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Vol. 18, Issue 3, 768-780, March 2007
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*Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104; Institut Curie, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 144, Paris 75248, France; Department of Basic Medical Sciences, St. George's Hospital Medical School, London, SW17 ORE, United Kingdom; Comparative Genetics Program, Texas A&M University, College Station, TX 77843; and ||Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA 90095
Submitted December 4, 2006;
Accepted December 7, 2006
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wei, 2006). HPS or a similar disorder in mice results from mutations in any of at least 15 genes (Wei, 2006). All of these genes are ubiquitously expressed, but their mutation in HPS affects mainly the generation and function of selected tissue-specific lysosome-related organelles (LROs; Bonifacino, 2004; Di Pietro and Dell'Angelica, 2005). Those LROs that are most severely affected in all forms of HPS—pigment cell melanosomes, platelet dense granules, and lung lamellar bodies—are unique in that they coexist with bona fide lysosomes in their respective cell types (Dell'Angelica et al., 2000; Marks and Seabra, 2001). The 15 known HPS-associated genes have been identified, and although the products of most are thought to participate in trafficking events that are uniquely required to form this class of LRO, the function of only a few is understood in detail.
The genes disrupted in human HPS-7 (Li et al., 2003) and HPS-8 (Morgan et al., 2006) and in the mouse HPS models pallid, muted, reduced pigmentation (rp), cappuccino, and sandy encode five of the eight known subunits of a stable protein complex known as biogenesis of lysosome-related organelles complex (BLOC)-1 (Falcon-Perez et al., 2002; Moriyama and Bonifacino, 2002; Ciciotte et al., 2003; Li et al., 2003; Gwynn et al., 2004; Starcevic and Dell'Angelica, 2004). To date, no specific subcellular function has been assigned to BLOC-1. A role for BLOC-1 in synaptic function is suggested by a putative, but disputed (Vites et al., 2004), interaction of its snapin subunit with the target membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) synaptosome-associated protein of 25 kDa (SNAP-25) and its nonneuronal paralogue SNAP-23 (Ilardi et al., 1999; Buxton et al., 2003), by the linkage of dysbindin subunit gene polymorphisms with schizophrenia (Straub et al., 2002; Numakawa et al., 2004), and by the copurificaton of BLOC-1 with synaptic vesicles from PC12 cells (Salazar et al., 2005). BLOC-1 in nonneuronal cells has been suggested to function on endosomes based on the interaction of its pallidin subunit with the endosomal tSNARE, syntaxin 13 (syn13; Huang et al., 1999; Moriyama and Bonifacino, 2002), and on altered intracellular distribution or cell surface accumulation of endosomal proteins in BLOC-1–deficient fibroblasts (Di Pietro et al., 2006; Salazar et al., 2006). In melanocytes, a cell type affected by HPS, BLOC-1 localizes at steady state to a subdomain of early endosomes and influences the cell surface flux of a melanosomal protein, tyrosinase-related protein-1 (Tyrp1; Di Pietro et al., 2006). Despite these observations, neither the pathway by which BLOC-1–dependent cargoes travel nor the specific transport step regulated by BLOC-1 is known. Furthermore, although BLOC-1 interacts physically with two other protein complexes—BLOC-2 and adaptor protein (AP)-3—that are defective in different forms of HPS (Di Pietro et al., 2006), and AP-3 regulates cargo transport in melanocytes (Huizing et al., 2001; Theos et al., 2005), a functional link between these complexes has not been well established. Here, we exploit primary and immortalized melanocytes from HPS model mice to identify a vesicular transport step regulated by BLOC-1 that is required for trafficking of selected cargo from early endosomes to maturing melanosomes. We also provide evidence that BLOC-2, but not AP-3, functions in the same pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies
Monoclonal antibodies, their targets, and sources were as follows: TA99 to Tyrp1 and 7G7.B6 to Tac were from American Type Culture Collection (Manassas, VA); YOL1/2 to tubulin was from Santa Cruz Biotechnology (Santa Cruz, CA); 1D4B to mouse lysosomal-associated membrane protein (LAMP)-1 and H4A3 to human LAMP-1 were from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); HMB-45 and HMB-50 to human Pmel17 were from Lab Vision (Fremont, CA); anti-mouse Tf receptor (CD71) was from BD Transduction Laboratories (San Diego, CA); H68.4 anti-human Tf receptor was from Zymed Laboratories (South San Francisco, CA); anti-HA.11 was from Covance Research Products (Berkeley, CA); and anti-
adaptin (Peden et al., 2004) was a gift from A. Peden (Cambridge University, Cambridge, United Kingdom). Polyclonal rabbit antisera to pallidin (Moriyama and Bonifacino, 2002) (a gift from J. Bonifacino, National Institutes of Health, Bethesda, MD), dysbindin and BLOS3 subunits of BLOC-1 (Starcevic and Dell'Angelica, 2004), syn13 (Prekeris et al., 1998), tyrosinase (Theos et al., 2005), and mouse Pmel17 (Theos et al., 2006) have been described previously. Goat anti-EEA1, rabbit anti-Myc and H-90 rabbit anti-Tyrp1 (used for immunoblotting) were from Santa Cruz Biotechnology. FITC-, rhodamine red-X–, and 7-amino-4-methylcoumarin-3-acetic acid–conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA); rabbit anti-Alexa-488–, anti-FITC–, and Alexa-488–conjugated secondary antibodies were from Invitrogen. Conjugation of Alexa-488 or R-phycoerythrin (R-PE) to monoclonal antibodies was performed using protein labeling kits from Invitrogen or Prozyme (San Leandro, CA), respectively.
DNA Constructs
Retroviral constructs encoding human HA11-epitope tagged pallidin (PaHA) and HA11-epitope tagged muted (MuHA) were generated by subcloning XhoI–NotI inserts from pXS vectors (Moriyama and Bonifacino, 2002), via pCI-neo intermediates, into pBMN-IRES-Hygro (a gift from R. Scheller, Genentech, San Francisco, CA). Untagged or Myc-epitope–tagged constructs were generated the same way, and untagged human BLOS3 was amplified from I.M.A.G.E. clone 3627463 (GenBank AY531266) and subcloned into the BamHI and XhoI sites of pBMN-IRES-Hygro. Mutagenesis of C-terminal amino acids L514A, L515A, and Y526A of Tyrp1 (Tyrp1 LLY), subcloned as a EcoRI fragment from pCDNA3-TRP-1 (a gift from Dr. W. Storkus, University of Pittsburgh, Pittsburgh, PA) in pCDM8.1 (Marks et al., 1996), was carried out using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All plasmid inserts were verified by DNA sequencing.
Cell Culture and Transgene Expression
Primary melanocytes were isolated from neonatal C57BL/6J (wild type) and pallid (B6.Cg-PldnPa/J, formerly C57BL/6J-pa) mice as described previously (Sviderskaya et al., 1997) and used within the first three passages. Immortal melanocyte cell lines melan-mu1, -2, and -3 and melan-pa1, -2, and -3 were grown from skins of neonatal muted (Mutedmu/mu) Ink4a-Arf–/– B6.CHMU/Le and pallid Ink4a-Arf–/– mice, respectively, as described previously (Sviderskaya et al., 2002). Melan-mu3 (referred to as melan-mu), melan-rp2 (Gwynn et al., 2004) (referred to as melan-rp; Bloc1s3rp/rp), melan-pa1 (referred to as melan-pa), melan-coa2 (Suzuki et al., 2001) (referred to as melan-coa; Hps3coa/coa), and melan-a (Bennett et al., 1987) were maintained as described previously (Sviderskaya et al., 2002). Retrovirus production from transiently transfected 293T cells and transduction of primary melanocytes and cell lines were carried out as described previously (Swift et al., 1999). Stable transductants were selected in medium containing 200–400 µg/ml hygromycin B. The 1011-mel cells were maintained as described previously (Berson et al., 2001), transiently transfected using FuGENE-6 with 3 µg of DNA in a six-well dish, and analyzed 48 h after transfection.
Immunofluorescence Microscopy and Immunoblotting
Cells were fixed with 2% formaldehyde, labeled with primary and fluorochrome-conjugated secondary antibodies as described previously (Berson et al., 2001), and analyzed on a DM IRBE microscope (Leica Microsystems, Wetzlar, Germany) equipped with an Orca digital camera (Hamamatsu, Bridgewater, NJ). Images were captured and manipulated using OpenLab software (Improvision, Lexington, MA) with the volume deconvolution package; regions were magnified for insets by using Adobe Photoshop (Adobe Systems, Mountain View, CA). To quantify marker overlap, deconvolved paired images were rendered binary by density slicing and the total area of overlap between them was calculated for objects containing more than 5 pixels, excluding the densely labeled perinuclear area. Eight to 14 cell profiles comprising >10,000 objects were quantified for each pairwise comparison. For cell surface labeling, cells on coverslips were incubated with medium containing Alexa-488–conjugated TA99 for 30–45 min at 4°C, washed with ice-cold PBS, and then fixed as described above. For immunoblots, cells were lysed in buffer (100 mM Tris-Cl, pH 7.6, 100 mM NaCl, 1% SDS, and protease inhibitor cocktail), sonicated for 1–2 min in a water bath sonicator, clarified by centrifugation, fractionated, and immunoblotted as described previously (Berson et al., 2001). Immunoblots were developed with enhanced chemo fluorescence (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and analyzed using a STORM PhosphorImager and ImageQuant software (GE Healthcare).
Electron Microscopy
Tf-FITC was internalized for 15 min as described previously (Theos et al., 2005). Cells were fixed with 2% (wt/vol) paraformaldehyde with or without 0.2% (wt/vol) glutaraldehyde, in 0.1 M phosphate buffer, pH 7.4, and processed for ultracryomicrotomy as described previously (Raposo et al., 1997). Ultrathin cryosections were prepared with an ultracryomicrotome Ultracut FCS (Leica, Vienna, Austria), and single or double immunogold labeled using protein A conjugated to 10- or 15-nm gold (PAG10, PAG15). Sections were analyzed under a Philips CM120 electron microscope (FEI, Eindoven, The Netherlands), and digital acquisitions were made with a numeric camera Keen View (Soft Imaging System, Munster, Germany). Quantification of Tyrp1 and tyrosinase distribution on the different intracellular compartments was performed as described previously (Theos et al., 2005).
Flow Cytometry
Cell Surface Levels.
Cells were harvested in Trypsin-EDTA, washed and suspended in growth medium (with 10% fetal bovine serum [FBS], 1% L-glutamine, and 25 mM HEPES, pH 7.4) containing saturating concentrations of unconjugated or Alexa-488–conjugated primary antibody on ice for 30–45 min. Cells were washed twice with ice-cold growth medium, incubated for 30 min on ice with Alexa-488-conjugated secondary antibody, and washed again where necessary, and then resuspended in ice-cold fluorescence-activated cell sorting (FACS) buffer (5% FBS, 1 mM EDTA, and 0.02% sodium azide in PBS) for analysis on a FACScan Plus by using CellQuest Pro software (BD Biosciences). Mean fluorescence intensity (MFI) was calculated by subtracting a background value obtained with 7G7.B6 anti-Tac antibody from the value of experimental samples. Melan-mu and melan-rp values were normalized to those of melan-mu:MuHA and melan-rp:BLOS3, respectively, within each experiment.
Endocytosis. Cells in suspension were incubated with growth medium containing unconjugated primary antibody on ice for 30 min, washed twice with ice-cold growth medium, and then incubated at 37°C to allow internalization. At each time point, samples were transferred to ice and Alexa-488–conjugated secondary antibodies were added to detect remaining cell surface molecules. Samples were washed twice with ice-cold medium, suspended in FACS buffer, and analyzed as described above. The % starting MFI for each experiment was calculated by dividing the MFI value of each time point with that of the zero time point. Endocytic rates were calculated from mean % starting MFI values within the first 2 min of internalization (for which the data of all samples could be reasonably fitted to a single exponential decay) by a log regression analysis by using LOGEST in Microsoft Excel (Microsoft, Redmond, WA).
Recycling.
Tyrp1 recycling was measured essentially as described previously (Peden et al., 2004). Briefly, cells were incubated with growth medium containing Alexa-488–conjugated TA99 antibody (or 7G7.B6 as a control) on ice for 30 min, washed twice with ice-cold growth medium, and incubated at 37°C for 5 min to internalize antibody-bound Tyrp1. Cells were then incubated on ice with rabbit anti Alexa-488 antibody to quench remaining cell surface fluorescence, washed twice with ice-cold growth medium, and divided into two pools. Cells were incubated for indicated times at 37°C in medium containing either rabbit anti-Alexa-488 or rabbit anti-Myc as a control, washed twice with ice-cold medium, suspended in FACS buffer, and analyzed as described above. The % starting MFI values at each time point for samples with anti-Alexa-488 were normalized to those with anti-Myc (normalized % MFI). Recycling rates were calculated from mean % starting MFI values within the first 10 min of recycling by log regression analysis. Identical results were obtained using a Fab fragment of TA99 generated with the ImmunoPure Fab preparation kit (Pierce Chemical, Rockford, IL).
For Tf receptor recycling, cells were washed, resuspended in serum-free medium containing 0.1% BSA, and starved for 30 min at 37°C. Cells were then incubated in the same medium containing 50–60 µg/ml Tf-FITC for 30 min at 37°C to allow Tf-FITC uptake, washed, and suspended in growth medium containing 10% FBS. Then, cells were incubated for the indicated times at 37°C to release the recycled Tf-FITC to the medium. Samples were washed twice with ice-cold medium, suspended in FACS buffer, and analyzed as described above.
Cell Surface Delivery. Cells were incubated with growth medium containing Alexa-488–conjugated TA99 on ice for 30 min, then additionally at 37°C for the indicated times. Cells were then washed twice with ice-cold medium, suspended in FACS buffer and analyzed as described above. In some experiments, cells were preincubated with unconjugated TA99 for 15 min on ice and 37°C for 20 min before washing twice and incubation with Alexa-488–conjugated antibody to saturate the cohort of recycling Tyrp1. For brefeldin A (BFA)-treated samples, 10 µg/ml BFA was added to medium during a 60-min preincubation at 37°C and to all incubation and wash media.
Endocytosis Competition.
The experiments were essentially as described previously (Marks et al., 1996). Briefly, 1011-mel cells were transfected with pCDM8.1, pCDM8.1-Tyrp1(WT), or pCDM8.1-Tyrp1(LLY). Cells were harvested 48 h after transfection and divided into two pools. Samples were incubated on ice for 30–45 min in growth medium containing R-PE–conjugated TA99 with either H4A3 anti-LAMP-1– or Alexa-488–conjugated HMB-50. Cells labeled with H4A3 were washed twice with growth medium and incubated in growth medium containing Alexa-488–conjugated isotype-specific anti-IgG1 antibody on ice for 30–45 min. All samples were washed twice with growth medium, resuspended in FACS buffer, and analyzed as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) melanocytes from wild-type C57BL/6J mice harbor highly pigmented melanosomes, visible by bright field microscopy. By contrast, nearly no pigment was observed in primary melanocytes from pallid mice, which lack the BLOC-1 subunit pallidin (Figure 1b), or in immortal melanocytes generated from muted (melan-mu; Figure 1d), pallid (melan-pa; see Figure 9c), or rp (melan-rp; our unpublished data) mice, which lack the BLOC-1 subunits muted, pallidin, and BLOS3, respectively. Modest pigmentation of primary pallid melanocytes developed only with increasing passage, likely due to senescence (Sviderskaya et al., 2002). On infection of early passage pallid melanocytes with recombinant retroviruses expressing hemagglutinin (HA)-epitope–tagged pallidin (PaHA), but not HA-tagged muted, 
10–15% of the cells developed pigment within 3–5 d (Figure 1c). Similarly, pigmentation developed in melan-mu cells stably reexpressing wild-type muted with an HA- (MuHA; Figure 1, e and f) or myc-epitope tag or no tag (our unpublished data), but not PaHA, and in melan-rp and melan-pa cells by stably reexpressing BLOS3 (our unpublished data) and PaHA, respectively (Supplemental Figure S4). MuHA expression in melan-mu cells stabilized the additional BLOC-1 subunits pallidin, dysbindin, and BLOS3 (Figure 1g), indicating reconstitution of the BLOC-1 holocomplex. These results establish that hypopigmentation of muted, pallid, and rp melanocytes is a direct consequence of the deficiency in BLOC-1 and can be rapidly restored upon restoration of the complex.
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) and of reconstituted BLOC-1 in melan-mu:MuHA cells to similar tubulovesicular structures near the Golgi, melanosomes, and endosomal vacuoles (Supplemental Figure S3, b and c).
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) and indicates that the BLOC-1 transport defect to melanosomes is cargo selective.
BLOC-1 Prevents Biosynthetic Delivery of Tyrp1 to the Cell Surface
Tyrp1 was enriched at the surface of BLOC-1–deficient melanocytes relative to controls, as shown by IFM analysis of primary melanocytes labeled for Tyrp1 before fixation (Figure 6a) and quantitated by flow cytometry of melan-mu and melan-rp cells relative to BLOC-1–reconstituted cells (Figure 6b; the more modest Tyrp1 surface expression in melan-rp relative to melan-mu likely reflects residual BLOC-1 activity in rp cells; Starcevic and Dell'Angelica, 2004; our unpublished data). Nevertheless, BLOC-1–deficient cells had lower total cellular Tyrp1 levels (60% that of BLOC-1–reconstituted cells; Figure 6c), indicating that the increased surface levels reflect Tyrp1 missorting and not increased expression; the decreased Tyrp1 cellular levels in these immortalized BLOC-1–deficient melanocytes may reflect dysregulated expression, because Tyrp1 half-life, measured by metabolic pulse/chase analysis, was unchanged compared with controls (our unpublished data). Moreover, the 7- and fourfold increases in surface expression in melan-mu and melan-rp cells, respectively, were specific to Tyrp1, because surface levels of another melanosomal protein (Pmel17) or the lysosomal protein LAMP-1 were unchanged or increased up to only twofold, respectively, relative to the corresponding BLOC-1–reconstituted cells (Figure 6b; tyrosinase levels could not be quantitated because the antibody recognizes only fixed or denatured protein). Tf receptor (TfR) surface levels were also dramatically increased but paralleled by an as yet unexplained increase in total cellular levels in BLOC-1–deficient cells (Figure 6, b–d) and thus were not attributed to missorting (Figure 7, c and f). Lower TfR levels in rescued cells reflected BLOC-1 recovery and not an artifact of transgene transduction, because stably transduced melan-mu cells expressing PaHA retained high cellular TfR levels (unpublished data). Comparison of the relative ratio of surface to total cellular levels shows a specific increase for Tyrp1, but not for TfR, in BLOC-1–deficient cells relative to BLOC-1–reconstituted cells (Figure 6d). Thus, Tyrp1 is specifically redistributed to the cell surface upon BLOC-1 depletion.
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If BLOC-1 were to facilitate biosynthetic Tyrp1 trafficking to melanosomes through endosomes, then BLOC-1 deficiency might increase Tyrp1 flux through the cell surface by "default" as a consequence of failing to withdraw Tyrp1 from endosomes. To monitor cell surface flux, we measured the rate of antibody uptake in cells exposed continuously to Alexa-488–conjugated anti-Tryp1 antibody. As predicted, melan-mu cells accumulated significantly more anti-Tyrp1 antibody than melan-mu:MuHA, reaching 15-fold higher levels by 2 h (Figure 7g). To limit the analysis primarily to biosynthetic Tyrp1 delivery, the experiment was repeated after preincubation with excess unlabeled anti-Tyrp1 antibody for 20 min, enough time to prebind most of the cell surface and endosomal recycling Tyrp1 pools (Figure 7e) and thus largely exclude them from the analysis. Preincubation dramatically reduced the accumulation of labeled antibody (Figure 7h), indicating that most of the cell surface flux of Tyrp1 reflects recycling from an endosomal pool. However, even after the preincubation, nearly fivefold more labeled antibody accumulated by 2 h in melan-mu than melan-mu:MuHA cells (Figure 7h, inset). Moreover, continuous antibody uptake after 20 min was dramatically inhibited by treatment of melan-mu, but not melan-mu:MuHA, with brefeldin A at a concentration sufficient to disrupt the Golgi complex (Figure 7i; in control experiments not shown, brefeldin A did not block Tyrp1 recycling). Together, these results indicate that biosynthetic Tyrp1 delivery to the cell surface is significantly increased in melan-mu cells. Given the balanced decrease in endocytic and recycling rates, biosynthetic delivery must account for the increased endosomal flux of Tyrp1 in BLOC-1–deficient cells. Moreover, the failure of brefeldin A to affect Tyrp1 cell surface flux in melan-mu:MuHA cells suggests that most surface Tyrp1 does not normally derive from biosynthetic delivery. Together, the data suggest that BLOC-1 facilitates diversion of biosynthetically delivered Tyrp1 out of endosomes and toward melanosomes.
BLOC-1 Deficiency Likely Reduces Endocytic Rates Indirectly by Increasing Endocytic Flux
How might the decreased endocytic rate for Tyrp1 and LAMP-1 in BLOC-1–deficient cells be explained? Interestingly, the endocytic rate for the overexpressed TfR in melan- mu cells was similarly decreased (Figure 7c; 39%/min for melan-mu:MuHA, 15%/min for melan-mu), whereas that of Pmel17 was not (Figure 7d). Endocytosis of LAMP-1 and TfR are mediated by cytoplasmic tyrosine-based signals (YxxØ class, where Ø represents a bulky hydrophobic amino acid and x is any amino acid) that bind to the µ subunits of AP-2 and related adaptors (Bonifacino and Traub, 2003). By contrast, Pmel17 endocytosis is mediated by a dileucine-based (D/ExxxLL class) signal (Theos et al., 2006), similar to those that engage different sites on adaptors (Bonifacino and Traub, 2003; Janvier et al., 2003) (Figure 8a). Endocytosis mediated by both signal classes is independently saturable (Marks et al., 1996). The Tyrp1 cytoplasmic domain contains both YxxØ and dileucine consensus motifs (Vijayasaradhi et al., 1995); thus, decreased endocytic rates in BLOC-1–deficient cells might reflect saturation of YxxØ- but not dileucine-dependent internalization due to increased surface expression of Tyrp1 and other proteins (such as TfR). Consistently, purposeful overexpression of Tyrp1 by transfection in wild-type 1011-mel melanocytic cells increased the surface expression of LAMP-1 (Figure 8, b–e), but not Pmel17 (Figure 8, h–k). Mutagenesis of both Tyrp1 consensus sorting signals ablated this effect (Figure 8, f, g, l, and m). Thus, decreased endocytosis is likely a consequence, not a cause, of the increased flux of Tyrp1, TfR, and other proteins through the surface of BLOC-1–deficient cells. A similar phenomenon may account for increased Tyrp1 surface expression in AP-3– and/or BLOC-2–deficient cells (our unpublished data).
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). To establish functional relationships between BLOC-1, BLOC-2, and AP-3 in Tyrp1 trafficking, intracellular Tyrp1 localization was assessed relative to various markers in BLOC-1–deficient melan-pa, BLOC-2–deficient melan-coa (lacking the HPS3 subunit; Suzuki et al., 2001; Gautam et al., 2004), and AP-3–deficient melan-pe cells (lacking the 
3A subunit; Theos et al., 2005) derived from pallid, cocoa, and pearl mice, respectively. Unlike in BLOC-1–deficient cells, pigmented melanosomes are detectable in melan-coa and melan-pe by light microscopy (Supplemental Figure S4, a–d), although they are not as dense as in control cells. As in melan-mu (Figure 3), Tyrp1 in melan-pa localized to discrete puncta that overlapped largely with syn13 in the cell periphery (44 ± 11%, compared with 12 ± 5% for reconstituted melan-pa:PaHA cells; Figure 9, a–c). In melan-coa, Tyrp1 distributed in larger, more discrete puncta that overlapped less (28 ± 7%) with syn13 (Figure 9, d and e), minimally (<10%) with the modestly pigmented melanosomes (Figure 9, d and f), and not at all with markers of late endosomes/lysosomes or stage II melanosomes (Supplemental Figure S4, e–g, k–m). By IEM, Tyrp1 localized primarily to tubulovesicular structures, only few of which labeled weakly for internalized Tf-FITC (Figure 9j and Supplemental Figure S5); many of these structures were closely apposed to or continuous with multivesicular bodies (MVBs) (Figure 9k and Supplemental Figure S5), suggesting that they are endosomal domains from which Tyrp1 may be delivered to late endosomes. Consistently, immunogold labeling density for Tyrp1 was lower in melan-coa than in the other cells analyzed, perhaps reflecting Tyrp1 degradation in late endosomes or lysosomes. These data suggest that BLOC-2 facilitates Tyrp1 trafficking to melanosomes from an endosomal domain that partially overlaps with but is largely distinct and downstream from that of BLOC-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous evidence has demonstrated selective endosomal protein sorting defects in AP-3–deficient melanocytes (Huizing et al., 2001; Theos et al., 2005), altered distribution of selected melanosomal cargo in BLOC-3– and BLOC-2–deficient human melanocytes (Boissy et al., 2005; Richmond et al., 2005), and enhanced cell surface flux of Tyrp1 in mouse melanocytes lacking AP-3, BLOC-1, or BLOC-2 (Di Pietro et al., 2006). We now show for the first time (to our knowledge) that BLOC-1–deficient melanocytes missort selected cargo destined for melanosomes from early endosomes. Our data further suggest that functional loss of BLOC-2 interferes with Tyrp1 delivery to melanosomes from an endosomal intermediate that is at least partially downstream of BLOC-1, with likely "default" targeting to late endosomes/lysosomes and consequent degradation as suggested in primary BLOC-2–deficient melanocytes (Di Pietro et al., 2006). At least two other protein complexes affected by mouse HPS models—HOPS, for which theVps33a subunit is deficient in buff mice (Suzuki et al., 2003), and rab geranylgeranyl transferase II, for which the 
subunit is deficient in gunmetal mice (Detter et al., 2000)—are also thought to impact early endosomal dynamics (Li et al., 2004; Richardson et al., 2004), and Rab38, mutated in a rat HPS model, regulates melanosomal transport through what are likely endosomal intermediates (Wasmeier et al., 2006). Thus, most forms of HPS reflect defective transport from early endosomes to selected LROs. These findings highlight the importance of early endosomes as intermediates in post-Golgi transport to LROs and the adaptability of the endosomal system in specialized cell types such as melanocytes.
Whereas both AP-3 and BLOC-1 function in sorting to melanosomes from endosomes, they regulate at least partially distinct cargoes. Although both complexes localize at steady state in melanocytic cells to tubular early endosomes, BLOC-1 is found on uncoated tubulovesicular domains distinct from the AP-3/clathrin-coated buds that concentrate tyrosinase (Theos et al., 2005; Di Pietro et al., 2006). Moreover, AP-3 deficiency dramatically alters intracellular tyrosinase distribution but only modestly affects that of Tyrp1 (Figure 9; Huizing et al., 2001; Theos et al., 2005), consistent with the differential binding of AP-3 
3/ hemicomplexes to the tyrosinase and Tyrp1 dileucine-based sorting signals (Theos et al., 2005). By contrast, the inhibition of melanosome transport in BLOC-1–deficient cells is only partial for tyrosinase but complete for Tyrp1. The severe hypopigmentation of BLOC-1–deficient cells contrasts with the brown pigmentation characteristic of Tyrp1 deficiency (Bennett et al., 1989; Boissy et al., 1996) and indicates that additional critical cargoes, besides Tyrp1, absolutely require BLOC-1 for melanosome transport. These additional cargoes do not likely require AP-3 to access melanosomes, because AP-3–deficient melanocytes are highly pigmented (Theos et al., 2005). Together, the data suggest that AP-3- and BLOC-1 regulate melanosome delivery of distinct cargoes through distinct early endosomal intermediates. That BLOC-1 and AP-3 regulate at least partially distinct cargo transport pathways is consistent with the observed synthetic effects of BLOC-1 and AP-3 deficiency on the structure and function of several LROs (Di Pietro et al., 2006; Gautam et al., 2006). We speculate that the copurification (Salazar et al., 2006) and physical interaction (Di Pietro et al., 2006) of AP-3 and BLOC-1 reflect a role for both complexes in regulating the trafficking of different classes of cargoes such as SNAREs, consistent with the mislocalization of syn13 to melanosomes in AP-3–deficient melanocytes (Figure 9).
Whether BLOC-1 interacts directly with its cargo or facilitates downstream membrane dynamics is not known. Although the primary endosomal sorting defect in BLOC-1–deficient melanocytes is cargo specific, the cells also exhibit a generalized decrease in the rate of clathrin-dependent endocytosis mediated by YxxØ but not dileucine motifs. Nevertheless, the endocytic flux for affected cargoes is significantly increased owing to the increased cell surface expression of Tyrp1, TfR, and likely other cargoes. As shown previously (Marks et al., 1996; Warren et al., 1997) and corroborated here, increased flux can saturate the endocytic machinery, resulting in reduced endocytic rates for proteins with related endocytic signals. Thus, decreased endocytic rates for Tyrp1 and other cargoes are most likely an indirect effect of the increased exocytic flux and/or expression of specific endocytic cargo induced by BLOC-1 deficiency. These results imply that defects in endosomal transport machinery can indirectly redistribute cargo to the cell surface, perhaps partially explaining the enhanced surface expression of several proteins in AP-3– and BLOC-2–deficient cells (Dell'Angelica et al., 1999; Le Borgne et al., 2001; Di Pietro et al., 2006; Salazar et al., 2006).
How does BLOC-1 facilitate trafficking from endosomes to melanosomes? The paucity of newly synthesized Tyrp1 that transits the plasma membrane in wild-type melanocytes suggests that Tyrp1 is normally routed directly from the trans-Golgi network (TGN) to endosomes and then to melanosomes (Figure 7i); indeed, this is corroborated by interfering with endosomal function in wild-type melanocytes (our unpublished data). The high flux of newly synthesized Tyrp1 through both the cell surface and early endosomes in BLOC-1–deficient cells is thus consistent with either of two models for BLOC-1 function. In the first model, BLOC-1 promotes fusion of TGN-derived Tyrp1-containing vesicles with early endosomes, such that these vesicles are mistargeted to the plasma membrane when BLOC-1 is absent. However, the high content of endosomal Tyrp1 and complete absence of Tyrp1 from melanosomes in these cells seems inconsistent with such a model, particularly given the localization of Tyrp1 to a different endosomal domain in BLOC-2–deficient cells or to melanosomes in AP-3–deficient cells, despite similar levels of cell surface—and hence internalized—Tyrp1. We thus favor a second model (Figure 10), in which BLOC-1 promotes the exit of cargo from early endosomal membranes toward melanosomes. Increased surface delivery and endosomal content of Tyrp1 would thus reflect futile cycles of endocytosis and recycling (of both internalized and biosynthetically derived cargo) resulting from the inability to "drain" endosomes toward melanosomes. This model best explains the complete absence of Tyrp1 from melanosomes, the accumulation of vacuolar endosomes, and the inefficient Tyrp1 recycling in BLOC- 1–deficient cells. The accumulation of Tyrp1 in a partially distinct and likely downstream compartment in BLOC-2–deficient cells would favor a model in which the BLOC- 1–dependent early endosome-derived membranes pass through another endosomal intermediate, from which BLOC-2 facilitates fusion with melanosomes (Figure 10); that this intermediate is contiguous with early endosomes is suggested by the partial accessibility of BLOC-2–labeled tubulovesicular structures in MNT-1 melanoma cells by internalized Tf (Di Pietro et al., 2006). This may be the same intermediate from which Rab38 regulates Tyrp1 and tyrosinase delivery to melanosomes (Wasmeier et al., 2006). We speculate that the interaction of BLOC-1 with syn13 (Huang et al., 1999; Moriyama and Bonifacino, 2002) and SNAP-23/25 (Ilardi et al., 1999; Buxton et al., 2003) might regulate the formation of these intermediates and/or their ability to fuse with melanosomes.
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; Di Pietro and Dell'Angelica, 2005). It is thus likely that the BLOC-1–dependent pathway that we have defined is ubiquitous in mammals, but it has been exploited by specialized cell types for delivery of specific cargo to LROs, such as melanosomes, platelet dense granules, and lung lamellar bodies, and/or to synaptic vesicles (McGarry et al., 2002; Talbot et al., 2004; Bray et al., 2005; Guttentag et al., 2005). Our data suggest that at least one critical cargo protein is targeted from early endosomes to each of these organelles in a BLOC-1–dependent manner.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Michael S. Marks (marksm{at}mail.med.upenn.edu)
Abbreviations used: BLOC, biogenesis of lysosome-related organelles complex; HPS, Hermansky-Pudlak syndrome; IEM, immunoelectron microscopy; IFM, immunofluorescence microscopy; LRO, lysosome-related organelle; MuHA, HA11-epitope tagged muted; PaHA, HA11-epitope tagged pallidin; rp, reduced pigmentation; syn13, syntaxin 13; Tf, transferrin; TfR, transferrin receptor; Tyrp1, tyrosinase-related protein-1.
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