS subunits Vps41–Myc and Vps39–GFP revealed that the signal overlap with coats and endosomal markers was far less pronounced as compared with the class C Vps proteins Vps33b/Vps16–HA. Nonetheless, the two HOPS-specific Vps subunits, Vps41–Myc and Vps39–GFP, were localized to rab5- and CHC-positive compartments (Figure 3J).

FIGURE 4:
Vps class C/HOPS subunits localize to discrete domains of enlarged early endosomes. HEK293T cells transiently expressing recombinant rab5Q79L–GFP alone (A) or rab5Q79L–GFP plus either Vps16–HA (B) or Vps41–Myc (C) were ...

The partial overlap between coats, Vps class C, and HOPS proteins suggests that domains of endosomes, rather than the whole organelle, contain these factors. We tested this hypothesis by enlarging rab5 compartments expressing the GTPase-deficient rab5Q79L. This tool has been successfully used to enlarge endosomes facilitating the identification of domains in the limiting membrane of endosomes by high-resolution optical microcopy (Raiborg et al., 2002 blue right-pointing triangle, 2006 blue right-pointing triangle; Craige et al., 2008 blue right-pointing triangle). We determined the distribution of coats, HOPS subunits, and class C Vps proteins by deconvolution immunofluorescence microscopy and volume rendering of digitally reconstructed enlarged endosomes (Figure 4 and Supplemental Movies 1 and 2). Single optical sections revealed that endogenous Vps33b, Vps16–HA, and Vps41–Myc were present in the limiting membrane of enlarged rab5 endosomes in discrete domains often partially overlapping with either CHC- or AP-3 δ–decorated patches (Figure 4). Fifty and thirty-five percent of all Vps33b domains present on the limiting membrane of rab5Q79L-enlarged endosomes were positive for CHC and AP-3 δ subunit, respectively (52.4 ± 25.7, n = 68; 34.2 ± 24.3, n = 103). Digital volume rendering of rab5Q79L-enlarged endosomes confirmed that the HOPS subunit Vps41–Myc was present in discrete domains that partially overlapped either with CHC (Supplemental Movie 1) or AP-3 δ (Supplemental Movie 2) at the limiting membrane of enlarged endosomes.

We isolated clathrin-coated vesicles from HEK293T cells or HEK293T cells stably expressing either Vps41–Myc or Vps16–HA and asked whether class C Vps/HOPS proteins were present in these organelles (Figure 5 and Supplemental Figure 2). Clathrin-coated vesicle isolation was monitored by the enrichment of CHC detected by Coomassie Blue dye (Figure 5A) or by immunoblot with antibodies CHC, AP-1 γ, AP-2 α, and AP-3 β3 subunits in clathrin-coated vesicle fractions (Figure 5B and Supplemental Figure 2, compare lanes 1 and 6). Clathrin-coated vesicles contained class C Vps proteins Vps33b and Vps16–HA and the HOPS subunit Vps41–Myc (Figure 5B and Supplemental Figure 2, compare lanes 1 and 6). Of these Vps proteins, Vps41–Myc and Vps16–HA were clearly enriched in clathrin-coated vesicle fractions. We isolated clathrin-coated vesicles from control or CHC RNAi–treated cells to define whether Vps class C and HOPS subunits cosedimenting with clathrin-coated vesicles correspond to true components of these carriers or just contaminants present on these membranes (Borner et al., 2006 blue right-pointing triangle). Coomassie Blue staining revealed that clathrin-coated vesicle fractions isolated from CHC down-regulated cells decreased the content of several polypeptides, including CHC (Figure 5A, asterisks). Down-regulation of CHC precluded formation of clathrin-coated vesicles as determined by the absence or decreased levels of adaptor subunits from clathrin-coated vesicles (Figure 5B; Supplemental Figure 2, compare lanes 6 and 6′; Figure 5C). Importantly, the levels of Vps33b, Vps16–HA, or Vps41–Myc were significantly reduced from clathrin-coated vesicles isolated from CHC RNAi–treated cells indicating that Vps33b, Vps16–HA, and Vps41–Myc specifically reside in clathrin-coated vesicles (Figure 5B; Supplemental Figure 2, compare lanes 6 and 6′; Figure 5C). Contamination of clathrin-coated vesicle fractions with early endosomes markers such as EEA1, rabaptin5, and Hrs was negligible (Figure 5B). Therefore these results indicate that clathrin, Vps class C, and HOPS subunits coreside both in coated domains of early endosomes as well as clathrin-coated vesicle carriers.

FIGURE 5:
Vps class C/HOPS subunits cosediment with clathrin-coated vesicles. (A and B) HEK293T cells were infected with scramble (lanes 1–6) or CHC directed shRNA lentiviruses (CHC, lanes 1′–6′). Cells were homogenized, and clathrin-coated ...

Acute perturbation of clathrin alters Vps class C/HOPS subunit subcellular distribution

We evaluated the effect of CHC shRNA on the distribution of Vps class C/HOPS subunits in rab5- and rab7b-positive endosomes. CHC down-regulation, which is achieved within several days of shRNA treatment, did not alter the distribution of Vps class C/HOPS subunits in rab5 or rab7b compartments, suggesting compensatory mechanisms controlling Vps class C/HOPS subunit subcellular distribution (unpublished data). To overcome possible compensatory mechanisms, we acutely perturbed clathrin function by using a chemical/genetic approach that uses chimeric CLCs carrying an oligomerization module, a modified FKBP 12 domain (Moskowitz et al., 2003 blue right-pointing triangle; Deborde et al., 2008 blue right-pointing triangle). FKBP domains selectively oligomerize upon addition of the cell-permeant bivalent chemical AP20187. CLC oligomerization that follows incubation with AP20187 halts vesicle formation from donor organelles, such as plasma membrane or trans-Golgi network by “freezing” clathrin onto membranes and aborted budding profiles (Moskowitz et al., 2003 blue right-pointing triangle; Deborde et al., 2008 blue right-pointing triangle). Thus we used FKBP–CLC chimeras to trap clathrin at membranes. Tagged FKBP–CLC chimeras incorporated into 60.7 ± 18.5% (n = 30) of all CHC-positive organelles in HEK293T cells as determined by deconvolution microscopy (Supplemental Figure 3A). Like endogenous CLC (Figure 2), chimeric mCherry-FKBP–CLC specifically coprecipitated class C Vps/HOPS subunits (Supplemental Figure 3D). Cell treatment with AP20187 for 2 h induced enlargement of CHC/tagged-FKBP–CLC dual-labeled organelles and the accumulation of endogenous and recombinant clathrin chains at the perinuclear region (Supplemental Figure 3, B and C). The perinuclear accumulation of CHC in HEK293T cells indicates that clathrin­-­dependent processes are rapidly inhibited by FKBP–CLC chimeras (Moskowitz et al., 2003 blue right-pointing triangle; Deborde et al., 2008 blue right-pointing triangle).

We next determined the effect of chemically perturbing clathrin function in HEK293T- and NGF-differentiated PC12 cells on Vps class C/HOPS subunits (Figures 69 and Supplemental Movies 3–6). We determined the speed of the AP20187-induced Vps class C redistribution by in vivo, time-lapsed confocal microscopy. mCherry-FKBP-CLC– and Vps18-GFP–expressing cells were imaged 25 min before drug addition and for 2 h in the presence of AP20187. mCherry-FKBP–CLC oligomerization induced by AP20187 quickly caused an accumulation of recombinant clathrin and Vps18–GFP in the perinuclear region. Concomitantly, Vps18–GFP organelles distributed in the cell periphery grew in number and size (Figure 6C and Supplemental Movie 5 and 6). Vps18–GFP redistributed in <30 min after AP20187 addition and reached a plateau by 60 min. Addition of either AP20187 to cells expressing solely Vps18–GFP (Figure 6A and Supplemental Movie 3) or ethanol vehicle to mCherry-FKBP-CLC and Vps18-GFP doubly expressing cells (Figure 6B and Supplemental Movie 4) did not alter the subcellular distribution of either fluorescently tagged protein. These results demonstrate a swift rearrangement of class C Vps proteins by acute chemical/genetic inhibition of clathrin-coated vesicle formation.

FIGURE 6:
In vivo chemical/genetic disruption of clathrin chains rapidly redistributes Vps18. HEK293T cells transiently expressing Vps18–GFP (A) and an mCherry-tagged (mCh, B and C) chimeric CLC carrying a modified FKBP 12, as an oligomerization module, ...
FIGURE 9:
Polarized distribution of clathrin and Vps class C proteins in neuronal cells. (A) Human cortical neuronal cells, HCN-1, were fixed and processed for indirect immunofluorescence microscopy. Cells were double labeled with antibodies against endogenous ...

The effects of AP20187-induced perturbation of clathrin function were not just restricted to Vps18–GFP. Analysis of images from fixed specimens showed that drug addition caused redistribution of endogenous Vps33b, Vps18–GFP, or Vps41–Myc into larger puncta preferentially found at the perinuclear region as was seen with Vps18–GFP during live imaging (Figure 7). This change in Vps class C/HOPS subunits distribution occurred concomitantly with an increased colocalization of Vps class C/HOPS subunits with either CHC or mCherry-FKBP–CLC (Figure 7A). Importantly, AP20187 increased the colocalization of clathrin chains with AP-3 as well as rab5 and rab7b, suggesting that AP20187 similarly traps clathrin chains on both endosomes (Figure 7, B and C). These results are consistent with the presence of clathrin in AP-3–, rab5-, and rab7b-positive compartments, as was observed at steady state in HEK293T cells (Figure 3J).

FIGURE 7:
Chemical/genetic disruption of clathrin chains affects Vps class C/HOPS subunits and coat distribution. HEK293T cells transiently expressing mCherry-FKBP–CLC were treated in the presence of ethanol vehicle (EtOH, 0.05% vol/vol) or AP20187 (50 ...

Vps class C/HOPS subunit binding to both clathrin–AP-3 and clathrin–Hrs coats suggest that Vps class C/HOPS subunit accumulation should be preferentially observed in early endosomes as compared with other intracellular compartments, such as late endosomes or the trans-Golgi network. To test this hypothesis, we analyzed the affects of AP20187 on the colocalization of rab5, rab7b, and AP-1 with CHC and/or endogenous Vps33b in mCherry-FKBP-CLC–expressing cells using deconvolution microscopy. The amount of CHC-positive puncta that were also positive for Vps33b doubled from 12.3 ± 5.3% (n = 120) to 20.8 ± 11.9% after drug addition (n = 120, p < 0.0001, Wilcoxon–Mann–Whitney test; Figure 8). AP20187 increased the colocalization of Vps33b with rab5 from 10.2 ± 5% to 20 ± 9.6% (n = 60, p < 0.0001, Wilcoxon–Mann–Whitney test; Figure 8; Supplemental Figure 4, compare A and B). In contrast, colocalization between rab7b and Vps33b modestly decreased after 2 h of clathrin perturbation (Figure 8; Supplemental Figure 4, compare C and D). Vps33b colocalization with clathrin in rab5-positive endosomes was selective because overlap between Vps33b and AP-1 γ remained at background levels before or after AP20187 incubation (Supplemental Figure 5 and Figure 8). Although AP20187 induced clathrin recruitment to rab5- and rab7b-positive compartments to a similar extent (Figure 8), the preferential association of Vps33b- to rab5-positive organelles after drug-induced clathrin perturbation supports the hypothesis that clathrin coats selectively regulate the localization of class C Vps proteins to early endosomes.

FIGURE 8:
Chemical/genetic disruption of clathrin affects Vps33b distribution in Rab5 and Rab7b endosomal compartments. This figure depicts the quantification of experiments as those in Supplemental Figure 3. HEK293T cells expressing mCherry-FKBP–CLC (+) ...

Clathrin and Vps class C proteins are targeted to neuronal processes by clathrin-dependent mechanisms

We tested whether class C Vps proteins present in clathrin-coated organelles undergo directional delivery in polarized cells. To this end, we used human cortical neurons HCN-1A, a cell type that extends processes (Ronnett et al., 1990 blue right-pointing triangle), as well as NGF-differentiated PC12 cells. HCN-1A cells were double-labeled with antibodies against endogenous Vps33b and CHC, and cells were imaged by high-resolution Delta deconvolution microscopy (Figure 9A). Vps33b was concentrated at the tip of neuronal processes where it preferentially colocalized with clathrin. This polarized distribution of a class C Vps/HOPS subunit was also observed by in vivo imaging of NGF-differentiated PC12 cells expressing recombinant mCherry-FKBP–CLC with either Vps39–GFP (unpublished data) or Vps18–GFP (Figure 9B).

We predicted that if class C Vps proteins associate with clathrin-positive organelles generated at the cell body for subsequent delivery to neurites, then inhibition of clathrin-dependent mechanisms by AP20187 should lead to a progressive decrease of mCherry-FKBP–CLC and Vps18–GFP fluorescent signals in the proximal segment of neurites. NGF-differentiated PC12 cells expressing mCherry-FKBP–CLC and Vps18–GFP were treated with vehicle or with AP20187 and continuously imaged by confocal microscopy for 2 h. The integrated fluorescence intensity per volume unit was measured in the proximal third of the neurite (Figure 9B, arrows). Addition of AP20187 decreased CLC and Vps18 fluorescence in the proximal segment of neurites after 2 h (Figure 9B). Quantification of CLC and Vps18 fluorescence intensity revealed a progressive reduction per unit of proximal neurite volume after AP20187 incubation (Figure 9C, open circles). This decrease was not observed in vehicle-treated cells (Figure 9C, closed circles). Changes in fluorescence intensity per voxel induced by drug incubation are due to neither changes in neurite length nor diameter. These results indicate that Vps18–GFP delivery to neurites of polarized neuroendocrine cells is sensitive to acute perturbation of clathrin function.

DISCUSSION

Mouse mutants in subunits of HPS protein complexes and Vps33a, a Vps class C tether complex subunit, share phenotypes. The shared phenotypes suggests that associations between Vps33a and other HPS protein complexes participate in the same pathway, delivering membrane proteins from early endosomes to late endosomes/lysosomes and lysosome-related organelles (Suzuki et al., 2003 blue right-pointing triangle; Li et al., 2004 blue right-pointing triangle). Defects in this route trigger HPS in humans (Li et al., 2004 blue right-pointing triangle; Di Pietro and Dell’Angelica, 2005 blue right-pointing triangle). Similarly, S. cerevisiae orthologues of Vps class C proteins and AP-3 subunits participate in the delivery of cargoes to the vacuole. Genetic and/or biochemical interactions between AP-3 and HOPS subunits in yeast and vertebrates provide mechanistic insight into the molecular organization of this genetic pathway (Angers and Merz, 2009 blue right-pointing triangle, 2010 blue right-pointing triangle; Nickerson et al., 2009 blue right-pointing triangle; Salazar et al., 2009 blue right-pointing triangle). Our results are the first evidence of vertebrate Vps class C/HOPS proteins interacting with the early endosomal coats clathrin and AP-3 and the clathrin-binding scaffold Hrs. Moreover, we provide biochemical evidence of an interaction between Vps33a and AP-3 predicted from the phenotypic similarities between Vps33a- and AP-3-deficient mice. Clathrin and AP-3 highlight fundamental differences in the way that yeast and mammalian Vps class C and coats interact. In contrast with yeast AP-3, the mammalian ortholog of this adaptor complex interacts with clathrin (Seeger and Payne, 1992 blue right-pointing triangle; Dell’Angelica et al., 1998 blue right-pointing triangle; Anand et al., 2009 blue right-pointing triangle). These observations suggest differences in the biochemical and functional architecture of mechanisms controlled by vertebrate Vps class C/HOPS–containing tethers and coats. Mammalian class C Vps proteins (Vps11, 16, 18, and 33a-b) and the HOPS-specific subunits Vps39 and Vps41 establish specific interactions with clathrin chains (Figure 2). We identified Vps class C/HOPS proteins in isolated clathrin-coated carriers, clathrin-positive domains of rab5-positive early endosomes, and rab7b-containing endosomes (Figures 35). The association of clathrin with Vps class C/HOPS proteins has at least two modalities: one occurring with the AP-3 complex and another in which Hrs participates. Clathrin–AP-3–Vps class C/HOPS and clathrin–Hrs–Vps class C/HOPS associations suggest vesicular and nonvesicular mechanisms controlling Vps class C/HOPS subunit subcellular distribution along the endocytic pathway, respectively.

We focused on the association between Vps class C/HOPS subunits and clathrin chains because quantitative fluorescence microscopy indicated a greater degree of overlap between Vps33b/Vps16 and CHC than with AP-3 (Figure 3J). The functionality of Vps class C/HOPS protein–clathrin interactions was demonstrated by acute perturbation of clathrin function (Moskowitz et al., 2003 blue right-pointing triangle; Deborde et al., 2008 blue right-pointing triangle). Chimeric CLC carrying the oligomerization module FKBP and AP20187 treatment rapidly redistributed CHC, class C Vps proteins (Vps18, Vps33b), and HOPS subunits (Vps39, Vps41) to organelles distributed throughout the cytoplasm and the perinuclear region (Figures 68). Quantitative immunofluorescence microscopy revealed that, upon clathrin function perturbation, Vps33b content preferentially increased in rab5-positive over either rab7b-containing endosomes or AP-1–positive organelles, such as the trans-Golgi (Figure 8 and Supplemental Figures 4 and 5). These findings support a model whereby clathrin-dependent mechanisms acutely define the subcellular distribution of Vps class C/HOPS-containing tethers. We directly tested this model in human cortical neurons and in NGF-differentiated PC12 cells, two polarized cellular models. We observed enrichment of class C Vps/HOPS subunits and clathrin at the tip of neurites (Figure 9A). Moreover, acute perturbation of clathrin function led to a progressive depletion of Vps18–GFP and mCherry-FKBP–CLC from the proximal segment of neurites imaged in vivo (Figure 9, B and C). We interpret this decrease in neurite Vps18–GFP and mCherry-FKBP–CLC fluorescence as the result of two concomitant processes: (1) inhibition of newly formed Vps18-clathrin–positive organelles entering the proximal neurite from the cell body plus (2) Vps18-clathrin–positive organelles already present in the neurite proximal segment at the time of drug addition progressively moving downstream toward the neurite tip.

Clathrin, AP-3, and Vps41 form a tripartite complex (Figure 2C). The association of Vps class C/HOPS subunits with clathrin, however, is independent of AP-3 expression levels (Figure 2D). Conversely, association of AP-3 and class C Vps/HOPS proteins is independent of clathrin expression (Figure 2E). These independent associations suggest that Vps class C/HOPS subunits establish multipronged interactions with clathrin and adaptors, such as AP-3 and/or that other clathrin-interacting molecules in early endosomes may mediate clathrin–Vps class C/HOPS subunit associations. We identified the early endosomal clathrin–Hrs flat coat participating in clathrin–Vps class C subunit interactions. Endogenous Hrs as well as recombinant Hrs coprecipitated endogenous clathrin and vps33b. Hrs and AP-3 mechanisms are likely independent because AP-3 is present in clathrin-coated vesicles yet Hrs is excluded from these coated carriers (Figure 5B). Therefore we propose that vesicular and nonvesicular clathrin–Vps class C/HOPS protein complexes regulate the subcellular distribution of class C Vps/HOPS subunits along the endocytic route. Structural predictions point to the presence of CHC homology domains in S. cerevisiae Vps 11, 18, 39, and 41, raising the possibility of multipronged associations between components of the coat and Vps class C/HOPS subunits (Darsow et al., 2001 blue right-pointing triangle; Nickerson et al., 2009 blue right-pointing triangle). Irrespective of whether multiple clathrin-binding molecules, such as AP-3 and Hrs, vesicular or nonvesicular mechanisms, or multipronged interactions between coats and Vps tethers exists, however, the redistribution of Vps class C/HOPS subunits upon of acute perturbation of clathrin function indicates that significant pools of Vps class C/HOPS tethers are under control of clathrin-dependent mechanisms.

Angers and Merz have put forward an attractive model in which interactions between a tether, HOPS, and the coat AP-3 mediate docking of vesicles with the vacuole in S. cerevisiae, a process that culminates with fusion of membranes. Donor compartment (Golgi) and incoming AP-3–coated vesicles are devoid of HOPS complex in this model. HOPS complexes reside in the acceptor vacuolar compartments where coat and tether encounters occur to facilitate vesicle consumption (Angers and Merz, 2009 blue right-pointing triangle, 2010 blue right-pointing triangle). Our data suggest that, in addition to this mechanism, mammalian Vps class C/HOPS tethers are included in clathrin- and clathrin-AP-3–coated carriers, suggesting a coat-dependent mechanism for delivering Vps class C/HOPS tethers. Perturbing clathrin function with FKBP-CLC/AP20187 caused an increase in the number and size of organelles positive for Vps class C/HOPS subunits and trapped Vps33b in rab5-positive endosomes (Figures 68; Supplemental Figure 4). We attribute these effects to inhibition of clathrin-coated vesicle budding, consistent with published data (Moskowitz et al., 2003 blue right-pointing triangle; Deborde et al., 2008 blue right-pointing triangle) and possibly to changes in the dynamic of flat clathrin–Hrs coats in early endosomes. Alternatively, FKBP-CLC/AP20187 could possibly cause promiscuous recruitment of cytoplasmic Vps class C/HOPS protein pools to membranes by oligomerized FKBP–CLC. We do not favor this alternative hypothesis, however, because we do not detect an increased association of Vps33b to rab7b- or AP-1–positive organelles (Figure 8; Supplemental Figures 4 and 5).

AP-3 budding occurs mainly, if not exclusively, from transferrin receptor–positive endosomes (Peden et al., 2004 blue right-pointing triangle; Theos et al., 2005 blue right-pointing triangle; Craige et al., 2008 blue right-pointing triangle). Quantitative electron microscopy indicates that half of these AP-3 budding profiles possess clathrin on them (Peden et al., 2004 blue right-pointing triangle; Theos et al., 2005 blue right-pointing triangle). Perhaps, the presence of clathrin allows coats and Vps class C/HOPS subunits to be recruited at early stages in the vesicle life cycle. In the absence of clathrin, tethers and coats could undergo late interactions at target organelles, as proposed previously for yeast (Angers and Merz, 2009 blue right-pointing triangle, 2010 blue right-pointing triangle). Early inclusion of Vps class C/HOPS tethers into clathrin-coated carriers could serve a role for long-range delivery of tethers to polarized domains in mammalian cells. This hypothesis is supported by our findings that endogenous Vps33b and recombinantly expressed Vps18 and Vps39 display polarized distribution in the tip of neurites in human cortical neurons or differentiated PC12 cells (Figure 9). An alternative yet nonexclusive model is that Vps class C/HOPS subunits could play a role in cargo selection either through indirect effects of Vps proteins on cargo recognition by coats or by direct association of class C Vps/HOPS proteins with SNAREs and nonSNARE membrane proteins. Support for this interpretation was obtained recently when ema/CLEC16A, which is a lectin-type membrane protein, was shown to directly bind to Vps16A in Drosophila melanogaster (Kim et al., 2010 blue right-pointing triangle).

At least four modalities of content delivery between stages of the endocytic route have been documented: vesicle-mediated (Stoorvogel et al., 1996 blue right-pointing triangle; Peden et al., 2004 blue right-pointing triangle), tubule-mediated transfer of cargoes (Delevoye et al., 2009 blue right-pointing triangle), kiss-and-run (Bright et al., 2005 blue right-pointing triangle), and endosome maturation (Stoorvogel et al., 1991 blue right-pointing triangle; Rink et al., 2005 blue right-pointing triangle; Poteryaev et al., 2010 blue right-pointing triangle). Endosome maturation is kinetically defined by the conversion over time of the same endosome membrane from a rab5- to a rab7-decorated compartment (Stoorvogel et al., 1991 blue right-pointing triangle; Rink et al., 2005 blue right-pointing triangle; Poteryaev et al., 2010 blue right-pointing triangle). This process depends on a switch mechanism in which a later acquisition of HOPS subunits promotes rab7 activation by interactors of the Vps39 subunit of HOPS (Nordmann et al., 2010 blue right-pointing triangle,; Poteryaev et al., 2010 blue right-pointing triangle). Presently, it is unknown whether rab5 compartments can mature into rab7b compartments. We focused on rab7b because class C Vps/HOPS proteins were found in very low (background) levels in rab7- or LAMP-1–containing late endosomes (Figure 3J), suggesting that Vps class C/HOPS subunit binding may be short-lived on them. In contrast, class C Vps/HOPS associates to rab7b compartments. Rab7b has been implicated in delivery of Toll-like receptors from the cell surface to lysosomes in macrophages as well as in retrograde transport between endosomes and Golgi complex in HeLa cells (Wang et al., 2007 blue right-pointing triangle; Progida et al., 2010 blue right-pointing triangle). The presence of clathrin in rab7b-positive compartments and the observation that down-regulation of rab7b increases the expression level of AP-3 (Progida et al., 2010 blue right-pointing triangle) suggest a role of rab7b in vesicle-mediated transport between early endosomes and late endosomes/lysosomes or in a specialized retrograde transport between endosomes and the Golgi complex. Irrespective of whether rab7b-clathrin compartments represent vesicles, endosomes, or a combination thereof, our findings suggest that maturation of clathrin-coated membranes in transit among endosomal compartments could occur.

S. cerevisiae class C Vps proteins (Vps11, 16, 18, and 33) form a core that incorporates into CORVET and HOPS complexes (Peplowska et al., 2007 blue right-pointing triangle; Nickerson et al., 2009 blue right-pointing triangle; Ostrowicz et al., 2010 blue right-pointing triangle; Wickner, 2010 blue right-pointing triangle). Vps8 and Vps3 constitute the CORVET complex whereas Vps39 and Vps41 establish specific interactions with the core defining the HOPS complex (Peplowska et al., 2007 blue right-pointing triangle; Markgraf et al., 2009 blue right-pointing triangle; Ostrowicz et al., 2010 blue right-pointing triangle). The organization and subcellular localization of HOPS has been partially characterized in metazoans (Kim et al., 2001 blue right-pointing triangle; Richardson et al., 2004 blue right-pointing triangle; Zhu et al., 2009 blue right-pointing triangle; Cullinane et al., 2010 blue right-pointing triangle). In contrast, metazoan CORVET has not been studied. Although putative human orthologues of CORVET subunits, Vps8/KIAA0804 and Vps3/TGFBRAP1, are present in databases, it remains unknown whether these gene products assemble with class C Vps proteins to form a mammalian CORVET complex. It is formally possible that mammalian class C Vps proteins, such as Vps33b and Vps16, identified in clathrin-containing organelles by biochemical and immunolocalization studies, may be part of both CORVET and HOPS complexes. The presence of class C vps subunits in both complexes might explain why there was a relatively higher colocalization between Vps33b/Vps16 (found in both CORVET and HOPS) and clathrin as compared with Vps41, Vps39, and clathrin (found only in HOPS; Figure 3). If this were the case, our data suggest that perturbing clathrin function affects Vps class C proteins along rab5 and rab7b compartments (Figure 8) by stalling maturation from a CORVET to a HOPS-positive-clathrin–coated vesicle or endosome.

Our results demonstrate a unique functional architecture of mechanisms controlled by vertebrate Vps class C/HOPS–containing tethers and clathrin coats. We postulate that clathrin-dependent mechanisms provide long-range and directional delivery of class C Vps/HOPS tethers to organelles and/or specialized domains of mammalian cells bearing complex architectures.

MATERIALS AND METHODS

Antibodies

The following antibodies were used in this study: polyclonal anti-myc (A190105A) and anti-HA (A190108A; Bethyl Laboratories, Montgomery, TX); polyclonal anti-GFP (cat# GFP-1020, Aves Labs, Tigard, OR, and cat# 132002, Synaptic Systems, Gottingen, Germany); monoclonal anti–Lamp H4A3, anti-SV2, and anti-AP3δ SA4 (Developmental Studies Hybridoma Bank, Iowa City, IA); monoclonal anti–rab7 ab50533 (Abcam, Cambridge, MA); monoclonal anti–rab7b clone 3B3 (Abnova, Taipei, Taiwan); monoclonal anti-rab5, anti–AP1γ adaptin, anti–rabaptin 5, and anti-EEA1 (610724, A36120, 610676, and 610456; BD Bioscience Transduction Laboratories, Pasadena, CA); monoclonal anti–CHC X22 (Calbiochem, San Diego, CA); monoclonal anti–CLC CON.1 (MMS423P; Covance, Berkeley, CA); polyclonal anti–mCherry dsRed (632496; Clontech, Mountain View, CA); monoclonal anti-AP2α adaptin and anti-actin (A4325 and A5441; Sigma, St. Louis, MO); polyclonal anti-AP3β1 (13384–1-AP; ProteinTech Group, Chicago, IL); monoclonal anti–GFP 3E6 (A11120; Molecular Probes, Eugene, OR); monoclonal anti–HRS A-5 (Enzo Life Sciences, Plymouth Meeting, PA); anti–synaptophysin clone SY38 (Millipore, Billerica, MA); and monoclonal anti–transferrin receptor H68.4 (136800; Zymed Laboratories, San Francisco, CA). Anti-Vps33b, anti-Spe39, and anti–AP3 β3 have been previously described (Faundez and Kelly, 2000 blue right-pointing triangle; Salazar et al., 2009 blue right-pointing triangle; Zhu et al., 2009 blue right-pointing triangle). Anti-VAMP7 was a gift from Andrew Peden (Department of Clinical Biochemistry, University of Cambridge, UK). Immunofluorescence secondary antibodies were Alexa Fluor 488, 555, 568, or 647 anti–rabbit, rat, chicken, or isotype specific mouse IgG (Molecular Probes). The secondary antibodies used on Western blots were horseradish peroxidase-goat anti-mouse or anti-rabbit (626420 and G21234; Invitrogen, Carlsbad, CA).

Plasmids, oligos, and peptides

Plasmids encoding C-terminally tagged murine Vps11–HA and Vps16–HA, and N-terminally tagged GFP-murine Vps18 and GFP–Vps39 were gifts from Robert Piper (Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA), Vps18–myc from Liping Wang (Human Nutrition Research Center, UC Davis, CA), N-terminally tagged myc–HRS from Harold Stenmark (Institute for Cancer Research; Oslo University Hospital) (Raiborg et al., 2002 blue right-pointing triangle), and N-terminally tagged GFP–Rab5Q79L from Laura Volpicelli (University of Pennsylvania) (Volpicelli et al., 2001 blue right-pointing triangle). Spe39-enhanced GFP and HA-tagged Vps33a and Vps33b have been described (Zhu et al., 2009 blue right-pointing triangle).

C-terminal Vps41–myc was created from human Vps41 cDNA clone (cat# SC111791; Origene, Rockville, MD) and the primers 5′ caccatggcggaagcagaggag and 3′ ttggagatgaaaaaagaacaaaaacttatttctgaagaagatctgtag using PCR. The PCR product was cloned into TOPO vector pcDNA 3.1 (Invitrogen), following the manufacturer's directions. The TOPO vector DNA was subsequently cut with enzymes BamHI and EcoRV, and the Vps41–myc fragment was subcloned into pIREShyg3. The resulting clone was confirmed as error-free by DNA sequencing.

An N-terminally tagged HA–FKBP–CLC construct was the gift of Enrique Rodriguez-Boulan (Deborde et al., 2008 blue right-pointing triangle). This construct was modified by replacing the HA tag with a fluorescent mCherry tag as follows. We designed PCR primers complementary to mCherry that were flanked by NheI 5′ and BglII 3′(5′ GCTAGCATGGTGAGCAAGGGC and 3′ AGATCTCTTGTACAGCTCGTCCATGC) sites to allow cloning from a pmCherry vector (Clontech) into the TOPO TA 2.1 vector (Invitrogen). The TOPO TA 2.1 mCherry vector and the HA-FKBP–CLC PCR product were each digested with NheI and BglII. Appropriately sized DNA bands were identified on an agarose gel, extracted, and ligated to create mCherry-FKBP–CLC. Coding sequence was verified as error-free by DNA sequencing. The ARGENT Regulated Homodimerization Kit containing FKBP plasmids and the dimerization drug AP20187 was purchased from ARIAD (www.ariad.com; Cambridge; MA).

All siRNA oligos were purchased from Dharmacon (Lafayette, CO). The siCONTROL Non-Targeting siRNA Pool #1 (D0012061305) was used for control knockdown. siGENOME human CLTC, NM_004859 siRNA was used for CHC knockdown (D00400102) sense: GCAAUGAGCUGUUUGAAGAUU, antisense: 5′ pUCUUCAAACAGCUCAUUGCUU. siRNA oligos were transfected as described later in the text. AP3δ (RHS4533-NM_003938) and CHC (RHS39799577067) shRNA in a pLKO.1 vector for lentiviral infection were obtained from Open Biosystems (Huntsville, AL). Control shRNA in pLKO.1 was obtained from Addgene (vector 1864; Cambridge, MA).

The peptide against the epitope for AP3δ SA4 (AQQVDIVTEEMPENALPSDEDDKDPNDPYRA) (Salazar et al., 2009 blue right-pointing triangle) was purchased from the Emory Microchemical Facility (Atlanta, GA) and Invitrogen (EvoQuest Team, Carlsbad, CA).

Cell culture, transfection, and lenti viral infection

HEK293T and HCN-1A cells (American Type Culture Collection [ATCC], Manassas, VA) in DMEM (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 100 μg/ml penicillin and streptomycin (Hyclone) and PC12 cells (ATCC) in DMEM supplemented with 5% FBS, 10% equine serum (Hyclone), and 100 μg/ml penicillin and streptomycin (Hyclone) were incubated at 37ºC with 10% CO2.

For recombinant DNA expression, HEK293T cells were transfected in six-well dishes with 0.5–2.0 μg DNA in 0.25% Lipofectamine 2000 (Invitrogen) diluted in Opti-Mem (Life Technologies, Grand Island, NY). Cells were transfected for 4 h followed by incubation in either culture medium alone or culture medium containing the selection drug(s) G418 (0.1 mg/ml) and/or hygromycin (0.1 mg/ml) for the maintenance of stable cell lines. PC12 cells were transfected by nucleofection with 3 μg of DNA using Amaxa Cell Line Nucleofector Kit V (cat# VCA-1003; Lonza Walkersville, Koeln, Germany, www.lonza.com), and were plated on Matrigel-coated glass-bottom culture dishes (Matek, Ashland, MA) in PC12 culture medium supplemented with 100 ng/ml NGF 2.5S (murine, natural) (cat# 13257-019; Invitrogen). PC12 cells were differentiated for 48–72 h at 37ºC with 10% CO2 For siRNA knockdown, HEK293T cells were transfected in six-well dishes with 50 nM oligo for 4 h, incubated for 20 h in culture medium, and transfected a second time with 50 nM oligo for 4 h followed by incubation for 3 d in culture medium. For lentiviral infection, HEK293T cells seeded in 10-cm plates were infected with 1 μl of high-titer lentivirus containing the shRNA constructs mentioned earlier in the text. Following a 24-h infection, cells were incubated for up to 6 d in culture medium supplemented with 4 μg/ml puromycin for selection. The Emory Neuroscience NINDS Viral Vector Core Facilities prepared all high-titer lentiviruses.

Cross-linking and immunoprecipitation

Cross-linking was performed as previously described using DSP (Craige et al., 2008 blue right-pointing triangle; Salazar et al., 2009 blue right-pointing triangle; Zlatic et al., 2010 blue right-pointing triangle). DSP is a homobifunctional reversible and cell-permeable cross-linker with a 12-Å spacer arm that stabilizes labile protein interactions (Lomant and Fairbanks, 1976 blue right-pointing triangle). Plates of confluent HEK293T cells were placed on ice, washed twice with ice-cold phosphate-buffered saline (PBS)/1 mM MgCl2/0.1 mM CaCl2, and then incubated with 1 mM DSP (cat# 22585; Thermo Scientific, Rockford, IL) or dimethyl sulfoxide control in PBS/1 mM MgCl2/0.1 mM CaCl2 for 2 h on ice. DSP was then quenched with 25 mM TRIS, pH 7.4, followed by two rinses with ice-cold PBS/1 mM MgCl2/0.1 mM CaCl2. Cells were lysed in Buffer A (150 mM NaCl, 10 mM HEPES, 1 mM EGTA, 0.1 mM MgCl2) + 0.5% Triton X-100 by incubation for 30 min at 4ºC. Any remaining cellular debris was scraped from plates and centrifuged at 16,000 × g for 10 min. The supernatant was collected, diluted to 1 μg/ul in Buffer A + 0.5% Triton X-100, and incubated with Dynal immunomagnetic precipitation beads (Dynal, Oslo, Norway) in the absence or presence of a 10 μM SA4 peptide competitor. Beads and lysate were incubated at 4ºC for 2 h and washed six times with Buffer A containing 0.1% Triton X-100 to remove nonspecifically bound material. The material that remained bound after these washes was then eluted by treatment with either the peptide antigen or SDS–PAGE sample buffer followed by incubation at 75ºC for 5 min. Immunoprecipitated material was analyzed on SDS–PAGE Western blot.

Despite reduced detection of δ in SA4 immunoprecipitates, all other AP-3 subunits are readily detectable in these immunocomplexes (Salazar et al., 2009 blue right-pointing triangle). Decreased detection of AP-3 δ by the SA4 mAb in immunoblots after DSP cross-linking likely reflects a chemical modification of the SA4 epitope by the cross-linking agent. The lysine and arginine present in this peptide are susceptible to modification by DSP. The SA4 antibody is used sequentially in immunoprecipitation and immunoblot, thus effectively magnifying the difference between (–) and (+) DSP samples.

Immunolocalization, microscopy, and quantification

Coverslips were prepared as previously described (Faundez et al., 1997 blue right-pointing triangle). Cells were seeded onto Matrigel (BD Bioscience, San Jose, CA)-coated glass coverslips, washed twice in PBS/1 mM MgCl2/0.1 mM CaCl2, and fixed using 4% paraformaldehyde in PBS. Cells were then permeabilized and blocked with 0.02% saponin (Sigma), 15% horse serum (Hyclone), 2% bovine serum albumin, and 1% fish skin gelatin (Sigma) in PBS. Blocked and permeabilized cells were incubated with the primary and secondary antibodies described earlier in the text and were mounted with Gelvatol onto slides. Fixed-cell confocal microscopy was performed on fixed cells as described (Deborde et al., 2008 blue right-pointing triangle) using an Axiovert 100M microscope (Carl Zeiss, Thornwood, NY) with Argon/HeNe (488/543) laser excitation. Images were captured using a Plan Apochromat 63×/1.4 oil DIC objective, BP 505–550/LP 560 filter set, and LSM 510 3.2.0.104 software (Carl Zeiss). Deconvolution microscopy was performed as described (Deborde et al., 2008 blue right-pointing triangle) with a 200M inverted microscope using 63×/1.4 and 100×/1.4 oil DIC objectives (Carl Zeiss) and a Sedat filter set. Images were collected using a scientific grade cooled charge-coupled Cool-Snap HQ camera with ORCA-ER chip on a multiwavelength, wide-field, three-dimensional microscopy system using Slidebook 4.0 OS X software (Intelligent Imaging Innovations, Denver, CO). Out-of-focus light was removed with a constrained iterative deconvolution algorithm (Swedlow et al., 1997 blue right-pointing triangle). Confocal and deconvolution images were processed and analyzed using LSM Image Browser 4.0.0.157 (Carl Zeiss), Metamorph software version 6.1 (Universal Imaging, Sunnyvale, CA), and Imaris 6.3.1 software (Bitplane, St. Paul, MN). Colocalization and puncta size were determined from three consecutive z-series focal planes per image. All channels were thresholded individually. Percent colocalization was determined by calculation of pixel area containing fluorescent signals from two channels per total pixel area for a single fluorophore. Puncta size was determined by integrated morphometric analysis of total pixel area within puncta as determined by image thresholding.

Clathrin-coated vesicle isolation

HEK293T or HEK293T cells stably expressing tagged HOPS or class C Vps proteins were used to prepare clathrin-coated vesicles as described previously (alternate protocol 2 in Girard et al., 2004 blue right-pointing triangle). Briefly, cells were grown to confluence and washed twice with PBS/1 mM MgCl2/0.1 mM CaCl2. Cells were lifted in CCV buffer (100 mM MES, 1.0 mM EGTA, 0.5 mM MgCl2, pH 6.5), transferred to a Potter Elvehjem glass-teflon homogenizer, and homogenized for 10 strokes at 1500 rpm on a Tri-R Stir-R variable speed laboratory motor (Model S63C; Tri-R Instruments, Rockville Center, NY). Homogenate was centrifuged in an SS-34 fixed-angle rotor for 20 min at 17,000 × g, 4ºC. The resulting supernatant was further centrifuged in a type 40 fixed-angle rotor for 60 min at 56,000 × g, 4ºC. The resulting pellet was resuspended, homogenized, and transferred to polyallomer centrifuge tubes (Beckman, Palo Alto, CA). The sample was then underlaid with D2O-sucrose solution (8% sucrose, 100 mM MES, 1.0 mM EGTA, 0.5 mM MgCl2, D2O) (D2O cat# 364312-10G; Sigma) and was centrifuged in a SW-55 swing-bucket rotor for 2 h at 116,000 × g, 4ºC. This final, clathrin-coated, vesicle-enriched pellet was resuspended and aliquoted along with reserves from previous fractionation steps and was run on SDS–PAGE gels for Western blotting or Coomassie stain.

Acute clathrin perturbation

HEK293T or HEK293T cells stably expressing tagged HOPS or class C Vps proteins and/or transiently expressing tagged FKBP–CLC were incubated for 2 h at 37ºC, 10% CO2 in culture medium supplemented with 50 nM AP20187 (ARIAD) or 0.05% ethanol vehicle control. Culture dishes were then placed on ice and processed for either immunolocalization or cross-linking followed by immunoprecipitation.

Live cell imaging

HEK293T or PC12 cells expressing Vps18–GFP and/or mCherry-FKBP–CLC were grown on Matrigel-coated glass-bottom culture dishes (Matek). Imaging medium consisted of Hank's balanced salt solution minus phenol red and NaHCO2 (Sigma) and supplemented with 10% FBS (Hyclone) and 20 mM HEPES for HEK293T, and with 10% Donor Equine Serum (Hyclone), 5% FBS (Hyclone), and 100 ng/ml NGF 2.5S (murine, natural, cat# 13257–019; Invitrogen) for PC12 cells. Live imaging was performed on an A1R Laser Scanning Confocal Microscope (Nikon, Melville, NY) equipped with a hybrid scanner, Perfect Focus, and an environmental chamber for regulation of temperature to 37ºC and 10% CO2. Fluorophores were alternately excited with 488 and 568 nm wavelength laser every 5 min for 2 h and 30 min. AP20187 (Ariad) or ethanol vehicle control was added to imaging medium at concentrations listed earlier in the text after the first 25 min of imaging. Images were captured with an APO TIRF 60×/1.49 oil DIC objective and 500–550/570–620 filter sets on NIS-Elements AR 3.1 (Nikon) software. NIS-Elements AR 3.0, Imaris 6.3.1 (Bitplane), and Image J 1.41(NIH) software were used for image analysis.

Statistical analysis

Experimental conditions were compared with the nonparametric Wilcoxon–Mann–Whitney Rank Sum Test using Synergy KaleidaGraph v4.03 (Reading, PA) or StatPlus Mac Built5.6.0pre/Universal (AnalystSoft, Vancouver, Canada). Data are presented as boxplots displaying the four quartiles of the data, with the “box” comprising the two middle quartiles, separated by the median. The upper and lower quartiles are represented by single lines extending from the box. Circles correspond to outlier points defined by the statistical software as beyond the upper or lower quartile plus 1.5 times the value of the 2–3 interquartile distance.

Supplementary Material

[Supplemental Materials]

Acknowledgments

This work was supported by grants from the National Institutes of Health to V.F. (NS42599 and GM077569) and to S.W.L. (GM082932) and by the University Research Committee. S.A.Z. was supported by T32 GM008367, National Institutes of Health, Training Program in Biochemistry, Cell, and Molecular Biology. We are indebted to the Faundez lab members for their comments. The work was also supported by the Neuronal Imaging and Lentiviral Cores of the Emory Neuroscience NINDS Core Facilities Grant P30NS055077.

Abbreviations used:

AP-3
Adaptor Protein Complex 3
CHC
clathrin heavy chain
CLC
clathrin light chain
DSP
dithiobissuccinimidyl propionate
FBS
fetal bovine serum
FKBP
FK506 binding protein
GFP
green fluorescent protein
HA
hemagglutinin
HOPS
HOmotypic fusion and Protein Sorting
HPS
Hermansky Pudlak Syndrome
Hrs
hepatocyte growth factor receptor substrate
Ig
immunoglobulin
mAb
monoclonal antibody
PBS
phosphate-buffered saline
SNARE
Soluble NSF Attachment Protein Receptor
VAMP7
vesicle-associated membrane protein 7

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10-10-0799) on March 16, 2011.

REFERENCES

  • Anand VC, Daboussi L, Lorenz TC, Payne GS. Genome-wide analysis of AP-3-dependent protein transport in yeast. Mol Biol Cell. 2009;20:1592–1604. [PMC free article] [PubMed]
  • Angers CG, Merz AJ. HOPS interacts with Apl5 at the vacuole membrane and is required for consumption of AP-3 transport vesicles. Mol Biol Cell. 2009;20:4563–4574. [PMC free article] [PubMed]
  • Angers CG, Merz AJ. New links between vesicle coats and Rab-mediated vesicle targeting. Semin Cell Dev Biol. 2011;22:18–26. [PMC free article] [PubMed]
  • Bonifacino JS, Glick BS. The mechanisms of vesicle budding and fusion. Cell. 2004;116:153–166. [PubMed]
  • Borner GH, Harbour M, Hester S, Lilley KS, Robinson MS. Comparative proteomics of clathrin-coated vesicles. J Cell Biol. 2006;175:571–578. [PMC free article] [PubMed]
  • Bright NA, Gratian MJ, Luzio JP. Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells. Curr Biol. 2005;15:360–365. [PubMed]
  • Craige B, Salazar G, Faundez V. Phosphatidylinositol-4-kinase type II alpha contains an AP-3 sorting motif and a kinase domain that are both required for endosome traffic. Mol Biol Cell. 2008;19:1415–1426. [PMC free article] [PubMed]
  • Cullinane AR, et al. Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization. Nat Genet. 2010;42:303–312. [PubMed]
  • Darsow T, Katzmann DJ, Cowles CR, Emr SD. Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains. Mol Biol Cell. 2001;12:37–51. [PMC free article] [PubMed]
  • Deborde S, Perret E, Gravotta D, Deora A, Salvarezza S, Schreiner R, Rodriguez-Boulan E. Clathrin is a key regulator of basolateral polarity. Nature. 2008;452:719–723. [PMC free article] [PubMed]
  • Delevoye C, et al. AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis. J Cell Biol. 2009;187:247–264. [PMC free article] [PubMed]
  • Dell’Angelica EC. AP-3-dependent trafficking and disease: the first decade. Curr Opin Cell Biol. 2009;21:552–559. [PubMed]
  • Dell’Angelica EC, Klumperman J, Stoorvogel W, Bonifacino JS. Association of the AP-3 adaptor complex with clathrin. Science. 1998;280:431–434. [PubMed]
  • Di Pietro SM, Dell’Angelica EC. The cell biology of Hermansky-Pudlak syndrome: recent advances. Traffic. 2005;6:525–533. [PubMed]
  • Faundez V, Horng JT, Kelly RB. ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells. J Cell Biol. 1997;138:505–515. [PMC free article] [PubMed]
  • Faundez V, Kelly RB. The AP-3 complex required for endosomal synaptic vesicle biogenesis is associated with a casein kinase I alpha-like isoform. Mol Biol Cell. 2000;11:2591–2604. [PMC free article] [PubMed]
  • Girard M, Allaire PD, Blondeau F, McPherson PS. Isolation of clathrin-coated vesicles. In: Bonifacino JS, Dasso M, Harford J, Lippincott-Schwartz J, editors. Current Protocols in Cell Biology. Vol. 1. K. Yamada, New York: John Wiley & Sons; 2004. pp. 3.13.11–13.13.31.
  • Kim BY, Kramer H, Yamamoto A, Kominami E, Kohsaka S, Akazawa C. Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7. J Biol Chem. 2001;276:29393–29402. [PubMed]
  • Kim S, Wairkar YP, Daniels RW, DiAntonio A. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation. J Cell Biol. 2010;188:717–734. [PMC free article] [PubMed]
  • Li W, Rusiniak ME, Chintala S, Gautam R, Novak EK, Swank RT. Murine Hermansky-Pudlak syndrome genes: regulators of lysosome-related organelles. Bioessays. 2004;26:616–628. [PubMed]
  • Lomant AJ, Fairbanks G. Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate) J Mol Biol. 1976;104:243–261. [PubMed]
  • Markgraf DF, Ahnert F, Arlt H, Mari M, Peplowska K, Epp N, Griffith J, Reggiori F, Ungermann C. The CORVET subunit Vps8 cooperates with the Rab5 homolog Vps21 to induce clustering of late endosomal compartments. Mol Biol Cell. 2009;20:5276–5289. [PMC free article] [PubMed]
  • Moskowitz HS, Heuser J, McGraw TE, Ryan TA. Targeted chemical disruption of clathrin function in living cells. Mol Biol Cell. 2003;14:4437–4447. [PMC free article] [PubMed]
  • Nickerson DP, Brett CL, Merz AJ. Vps-C complexes: gatekeepers of endolysosomal traffic. Curr Opin Cell Biol. 2009;21:543–551. [PMC free article] [PubMed]
  • Nordmann M, Cabrera M, Perz A, Brocker C, Ostrowicz C, Engelbrecht-Vandre S, Ungermann C (2010). The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Curr Biol. 20:1654–1659. [PubMed]
  • Odorizzi G, Cowles CR, Emr SD. The AP-3 complex: a coat of many colours. Trends Cell Biol. 1998;8:282–288. [PubMed]
  • Ostrowicz CW, Brocker C, Ahnert F, Nordmann M, Lachmann J, Peplowska K, Perz A, Auffarth K, Engelbrecht-Vandre S, Ungermann C. Defined subunit arrangement and Rab interactions are required for functionality of the HOPS tethering complex. Traffic. 2010;11:1334–1346. [PubMed]
  • Ostrowicz CW, Meiringer CT, Ungermann C. Yeast vacuole fusion: a model system for eukaryotic endomembrane dynamics. Autophagy. 2008;4:5–19. [PubMed]
  • Peden AA, Oorschot V, Hesser BA, Austin CD, Scheller RH, Klumperman J. Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins. J Cell Biol. 2004;164:1065–1076. [PMC free article] [PubMed]
  • Peplowska K, Markgraf DF, Ostrowicz CW, Bange G, Ungermann C. The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis. Dev Cell. 2007;12:739–750. [PubMed]
  • Poteryaev D, Datta S, Ackema K, Zerial M, Spang A. Identification of the switch in early-to-late endosome transition. Cell. 2010;141:497–508. [PubMed]
  • Progida C, Cogli L, Piro F, De Luca A, Bakke O, Bucci C. Rab7b controls trafficking from endosomes to the TGN. J Cell Sci. 2010;123:1480–1491. [PubMed]
  • Raiborg C, Bache KG, Gillooly DJ, Madshus IH, Stang E, Stenmark H. Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat Cell Biol. 2002;4:394–398. [PubMed]
  • Raiborg C, Wesche J, Malerod L, Stenmark H. Flat clathrin coats on endosomes mediate degradative protein sorting by scaffolding Hrs in dynamic microdomains. J Cell Sci. 2006;119:2414–2424. [PubMed]
  • Rehling P, Darsow T, Katzmann DJ, Emr SD. Formation of AP-3 transport intermediates requires Vps41 function. Nat Cell Biol. 1999;1:346–353. [PubMed]
  • Richardson SC, Winistorfer SC, Poupon V, Luzio JP, Piper RC. Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Mol Biol Cell. 2004;15:1197–1210. [PMC free article] [PubMed]
  • Rink J, Ghigo E, Kalaidzidis Y, Zerial M. Rab conversion as a mechanism of progression from early to late endosomes. Cell. 2005;122:735–749. [PubMed]
  • Ronnett GV, Hester LD, Nye JS, Connors K, Snyder SH. Human cortical neuronal cell line: establishment from a patient with unilateral megalencephaly. Science. 1990;248:603–605. [PubMed]
  • Salazar G, Craige B, Wainer BH, Guo J, De Camilli P, Faundez V. Phosphatidylinositol-4-kinase type II {alpha} is a component of adaptor protein-3-derived vesicles. Mol Biol Cell. 2005;16:3692–3704. [PMC free article] [PubMed]
  • Salazar G, Zlatic S, Craige B, Peden AA, Pohl J, Faundez V. Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and nonneuronal cells. J Biol Chem. 2009;284:1790–1802. [PubMed]
  • Seeger M, Payne GS. A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO J. 1992;11:2811–2818. [PubMed]
  • Stoorvogel W, Oorschot V, Geuze HJ. A novel class of clathrin-coated vesicles budding from endosomes. J Cell Biol. 1996;132:21–33. [PMC free article] [PubMed]
  • Stoorvogel W, Strous GJ, Geuze HJ, Oorschot V, Schwartz AL. Late endosomes derive from early endosomes by maturation. Cell. 1991;65:417–427. [PubMed]
  • Suzuki T, Oiso N, Gautam R, Novak EK, Panthier JJ, Suprabha PG, Vida T, Swank RT, Spritz RA. The mouse organellar biogenesis mutant buff results from a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation. Proc Natl Acad Sci USA. 2003;100:1146–1150. [PubMed]
  • Swedlow JR, Sedat JW, Agard DA. Deconvolution in optical microscopy. In: Jansson PA, editor. Deconvolution of Images and Spectra. San Diego, CA: Academic Press; 1997. pp. 284–307.
  • Theos AC, et al. Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes. Mol Biol Cell. 2005;16:5356–5372. [PMC free article] [PubMed]
  • Volpicelli LA, Lah JJ, Levey AI. Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies. J Biol Chem. 2001;276:47590–47598. [PubMed]
  • Wang Y, Chen T, Han C, He D, Liu H, An H, Cai Z, Cao X. Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4. Blood. 2007;110:962–971. [PubMed]
  • Wickner W. Membrane fusion: Five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. Annu Rev Cell Dev Biol. 2010;26:115–136. [PubMed]
  • Zhu GD, Salazar G, Zlatic SA, Fiza B, Doucette MM, Heilman CJ, Levey AI, Faundez V, L’Hernault S W. SPE-39 family proteins interact with the HOPS complex and function in lysosomal delivery. Mol Biol Cell. 2009;20:1223–1240. [PMC free article] [PubMed]
  • Zlatic SA, Ryder PV, Salazar G, Faundez V. Isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography. J Vis Exp. 2010:1855. [pii] 1810.3791/1855. [PMC free article] [PubMed]

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