Physical Interactions Between BLOC-1, BLOC-2, and AP-3
To test for stable physical association between AP-3 and the BLOCs, each endogenous complex was immunoprecipitated under mild, nondenaturing conditions from HeLa cell extracts, and the washed immunoprecipitates were analyzed by immunoblotting using antibodies against representative subunits of each complex (see and Materials and Methods
for further details). Because the AP-3 complex and all BLOCs exist in both soluble and membrane-associated pools (reviewed by Dell’Angelica, 2004
), parallel experiments were carried out using cytosolic and solubilized membrane extracts. As shown in A, AP-3 and the three BLOCs were detected and immunoprecipitated from each extract. Significantly, BLOC-1 was also detected in BLOC-2 and AP-3 immunoprecipitates obtained from solubilized membrane extracts, but not from those obtained from cytosol (A). In addition, BLOC-2 and AP-3 were detected in BLOC-1 immunoprecipitates obtained from solubilized membranes but not from cytosol (A). Coimmunoprecipitation of BLOC-2 and AP-3 with BLOC-1 was observed upon immunoprecipitation using antibodies against two different BLOC-1 subunits (B). Under these conditions, no specific association between AP-3 and BLOC-2, or between BLOC-3 and any of the other complexes, was detected (A). Additional experiments were performed in which BLOC-1 was immunoprecipitated from membrane extracts prepared in the presence of GDP instead of the nonhydrolyzable GTP analog, GTPγS. Although comparable amounts of BLOC-2 were associated to BLOC-1 immunoprecipitates regardless of the guanosine nucleotide used, the association of AP-3 to BLOC-1 was significantly compromised, albeit not completely disrupted, upon substitution of GDP for GTPγS (B). Therefore, these results suggest that BLOC-1 interacts, either directly or indirectly, with AP-3 and BLOC-2, and that the two interactions may be differentially regulated.
Figure 1. Coimmunoprecipitation of BLOC-1 with BLOC-2 and AP-3. (A) HeLa cells were homogenized in the absence of detergents and presence of GTPγS, and the homogenate was centrifuged to yield cytosolic and membrane fractions. The membrane fraction was solubilized (more ...)
Next, coimmunoprecipitation experiments were carried out using liver cytosolic and membrane extracts from various mouse strains. Of interest was the analysis of tissue samples from homozygous pallid, cocoa, and pearl mice carrying mutations in genes encoding subunits of BLOC-1, BLOC-2, and AP-3, respectively (; reviewed by Li et al., 2004
). In agreement with the results described above, immunoprecipitation of BLOC-1 from wild-type membrane extracts resulted in coimmunoprecipitation of the AP-3 complex and BLOC-2, and immunoprecipitation of the AP-3 complex or BLOC-2 from the same extracts resulted in coimmunoprecipitation of BLOC-1 (C and unpublished data). Again, no coimmunoprecipitation between these complexes was observed using cytosolic extracts. Importantly, no AP-3 was detected upon subjecting extracts prepared from BLOC-1–deficient mice to a “mock” immunoprecipitation using an antibody to a BLOC-1 subunit, and no BLOC-1 was detected upon mock immunoprecipitation of extracts prepared from AP-3–deficient mice using an antibody to AP-3 σ3 (C), implying that the observed coimmunoprecipitation was not due to antibody cross-reactivity during the immunoprecipitation step. Similarly, control immunoprecipitations performed in extracts prepared from BLOC-1– or BLOC-2–deficient mice yielded the expected negative results for the BLOC-1·BLOC-2 interaction (unpublished data). Interestingly, the association between BLOC-1 and AP-3 was observed in liver membrane extracts prepared from BLOC-2–deficient mice, and that between BLOC-1 and -2 was observed in extracts prepared from AP-3–deficient mice (C and unpublished data). Together, these results provide support to the idea that the interactions between BLOC-1 and BLOC-2 or AP-3 are specific and suggest that BLOC-1 can associate with BLOC-2 independently of AP-3, and likewise, it can associate with AP-3 independently of BLOC-2 function.
Genetic Interactions Between BLOC-1, BLOC-2, and AP-3
As a complementary approach to understand the functional relationships between AP-3 and the BLOCs, we tested for epistatic interactions through generation of homozygous double mutant mice simultaneously deficient in pairs of these protein complexes. Although the coat color phenotype of mice simultaneously deficient in BLOC-1 and BLOC-2 (pallid/cocoa) was virtually indistinguishable from that of single mutant mice deficient in BLOC-1, that of mice simultaneously deficient in BLOC-1 and AP-3 (pearl/pallid) was more severe than that of age-matched single mutants (Supplementary Figure 1). In addition, mice simultaneously deficient in AP-3 and BLOC-1 were very difficult to breed as double homozygous, unlike the corresponding single mutants or mice simultaneously deficient in BLOC-1 and BLOC-2 (unpublished data). Strikingly, the coat color phenotype of double mutant mice deficient in AP-3 and BLOC-2 was not only more severe than that of the corresponding single mutants but highly similar to that of BLOC-1–deficient mice (Supplementary Figure 1). The simplest interpretation of these results is that AP-3 and BLOC-2 can function independently of each other and that the impact of BLOC-1 deficiency on pigmentation can be mimicked by simultaneous deficiencies in AP-3 and BLOC-2.
Functional Interactions between BLOC-1, BLOC-2, and AP-3 as Evidenced by Regulation of Membrane Association
As a first step to address the biological significance of the observed interactions between BLOC-1, BLOC-2, and AP-3, we tested whether membrane association of each complex was affected by deficiencies in its interacting partner(s). To this end, cytosolic and membrane fractions were obtained from skin fibroblast lines derived from wild-type and mutant mice, and the relative amounts of each complex in the membrane fraction were estimated by quantitative immunoblotting. In an attempt to minimize dissociation from membranes during the fractionation procedure, we used a sucrose-containing buffer that had been shown to help stabilize the membrane-associated forms of the three complexes (Dell’Angelica et al., 1997a
; Falcón-Pérez et al., 2002
; Di Pietro et al., 2004
) and we significantly reduced the duration of the ultracentrifugation step (see Materials and Methods
). Under these conditions, about half of BLOC-1, ~20% of BLOC-2, and ~40% of AP-3 were recovered from the membrane fractions obtained from wild-type mouse fibroblast lines (, A and B). Interestingly, the relative amounts of BLOC-1 recovered from membranes were reduced to <20% in AP-3–deficient cells (or in cells simultaneously deficient in AP-3 and BLOC-2), and those of BLOC-2 and AP-3 were increased in BLOC-1–deficient cells (). Membranes isolated from BLOC-2–deficient fibroblasts contained relative amounts of BLOC-1 and AP-3 that were comparable to those of wild-type cells, and membranes isolated from AP-3–deficient fibroblasts contained normal amounts of BLOC-2 (). In another set of experiments, knockdown of AP-3 expression by siRNA treatment of human M1 cells (see below) resulted in a ~40% decrease in the relative amount of membrane-associated BLOC-1 without affecting the membrane-associated pool of BLOC-2 (unpublished data), thus in agreement with the results obtained using immortalized fibroblasts from mutant mice. As judged from immunofluorescence analysis of fixed/permeabilized fibroblasts, neither the overall distribution of AP-3 nor its degree of colocalization with TfR was noticeably affected by deficiencies in BLOC-1 or -2 (Supplementary Figure 2). Together, these results suggest that AP-3 and BLOC-1 can regulate the membrane association/dissociation of each other, albeit without significantly altering AP-3 distribution, and that BLOC-1 can also regulate membrane association/dissociation of BLOC-2.
Figure 2. Membrane-associated pools of BLOC-1, BLOC-2 and AP-3 in fibroblasts from mutant mice. (A) Immortalized fibroblasts derived from the skin of C57BL/6J mice (WT), homozygous pallid (pa), cocoa (coa), and pearl (pe) mice deficient in BLOC-1, BLOC-2, and AP-3, (more ...)
Localization of Endogenous BLOC-1 and BLOC-2 to Endosomes
It is well established that AP-3 associates with clathrin-coated buds on early endosome-associated tubules (Dell’Angelica et al., 1998
; Peden et al., 2004
; Theos et al., 2005
), although the existence of a pool of AP-3 associated with pericentriolar endosomal membranes devoid of clathrin has also been noted. In this study, we sought to determine the localization of BLOC-1 by immunoelectron microscopy on ultrathin cryosections of MNT-1, a highly pigmented human melanoma line in which we have characterized the melanosomal maturation stages, the compartments of the endocytic pathway, and the localization of AP-3 (Raposo et al., 2001
; Theos et al., 2005
). Two of our affinity-purified polyclonal antibodies against BLOC-1 subunits, those against pallidin and dysbindin, resulted in immunogold labeling that was deemed to be specific on the basis of 1) low background labeling in compartments such as nuclei or mitochondria and 2) essentially the same labeling pattern obtained using both antibodies. The antibodies labeled mainly tubulovesicular elements that were distributed throughout the cytoplasm, albeit concentrated in the vicinity of melanosomes and the perinuclear cytoplasmic region that harbors the Golgi apparatus (, A–C). Occasional labeling of the melanosomal membrane was also observed. We counted 211 gold particles in randomly selected portions of three independent grids containing ultrathin cryosections of MNT-1 cells labeled with anti-dysbindin. Of them, 148 (70%) were on tubulovesicular elements in the vicinity of the Golgi apparatus and/or pigmented (stage IV) melanosomes, 27 (13%) on vesicles dispersed throughout the cell periphery, 23 (11%) on the melanosomal limiting membrane, 13 (6%) on mitochondria, and none at the plasma membrane or nuclei. This labeling pattern was reminiscent of proteins localized to early endosomes in MNT-1 cells (Raposo et al., 2001
; Theos et al., 2005
). In fact, BLOC-1 labeling was observed in tubulovesicular elements that also contained internalized Tf-biotin (C). We then used the “whole-mount” electron microscopy technique (Stoorvogel et al., 1996
) to test for the presence of BLOC-1 on endosomes that had been loaded with internalized Tf-HRP and selectively fixed by cross-linking. Importantly, we observed labeling for endogenous BLOC-1 on cross-linked endosomal tubules that also contained EEA1 (D) and AP-3 (E). Although labeling for BLOC-1 was observed more frequently on endosomal tubules (and, occasionally, vacuoles; see inset in D) and that for AP-3 on associated buds, in some instances both complexes were detected in close proximity (e.g., E, arrow).
Figure 3. Localization of endogenous BLOC-1 in human MNT-1 cells as determined by immunoelectron microscopy. (A–C) Ultrathin cryosections of MNT1 cells were labeled with polyclonal antibodies against the pallidin (A) or dysbindin (B and C) subunits of BLOC-1 (more ...)
Additional experiments were performed to localize BLOC-2 by immunoelectron microscopy on ultrathin cryosections of MNT-1 cells. Two affinity-purified antibodies against the BLOC-2 subunits, HP3c anti-HPS3, and HP6d anti-HPS6, labeled tubulovesicular elements that, like those labeled for BLOC-1, were often found in the vicinity of the Golgi apparatus or pigmented melanosomes and were accessible to internalized Tf-biotin (, A–C). Although for both antibodies the labeling efficiency was low, again it was deemed to be specific based on 1) negligible background labeling of mitochondria and nuclei and 2) basically the same labeling pattern obtained with both antibodies. Labeling for BLOC-2 was best appreciated using the whole-mount technique, which resulted in detection of BLOC-2 associated with endosomal tubules that also contained EEA1 (D). The fact that the labeling efficiencies for BLOC-1 and -2 were drastically reduced upon the glutaraldehyde fixation step required for double immunogold labeling experiments (unpublished data) hampered our attempts to test directly for colocalization between these two binding partners.
Figure 4. Localization of endogenous BLOC-2 in human MNT-1 cells as determined by immunoelectron microscopy. (A–C) Ultrathin cryosections of MNT-1 cells were labeled with polyclonal antibodies against the HPS3 and HPS6 subunits of BLOC-2, followed by protein (more ...)
Taken together, these results indicate that BLOC-1 and -2 localize, at least in part, to early endosomes and their associated tubules.
Knockdown of BLOC-1 from Human Fibroblasts Leads to Cell Surface Accumulation of CD63
The observed interaction between AP-3 and BLOC-1 was surprising given previous data that had suggested that mutant mouse fibroblasts deficient in BLOC-1 do not display a characteristic phenotype of AP-3–deficient cells, i.e., enhanced trafficking of LAMP1 through the cell surface (Dell’Angelica et al., 2000
; Gwynn et al., 2000
; Martina et al., 2003
). We first attempted to address this issue by performing flow cytometric analyses of LAMP1 surface levels and internalization, using several independent lines of immortalized fibroblasts derived from mutant mice deficient in each complex. Although our results were suggestive of enhanced LAMP1 trafficking through the surface of mutant fibroblasts deficient in BLOC-1, they failed to reach statistical significance owing to high variability in the results obtained using different cell lines derived from each mouse strain (unpublished data). Similar experiments performed by Salazar et al. (2006)
, however, succeeded in demonstrating enhanced surface levels of endogenous LAMP1 is BLOC-1–deficient mouse fibroblasts, notwithstanding the variability between cell lines. Here, we adopted an alternative experimental approach that was based on acute knockdown of BLOC-1 expression in the human M1 fibroblastoid cell line by siRNA, followed by analysis of the endogenous CD63/LAMP3 protein by indirect immunofluorescence and flow cytometry. Among the advantages of this approach were the use of a single immortalized cell line analyzed in parallel upon different siRNA treatments, as opposed to a comparison between independent mutant cell lines, and the use of endogenous CD63 as a marker for AP-3–dependent trafficking, which facilitated quantitative analyses with improved signal-to-noise ratio (Dell’Angelica et al., 1999b
; Janvier and Bonifacino, 2005
). We identified two independent siRNA duplexes that were able to significantly knockdown expression of BLOC-1, two for efficient knockdown of AP-3, and one siRNA duplex to knockdown expression of each of BLOC-2 and -3 (A). In agreement with published data (Janvier and Bonifacino, 2005
), knockdown of AP-3 led to a significant accumulation of CD63 at the cell surface, which was readily detected by indirect immunofluorescence (D) and flow cytometry (E). Interestingly, knockdown of BLOC-1 with either siRNA duplex elicited a similar effect, albeit to a lesser extent, as judged by both methods (, C, E, and G). On the other hand, the surface levels of CD63 were not noticeably affected upon knockdown of BLOC-2 or -3 (G). Like in AP-3–deficient cells, the surface levels of TfR were not affected by knockdown of BLOC-1 (F). These observations suggest that BLOC-1 plays a role in the trafficking of CD63/LAMP3, a well-known AP-3 cargo.
Figure 5. Knockdown of AP-3 and BLOC-1 in human fibroblasts elicits surface accumulation of CD63. (A) Human M1 fibroblasts were either mock-treated (No siRNA) or treated with siRNA duplexes to target the δ subunit of AP-3, the pallidin subunit of BLOC-1, (more ...)
Abnormal Tyrp1 Trafficking and Steady State Levels in Melanocytes Deficient in BLOC-1, BLOC-2, and AP-3
In a recent study, we have found that melanocytes deficient in BLOC-1 display abnormal accumulation of Tyrp1 in early endosomes (positive for EEA1, syntaxin 13, and internalized Tf), with increases in the amounts of Tyrp1 trafficking to the plasma membrane and undergoing internalization (S.R.G. Setty, M. Starcevic, D. Tenza, S. T. Truschel, E. Chou, A. C. Theos, E. V. Sviderskaya, M. L. Lamoreux, D. C. Bennett, E. C. Dell’Angelica, G. Raposo, and M. S. Marks, unpublished results). Here, we sought to determine the extent to which BLOC-2 and AP-3 contribute to the role of BLOC-1 in trafficking of Tyrp1 to melanosomes. To this end, we have obtained and analyzed primary skin melanocyte cultures deficient in AP-3 and/or the BLOCs. Our decision to use primary melanocytes endogenously expressing Tyrp1 was aimed at avoiding concerns associated with the use of transformed melanocyte lines or with experiments involving expression of Tyrp1 by transfection, although the yields of primary melanocytes were not sufficient to allow flow cytometric or biochemical analyses. Here, live cells were allowed to simultaneously internalize antibodies to the luminal domains of Tyrp1 and TfR, and they were subsequently fixed/permeabilized and incubated with species-specific secondary antibodies to reveal both primary antibodies internalized by the same cell. A strong signal corresponding to internalized anti-Tyrp1 (as well as some antibody bound to Tyrp1 at the cell surface) was observed in BLOC-1-deficient melanocytes (I) under conditions in which the corresponding signal was barely detectable in wild-type melanocytes (A). In contrast, both wild-type and mutant cells displayed comparable amounts of internalized anti-TfR (, B and J). Interestingly, internalization of anti-Tyrp1, but not of TfR, was also enhanced in melanocytes from AP-3– or BLOC-2–deficient mice (, C–F). To test whether the observed differences were statistically significant, several independent primary culture preparations were analyzed for each strain. The average fluorescence signal per cell for each preparation of each strain were determined using a “blinded” approach, averaged and normalized to that of AP-3–deficient melanocytes analyzed in parallel (we chose to normalize the data to that of AP-3–deficient melanocytes owing to the low signal of anti-Tyrp1 obtained for wild-type melanocytes). The results of these analyses are shown in K. Relative to AP-3–deficient melanocytes, the amounts of internalized Tyrp1 were significantly lower in wild-type and BLOC-3–deficient melanocytes, comparable in BLOC-2–deficient melanocytes, and significantly higher in BLOC-1–deficient melanocytes. In contrast, the amounts of internalized anti-TfR were either comparable or modestly increased in wild-type and BLOC-3–deficient melanocytes, relative to those of AP-3 mutant melanocytes. Of note was the phenotype of melanocytes cultured from homozygous AP-3/BLOC-2 double mutant mice (pe/coa), which was strikingly similar to that of BLOC-1–deficient melanocytes (, G–K).
Figure 6. Enhanced flux of Tyrp1 internalization in primary skin melanocytes from mutant mice deficient in AP-3, BLOC-1 or BLOC-2. (A–K) Skin melanocytes were cultured from mice of the control C57BL/6J strain (WT), the homozygous mutant strains pearl ( (more ...)
Possible caveats to the antibody internalization assay were considered. First, the possibility that differences in mean fluorescence intensity per cell could be secondary to differences in cell size was ruled out through comparison of areas of the melanocyte images used for quantification (unpublished data). Second, the possibility that the observed increases in surface/internalized Tyrp1 could be secondary to increases in total Tyrp1 expression levels was addressed by immunofluorescence staining of fixed/permeabilized cells. Surprisingly, not only was the immunofluorescence signal corresponding to endogenous Tyrp1 not higher in mutant melanocytes, compared with that of wild-type melanocytes, but it was dramatically reduced (, A–F). The differences were statistically significant, as determined by “blinded” analysis of independent primary melanocyte preparations for each strain (G). Again, the phenotype was more severe in melanocytes deficient in BLOC-1 or simultaneously deficient in AP-3 and BLOC-2 (pe/coa) than in cells deficient in either AP-3 or BLOC-2 alone. As expected, the immunofluorescence signal of an irrelevant membrane protein, PMP70, was not reduced in mutant melanocytes relative to wild-type melanocytes (G). We reasoned that missorting to lysosomes could in part contribute to the reduced steady state levels of Tyrp1, especially if the lumenal epitope recognized by the MEL-5 mAb was sensitive to lysosomal degradation. To address this point, primary cultures of wild-type, AP-3–deficient, and BLOC-1–deficient melanocytes were divided into two aliquots: one aliquot was incubated with medium containing the lysosomal protease inhibitor, leupeptin, and the other with medium alone, for 6 h before cell fixation and immunofluorescence staining. Fluorescence images of randomly selected cells were acquired under identical conditions and quantified. As shown in H, treatment with leupeptin led to relatively higher Tyrp1 staining intensities in both AP-3– and BLOC-1–deficient melanocytes, but not in wild-type melanocytes. Moreover, the Tyrp1 staining of leupeptin-treated, AP-3–deficient melanocytes was comparable to that of wild-type melanocytes (H). Similar results were obtained in another experiment using an independent preparation of AP-3–deficient melanocytes as well as BLOC-2–deficient melanocytes (p < 0.001, leupeptin-treated vs. control cells).
Figure 7. Decreased steady-state Tyrp1 protein levels in primary skin melanocytes from mutant mice deficient in AP-3, BLOC-1, or BLOC-2. Skin melanocytes were cultured from mice of the control C57BL/6J strain (WT), the homozygous mutant strains pearl (pe), pallid (more ...)
Taken together, these results suggest that the endosomal trafficking of Tyrp1 is abnormal in melanocytes deficient in AP-3 or BLOC-2, and even more so in melanocytes deficient in BLOC-1 or simultaneously deficient in AP-3 and BLOC-2.