BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles

Abstract

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.

FIGURE 1.

Rab32 and Rab38 coimmunoprecipitate with BLOC-2, AP-3, and AP-1 but not with AP-2. MNT-1 cells were homogenized in the absence of detergents, and the homogenate was centrifuged to yield cytosolic and membrane fractions. The membrane fraction was solubilized in buffer containing 1% Triton X-100, and the same concentration of detergent was added to the cytosol to match the buffer compositions (see “Experimental Procedures”). Both cytosolic and solubilized membrane fractions were divided into aliquots and subjected to immunoprecipitation (IP) using irrelevant IgG, anti-Rab32, or anti-Rab38 rabbit antibodies. The immunoprecipitates, together with an aliquot of the input extracts corresponding to 1% of the material available for immunoprecipitation, were analyzed by immunoblotting (IB) using antibodies to the HPS6, μ3, γ, and-α subunits of BLOC-2, AP-3, AP-1, and AP-2, respectively.

FIGURE 2.

GTP-dependent interaction of Rab32 and Rab38 with BLOC-2, AP-3, and AP-1. GST pulldown using cytosolic extracts from MNT-1 cells and ∼20 μg each of GST, GST-Rab11, GST-Rab32, or GST-Rab38 immobilized on glutathione-Sepharose and loaded with GDP or the stable GTP analog, GTPγS. The washed glutathione-Sepharose beads, together with an aliquot of the input cytosol corresponding to 1 or 20% of the material available for pulldown, were analyzed by immunoblotting (IB) using antibodies to the HPS6, β3, γ, and α subunits of BLOC-2, AP-3, AP-1, and AP-2, respectively. The bottom panel corresponds to a Coomassie Blue-stained gel and shows the GST fusion proteins.

FIGURE 3.

Rab38 association to membranes is altered by AP-3 and BLOC-2 deficiency but only BLOC-2 knockdown affects Rab38 stability. A, MNT-1 cells deficient for AP-1, AP-3, or BLOC-2 together with control cells were subjected to a quick homogenization and ultracentrifugation procedure to yield postnuclear membrane (M) and cytosolic (C) fractions (see “Experimental Procedures”). The samples were analyzed by immunoblotting (IB) using antibodies to Rab32 or Rab38. The graph shows the percentage of total Rab38 or Rab32 that was found in the membrane fraction in at least three independent experiments (means ± S.D.). For each Rab protein, the data corresponding to AP-1-, AP-3-, or BLOC-2-deficient cells were compared with that of control cells by means of a t test. B, quantification of the total amount of Rab32 and Rab38 present in extracts of MNT-1 cells treated with the indicated siRNAs relative to control cells. Bars represent means ± S.D. of at least three independent experiments. One sample t test was used to compare the results obtained from depleted cells with the reference value of 1 set for control cells. C, MNT-1 cells deficient for Rab32 or Rab38 together with control cells were processed as described in A. The samples were analyzed by immunoblotting using antibodies to BLOC-2. The graph shows the percentage of total BLOC-2 that was found in the membrane fraction in three independent experiments (means ± S.D.). The data corresponding to Rab32- and Rab38-deficient cells was compared with that of control cells by means of a t test. *, p < 0.05; **, p < 0.01.

FIGURE 4.

Rab38 partially colocalizes to proteins required in the trafficking from specialized early endosomal domains to melanosomes. MNT-1 cells were fixed/permeabilized and costained with antibodies against Rab38 (A, D, G, and J) and the δ subunit of AP-3 (B), the γ subunit of AP-1 (E), the HPS6 subunit of BLOC-2 (H), or the clathrin heavy chain (K). Cells were imaged by confocal fluorescence microscopy, and Rab38 was found both on small structures and in diffuse staining distributed throughout the cells. A significant number of AP-3- (B), AP-1- (E), BLOC-2- (H), and clathrin (K)-labeled structures show colocalization with Rab38 in the merged images (C, F, I, and L). Boxed areas are shown in the magnified insets, where arrowheads indicate sites of colocalization. Scale bars indicate 10 μm.

FIGURE 5.

Rab38 and Rab32 do not colocalize to proteins that label early endosome vacuolar domains or the retrieval pathway to the trans-Golgi network. MNT-1 cells were fixed/permeabilized and costained with antibodies against Rab38 (A and G) or Rab32 (D and J) and Early Endosome Antigen 1, EEA1 (B and E), or the retromer subunit Sortin Nexin 1, SNX1 (H and K). Cells were imaged by confocal fluorescence microscopy, and only background levels of colocalization were detected in the merged images (C, F, I, and L). Boxed areas are shown in the magnified insets. Scale bars indicate 10 μm.

FIGURE 6.

Rab38 and Rab32 partially colocalize to melanosomes but not to lysosomes. MNT-1 cells were fixed/permeabilized and costained with antibodies against Rab38 (A) or Rab32 (D) and the melanosomal protein Tyrp1 (B and E). Alternatively, cells were allowed to internalize dextran/Alexa-647, followed by a chase period of 4 h to ensure specific labeling of mature lysosomes (H and K), fixed/permeabilized, and stained with antibodies against Rab38 (G) or Rab32 (J). Cells were imaged by confocal fluorescence microscopy, and both Rab38 and Rab32 were found to partially colocalize with the melanosome marker Tyrp1 (C and F) but not with lysosomes (I and L) in the merged images. Boxed areas are shown in the magnified insets. Scale bars indicate 10 μm.

FIGURE 7.

Knockdown of Rab32 or Rab38 causes mistrafficking of Tyrp1. Live MNT-1 control cells or cells deficient for AP-3, AP-1, Rab32, Rab38, or both Rab32 and Rab38 were incubated in media containing a mouse anti-Tyrp1 antibody for 20 min at 37 °C and subsequently fixed, permeabilized, and immunostained with an Alexa-488-conjugated anti-mouse IgG. Cells were imaged using an epifluorescent microscope, and the relative amounts of internalized anti-Tyrp1 antibody were estimated as the average fluorescence intensity per cell determined with ImageJ and normalized to control cells (means ± S.D.). Result from at least three independent experiments, n > 48 for each treatment, were compared with control cells (or between the Rab32/Rab38 double knockdown and the corresponding single knockdowns) by means of a t test, *, p < 0.05.

FIGURE 8.

Rab32 and Rab38 are required for normal steady state levels of tyrosinase, Tyrp1, and Tyrp2 and are not fully redundant in overall melanosome biogenesis. A, immunoblotting analysis of total cell extracts from control MNT-1 cells and cells deficient for Rab32, Rab38, or both Rab32 and Rab38 was performed and quantified to determine the total abundance of tyrosinase (black), Tyrp1 (gray), and Tyrp2 (white) relative to control cells. Results correspond to three independent experiments normalized to number of cells, and compared using the t test, *, p < 0.05; **, p < 0.01. B, melanin was extracted from MNT-1 control cells or cells deficient for AP-1, Rab32, Rab38, or both Rab32 and Rab38 and quantified by a spectrophotometric method. Results correspond to at least three independent experiments normalized to number of cells and abundance of melanin in control cells, *, p < 0.05.

FIGURE 9.

Cells deficient for BLOC-3 display abnormal Tyrp2 steady state levels. Immunoblotting (IB) analysis of total cell extracts from control MNT-1 cells and cells deficient for BLOC-3 or BLOC-1 was performed and quantified to determine the total abundance of Tyrp2 relative to control cells. Results correspond to three independent experiments normalized to number of cells and compared using the t test, *, p < 0.05.

FIGURE 10.

Model for AP-3, AP-1, and BLOC-2 cooperation with Rab38 and Rab32 to mediate transport to maturing melanosomes. Rab38 and Rab32 interact with AP-3, AP-1, and BLOC-2 at early endosome membrane domains where cargo such as the tyrosinases are concentrated and packaged into transport intermediates. Upon budding, some components of the coat (AP-3, AP-1, and clathrin) dissociate from the vesicle but others remain bound (Rab32, Rab38, and possibly BLOC-2) to mediate further transport, tethering, and fusion with maturing melanosomes. During melanosome biogenesis, transition between stage II and stage III occurs upon incorporation of the melanogenic enzymes with the concomitant beginning of melanin synthesis, and thus vesicles defined by Rab32 and Rab38 likely target this melanosome maturation stage. Deficiency in different components of these pathways elicits cargo accumulation in the early endosomes that eventually leaks into other pathways such as the recycling pathway toward the plasma membrane or the late endosome/multivesicular body (MVB) degradative pathway.