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Evidence for Distinct Membrane Traffic Pathways to Melanosomes and Lysosomes in Melanocytes
Evidence for Distinct Membrane Traf®c Pathways to Melanosomes and Lysosomes in Melanocytes Hideaki Fujita,1 Emi Sasano,1 Kumiko Yasunaga, Koh Furuta,* Sadaki Yokota,² Ikuo Wada,³ and Masaru Himeno
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; *University of Occupational and Environmental Medicine, Kita-Kyushu, Japan; ²Yamanashi Medical University, Yamanashi, Japan; ³Sapporo Medical University, Hokkaido, Japan
stage IV melanosomes. Terminal lysosomes loaded with TR-dextran are also distinct from melanosomes, thus indicating that melanosomes are isolated from the endocytic pathway that is a representative route to lysosomes. Because AP-3 mutation leads to mistargeting of both melanosome and lysosome membrane proteins, we propose that there is a late sorting step for melanosomes and lysosomes in melanocytes after AP-3 sorting. Key words: protein sorting/ targeting signal/AP-3. Journal of Investigative Dermatology Symposium Proceedings 6:19±24, 2001
We report here morphologic and biochemical evidence that melanosomes are distinct from lysosomes. Immuno¯uorescence analysis revealed that TRP-1, a melanosomal membrane protein, did not colocalize with lysosomal membrane proteins LAMP1 and LGP85 in melan-a cells. Wortmannin treatment of melanocytes enhanced the distinct compartmentalization of these melanosomal/lysosomal membrane proteins by the swelling of the endosomal-lysosomal systems. The heavily melanized melanosomes did not have an altered shape, which suggests a lesser degree of membrane dynamics of
L
targeting signals in their cytoplamic tails. Tyrosinase and TRP-1, melanosomal membrane proteins, contain more than two potential targeting motifs (Simmen et al, 1999; Vijayasaradhi et al, 1995), whereas lysosomal membrane proteins have one of each. Currently it is not clear whether melanosomal membrane proteins require two targeting motifs or if one of each is suf®cient for targeting. Genetic analysis revealed that gene products of coat color mutation in mice and eye color mutation in the fruit ¯y, Drosophilla, encoded various membrane transport factors (Lloyd et al, 1998; Odorizzi et al, 1998), mutation in one of the AP-3 subunits was identi®ed as mocha in mice and garnet in the ¯y (Simpson et al, 1997; Kantheti et al, 1998). Moreover, VPS41 (light in ¯y) (Warner et al, 1998), pallidin (syntaxin 13 binding protein; pallid in mice) (Huang et al, 1999), and myosin-V (dilute in mice) (Wu et al, 1998) are possibly implicated in vesicle transport and sorting to lysosomes and melanosomes. AP-3 recognizes the Di-leucine-based motif of membrane proteins (Honing et al, 1998) and myosin-V is required for melanosomes interactions with actin ®laments at the peripheral site of melanocytes (Wu et al, 1998). This genetic and biochemical evidence suggests that both melanosomes and lysosomes utilize the same mechanism for membrane transport; however, there are speci®c features in melanosomes, melaninsynthesizing enzymes, and matrix ®laments or the laminar matrix inside the membrane. Furthermore, melanosomes are transferred to adjacent cells, keratinocytes, by cytophagocytosis (Okazaki et al, 1976). Therefore, it is likely that melanosomes differ from lysosomes and have a speci®c protein-targeting mechanism. Our immunolocalization analysis revealed that melanosomes are distinct from lysosomes and we propose that melanosomes are not just another lysosome but have a speci®c membrane transport route.
ysosomes are general organelles that are expressed in most cells, while melanosomes are speci®cally expressed in melanocytes. There are many common features between lysosomes and melanosomes, such as acidic compartments and with acid hydrolases (Boissy et al, 1987; Diment et al, 1995), and it has been suggested that melanosomes are specialized lysosomes (Orlow, 1995, 1998). Morphologic analyzes suggest the melanosomes biogenesis model, in which the membrane of melanosomes originate from smooth ER, and this compartment receives melanin-synthesizing enzymes from the TGN-derived vesicles (Jimbow et al, 1997; Orlow, 1998). Immature melanosomes gradually mature into heavily melanized melanosomes and are ®nally transferred to adjacent cells, keratinocytes, by cytophagocytosis (Okazaki et al, 1976); however, there is little biochemical evidence to support this hypothesis and the origin of the membrane and how the melanosomes construct the ®lamentous matrix inside of the membrane during maturation are not clear. Other researchers reported that membrane proteins are targeted to the lysosomes and melanosomes using the same targeting motif, socalled tyrosine or di-leucine based motifs (Williams and Fukuda, 1990; Vega et al, 1991a, b; Vijayasaradhi et al, 1995; Calvo et al, 1999). Both lysosomal and melanosomal membrane proteins contain Manuscript received June 14, 2001; accepted for publication June 14, 2001. Reprint requests to: Dr. Masaru Himeno, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan. Email: [email protected] Abbreviations: AP, adaptor protein; GFP, green ¯uorescent protein; LAMP1, lysosome-associated membrane protein-1; LGP, lysosomal glycoprotein; PFA, paraformaldehyde; PLC, prelysosomal compartment; TGN, trans-Golgi network; TRP-1, tyrosinase-related protein-1. 1 These authors contributed equally to this work. 1087-0024/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 19
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MATERIALS AND METHODS Cell culture Murine melanoma, melan-a cells were maintained in DMEM supplemented with 10% fetal bovine serum and phorbol 12myristate 13-acetate (Life Technology, Gaithersburg, MD). For immuno¯uorescence experiments, the cells were grown on the poly lysine (10 mg per ml) coated coverslips. Immuno¯uorescence analysis Cells were ®xed with 4% PFA in phosphate-buffered saline (PBS) and then permeabilized with methanol at 4°C. Fixed cells were blocked with PBS containing 1% (wt/vol) bovine serum albumin and processed for indirect immuno¯uorescence using the following primary antibodies: anti-TRP-1 tail antibody (af®nity puri®ed rabbit polyclonal, 5 mg per ml), anti-LGP85 tail antibody (af®nity puri®ed rabbit polyclonal, 5 mg per ml), antirat cathepsin L antibody (af®nity puri®ed rabbit polyclonal, 25 mg per ml), antimouse LAMP1 antibody (rat monoclonal ascites, 1:100 dilution). All the secondary antibodies (Cy3-goat antirat, Alexa 488-goat antirabbit) were used at 1:200±300 dilution. Antibodies Synthetic peptide corresponding to the middle of the deduced cytoplasmic portion of mouse TRP-1 (509±528: EANQPLLTDHYQRYAEDY EEC) and LGP85 tail (457±477: CRGQGSTDEGTADERAPLIRT, the tail sequences are identical among mouse, rat, and human) were synthesized and coupled to bovine serum albumin as immunogen (Sawady Technology, Tokyo, Japan). Rabbit polyclonal antibodies against each peptide were generated by KBT Oriental (Tosu, Japan) and af®nity puri®cation was performed with the peptide column, respectively. Rabbit polyclonal anti-rat cathepsin L was generated with GST-mature cathepsin L fusion protein as the immunogen and the speci®c antibody was prepared by the sequential af®nity puri®cation with GST and a GST-fusion one coupled to Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden). Rat monoclonal antimouse LAMP1 antibody (1D4B) was a generous gift from Dr. T. August (Johns Hopkins University, Baltimore, MD). Secondary antibodies coupled with Cy3 or Alexa488 were purchased from Jackson (West Grove, PA) and Molecular Probes (Eugene, OR), respectively. Plasmid construction and transfection Plasmid encoding the chimeric protein, human TRP-1, and GFP was generated by the polymerase chain reaction (PCR) with oligonucleotides corresponding to the amino- and carboxyl-terminal of human TRP-1. Primers (Hokkaido System Science, Sapporo, Japan) were designed to obtain an Eco RI site at the 5¢ end (5¢-CTCTGAATTCAAGCAGAATGAGTGCTCC-3¢) and Sal I site at the 3¢ end (5¢-CTGGAGTCGACCATATTCTTTCTTCAGC-3¢). The PCR fragment was directionally subcloned into pHSG399 (TAKARA, Kyoto, Japan), sequenced, and re-cloned into pEGFP-N1 vector (Clontech, Palo Alto, CA). Plasmid transfection was performed with Fugen6 Transfection Reagent (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's protocol. Imaging Immuno¯uorescence images were observed on a Leica DMRB microscope (Wetzlar, Germany), acquired through cooled CCD camera, MicroMAX (Princeton Instruments, Trenton, NJ) and digitally
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processed using IPlab Software (Scanalytics, Fairfax, VA). All images from the real time movie, immuno¯uorescence microscopy, and western blotting were subsequently assembled and labeled using Adobe Photoshop (Adobe Systems, Mountain View, CA). Immunoisolation of melanosomes and lysosomes Preparation of heavy granular fractions was done essentially according to Gahl's method from cultured melan-a cells (Gahl et al, 1995). Granular fractions were incubated with af®nity puri®ed tail speci®c antibodies (anti-TRP-1 tail or anti-LGP85 tail) or control IgG (anti-GST) at 4°C for 16 h. Subsequently, bovine serum albumin blocked protein A agarose (Boehringer Mannheim, Indianapolis, IN) was added and followed by a further incubation for 3 h. The sedimented beads were washed four times with 0.25 M sucrose/Hepes/EDTA buffer and bound organelle was solubilized with Laemmli's sample buffer for the western blotting. TR-dextran labeling of melan-a Melan-a cells were labeled with 2 mg per ml of TR-dextran (Molecular Probes) for 15 min at 37°C, chased for the indicated time, and then ®xed for immuno¯uorescence.
RESULTS Wortmannin-induced vacuole formation in melan-a cells Wortmannin, a phosphatidyl inositide-3-kinase inhibitor, causes vacuolation of endosomes and lysosomes because of transport inhibition from prelysosomal compartments (PLC) to lysosomes (Reaves et al, 1996). We examined the effect of wortmannin on melanosomes in mouse melanocyte, melan-a cells. Wortmannin (10 mM)-treated melan-a cells were monitored every 3 min in living cells (Fig 1). Small black granules (thin arrows at 0 min) within the cytoplasm are melanosomes. Spherical small vacuoles began to appear near the nucleus 6 min after wortmannin treatment (thick arrows) and these small vacuoles moved around, increased in size, and sometimes fused; however, the small black granules (melanosomes) never got into the vacuoles. This ®nding suggested that although vacuolation of endosomes and lysosomes had been observed in the melanocytes, the heavily melanized melanosomes (stage IV) did not change in shape and never reached wortmannin-induced vacuoles in melan-a. TRP-1 did not colocalize with either lamp1 or LGP85 in melanocytes The staining of melanosomes with anti-TRP-1 antibody had the cytoplasmic punctuate pattern with strong concentration at the perinuclear in melan-a cells (Fig 2B). Monoclonal antibody against Lamp1, a lysosomal membrane protein, also showed strong perinuclear staining representing both late endosomes and lysosomes, in addition to the peripheral cytoplasmic signals indicating early endosomes (Fig 2A). Although they shared a partial overlap at the perinucleous region (probably at the late endosomes), cytoplasmic and peripheral staining was distinct (Fig 2C). In the presence of wortmannin, both
Figure 1. Wortmannin-induced vacuole formation in melan-a cells. Wortmannin (10 mM)-treated melan-a cells were monitored every 3 min in living cells. Small black granules (thin arrows at 0 min) within the cytoplasm are melanosomes. Spherical small vacuoles begin to appear near the nucleus at 6 min after wortmannin treatment (thick arrows) and these small vacuoles moved around, increased in size, and sometimes fused.
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melanosomes and lysosomes were swollen, probably because of an imbalance of membrane retrieval or due to inhibition of the fusion (Fig 2D, E). There were few vacuoles stained with both TRP-1 and LAMP1 but many vacuoles were positive for one but not the
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other (Fig 2F). The wortmannin-induced vacuolation had an exaggerated distinctive compartmentalization in the cytoplasm. LGP85, another lysosomal membrane protein (Fujita et al, 1991), revealed perfect colocalization with LAMP1 (data not shown),
Figure 2. Localization of LAMP1 and TRP-1 in melan-a cells. Cells were treated without (A±C) or with (D±F) 10 mM wortmannin for 1 h at 37°C. TRP-1 did not colocalize with LAMP1. Large arrows indicate solely LAMP1-positive structures and small arrows represent TRP-1-speci®c compartments, respectively. Wortmannin-induced vacuolation exaggerated the distinct compartmentalization of each membrane protein.
Figure 3. Localization of hTRP-1-GFP and LGP85 in melan-a cells. hTRP-1-GFP plasmid was transfected and the cells were treated without (A±C) or with (D±F) 10 mM wortmannin for 3 h at 37°C. Small arrows indicate solely TRP-1-GFP-positive structures and large arrows represent LGP85-speci®c compartments, respectively. Both proteins were distributed into the distinct vacuole in the presence of wortmannin.
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thereby suggesting that the distinct staining pattern between TRP1 and LAMP1 was speci®c and signi®cant. Interestingly, GFPtagged TRP-1 behaved in the same way as endogenous TRP-1 in melanocytes (data not shown), but did not colocalize with LGP85 in the absence or presence of wortmannin (Fig 3A±F). Immunoseparation of lysosomes from melanosomes suggesting the distinct but partial overlap of melanosomes with lysosomes To observe the distinct compartmentalization of melanosomes and lysosomes in melanocytes, we wanted to immunoseparate melanosomes from lysosomes, using tail-speci®c antibodies, rabbit polyclonal antibodies against the cytoplasmic tails of both LGP85 and TRP1, respectively, following protein-A
Figure 4. Immunoisolation of melanosomes and lysosomes using tail-speci®c antibodies. LGP85-positive compartments and TRP-1positive compartments were immunoisolated with cytoplasmic tailspeci®c antibodies, respectively. The isolated fractions were analyzed by western blotting. LGP85-positive compartments are devoid of TRP-1, whereas TRP-1-positive fractions contained signi®cant amounts of LGP85.
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agarose beads sedimentation. The isolated fractions were analyzed by western blotting (Fig 4). The subcellular fraction isolated with the antibody to LGP85 tail was devoid of TRP-1, which suggested that the isolated fraction was highly enriched in lysosomes but not in melanosomes, i.e., melanosomes were distinct from lysosomes. The reciprocal experiment, the separation of melanosomes from lysosomes with the antibody against TRP-1, was not successful. TRP-1 positive fractions contained signi®cant amounts of LGP85, which may mean that lysosomes were more abundant than melanosomes in melan-a. Therefore, the anti-LGP85 antibody was primarily saturated with lysosomes, whereas the TRP-1 antibody fully interacted with both melanosomes and lateendosomes, the sorting place of TRP-1 and LGP85. Melanosomes are distinct from terminal lysosomes We then asked if melanosomes are the terminal of the endocytic pathway as lysosomes. In melan-a cells, TR-dextran was internalized and chased for the indicated time, then the ®xed cells were immunostained with anti-LAMP1, cathepsin L, and TRP-1 antibodies, respectively. Cathepsin L is one of the major acid hydrolases in lysosomes and is well characterized as the marker enzyme of terminal lysosomes with dense and acidic compartments. At an early time point (30 min or 1 h), TR-dextran was localized in the early endocytic compartments and did not colocalize with LAMP1 or TRP-1 (Fig 5A, E). TR-dextran colocalized with cathepsin L at 24 h, which suggested that TRdextran was delivered to the terminal lysosomes in melanocytes at this time point (Fig 5D). LAMP1 colocalized with TR-dextran at later time points, 8 and 24 h after the chase period; however, the extent of colocalization was slightly less than that seen with cathepsin L at 24 h (Fig 5B, C). This may mean that LAMP1 localizes on not only lysosomes but also the late/early endosomes. In contrast, there was no colocalization of TR-dextran with TRP1 positive compartments at any time point (Fig 5E±G). These ®ndings suggested that melanosomes are not the terminal of the endocytic pathway as lysosomes. The endocytic molecules never encountered the biosynthetic route of melanosomes.
Figure 5. Relationship between the endocytic routes and melanosomes. Melan-a cells were labeled with TR-dextran, chased for the indicated times at 37°C, and then processed for the immuno¯uorescence analysis with antibodies against LAMP1 (A±C), cathepsin L (D), and TRP-1 (E±G), respectively. At an early time point, TR-dextran was internalized via ¯uid phase endocytosis and delivered to early endosomes those are devoid of LAMP1 (A). At a later time point, TR-dextran reached late endosomes and lysosomes and colocalized with LAMP1 (B, C) and cathepsin L (D); however, TRP-1 never colocalized with TR-dextran at any time point (E±G).
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Figure 6. Model of the membrane traf®c to melanosomes and lysosomes in melanocytes. In biosynthetic pathways, the traf®c route of melanosome membrane protein (TRP-1) partially overlaps but is distinct from that of the endo/ lysosomal one (LAMP1, LGP85). The melanosome pathway is separated from the lysosome one after the AP-3-dependent sorting steps, probably at late endosomes and/or PLC. Melanosomes gradually maturate into heavily melanized melanosomes (from stage I to stage II±IV) with the accumulation of melanin inside the membrane. In the presence of wortmannin, there is membrane retrieval from the lysosomes or immature melanosomes, but not from the heavily melanized melanosomes. The melanosome pathway is also segregated from the endocytic pathway (TR-dextran).
DISCUSSION Melanosomes and lysosomes are highly related organelles and the protein targeting to these compartments is based on the vesicular transport. Accumulating evidence suggests targeting signals and molecular mechanisms that regulate the vesicle formation, transport, and fusion are common; however, it is not clear to what extent melanosomes are related to lysosomes in melanocytes. To characterize the membrane property of melanosomes we applied wortmannin to melanocytes. Wortmannin treatment (10 mM) caused the appearance of the spherical vacuoles as with other types of cells; however, the black granules (heavily melanized melanosomes) never got into these vacuoles. Ultrastructural analysis also indicated that the heavily melanized melanosomes did not change shape and size in the presence of this drug (data not shown). As they have an organized internal matrix (i.e., ®laments) within a membrane and are rigid, they have little membrane retrieval. When melanosomes have matured, they are separated from any membrane transport pathway and lose the membrane dynamics. We have found that melanosomes and lysosomes are distinct in melanocytes and wortmannin-induced vacuolation exaggerated the distinct compartmentalization of each membrane protein. LAMP1 and LGP85 colocalized either in the presence or in the absence of wortmannin, whereas TRP-1 indicated a little overlap with lysosome markers only at the perinuclear region where TGN or late endosomes are located. The appearance of swollen TRP-1speci®c vacuoles in the presence of wortmannin suggests that TRP-1 is not restricted on the mature melanosomes and has signi®cant distribution on the immature melanosomes, which are wortmannin. Interestingly, TRP-1 redistributed into the distinct vacuoles from that of lysosomes in the presence of wortmannin. A likely explanation for the appearance of swollen TRP-1-speci®c vacuoles is that TRP-1 is not restricted on the mature melanosomes and has signi®cant distribution on the immature melanosomes, where they are wortmannin sensitive but distinct from both heavily melanized melanosomes and lysosomes. This compartment re¯ects the existence of premelanosomal compartments, which are equivalent to PLC in nonmelanocytes. Orlow et al have shown that TRP-1 is limited to a less dense melanosomal compartment in contrast to tyrosinase, which is eventually localized to heavily melanized melanosomes. Their observation also supports the heterogeneity of melanosomes and the existence of the premelanosomal compartments (Orlow et al, 1993); however, they did not distinguish melanosomes from lysosomes and indicated the colocalization of LAMP1 and TRP-1 in melanocytes. The discrepancy between our results and theirs might be explained by the difference of cell types or the degree of melanization. Because
TRP-1 and LAMP-1/LGP85 are already segregated at the premelanosomal compartment, the ®nal step of the sorting could be taking place before this, possibly at late endosomes. Lysosomes are regarded as the terminal compartment of the endocytic pathway, which contain acid hydrolases but have less membrane retrieval in the presence of wortmannin (Reaves et al, 1996; Bright et al, 1997). The PLC is thought to be an intermediate compartment between lysosomes and late endosomes. Transiently, this compartment is formed by direct fusion of lysosomes with late endosomes. Contents are rapidly mixed and the membranes are retrieved and redistributed to their original positions. Therefore PLC, so-called late endosome-lysosome hybrid organelle, is very dynamic and unstable (Mullock et al, 1998; Luzio et al, 2000; Pryor et al, 2000). Wortmannin inhibits antetrograde movement from late endosomes to lysosomes, and consequently enhances formation of hybrid compartments. We assume that in melanocytes there are both PLC and pre-melanosomal compartments, whereas nonmelanocytes contain only PLC. In melanocytes, wortmannin may induce vacuolation of both compartments. Immunoelectron microscopic analysis strongly supported our assumption, as it is clear that heavily melanized melanosomes are devoid of both LGP85 and LAMP1 (data not shown). We also found a colocalization of both TRP-1 and LAMP1 in immature melanosomes and late endosomes (data not shown), which might be the sorting place for the melanosome proteins. A selective isolation of the subset of lysosomes from melanosomes indicates direct evidence that lysosomes are distinct from melanosomes; however, it is controversial as to whether the TRP1-positive compartment contains signi®cant amounts of LGP85. One interpretation of this discrepancy is that the number of lysosomes is more abundant than that of melanosomes in melanocytes. Therefore, the LGP85 tail antibody easily reacts and is saturated with lysosomes, whereas the TRP-1 tail antibody pulls down the mixture of melanosomes and late endosomes, which are the sorting sites of melanosomes and lysosomes. Further careful analysis needs to be carried out to support this. Based on the endocytic analysis in melanocytes, melanosomes are also distinct from the endocytic pathway. When terminal lysosomes were labeled with TR-dextran for 24 h chase, however, TRP-1 never colocalized with endocytic markers at any time point. Therefore, we propose that melanosomes are segregated from the endocytic route. Because heavily melanized melanosomes have little membrane retrieval, it is unlikely that they receive endocytic molecules from the outside of the cells. Interestingly the endocytosed TR-dextran colocalized well with LAMP-1 at an early time point, such as 8 h after the internalization, whereas they took longer to reach the cathepsin L-positive terminal lysosomes.
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This result also represents the existence of the subpopulation and the heterogeneity of lysosomes. LAMP1 is known as a recycling molecule between lysosomes and plasmamembrane, therefore this colocalization is found at early and late endosomes rather than at lysosomes. There is evidence for retrograde traf®c between terminal lysosomes and PLC (Reaves et al, 1996); however, there is no evidence for the presence of premelanosomal compartments. Our results are a demonstration of the presence of the intermediate compartments and the membrane. In summary, we propose the following model of membrane protein transport to the lysosomes and melanosomes in melanocyte (Fig 6). Both melanosome and lysosome membrane proteins follow the AP-3 pathway until the late endosomes and/or PLC, because AP-3 mutation causes mistargeting of both membrane proteins to the cell surface (Le Borgne et al, 1998); however, after leaving the late endosome and/or PLC, they must take different routes to their ®nal destinations. Whereas lysosome membrane proteins are delivered to dense lysosomes, melanosome membrane proteins reach to the immature melanosomes (stage I). At this stage, it is likely that melanosome membrane protein, TRP-1 can be retrieved from there to the former sorting stations (PLC and/or late endosomes). Stage I melanosomes gradually maturate into heavily melanized melanosomes (stages II, III, and IV), lose the membrane dynamics, and then are transferred to the adjacent cells, keratinocytes by the cytophagocytosis. It is reasonable that melanosomes and lysosomes have different contributions to the cell homeostasis; melanosomes are for the pigmentation and lysosomes are for the degradation. To perform these distinct roles, they need to have a speci®c protein-sorting and transport system. Our ®ndings support the idea that the melanocyte has a speci®c sorting place and a mechanism to have melanosomes independent from lysosomes. Furthermore, heavily melanized melanosomes have unique membrane structures and proteins/lipids composition to be speci®cally transferred to the adjacent cells. This is the only example of the intercellular organelle transfer phenomena. Identi®cation the molecular mechanism of this phenomenon is very important to understand the pigmentation in melanocytes. How is the sorting of melanosome and lysosome performed or what kind of molecules are implicated in these steps? The genetic analysis of coat color mutation in mouse (Odorizzi et al, 1998) and eye color mutation in the fruit ¯y Drosophilla (Lloyd et al, 1998) may give us the answer to these questions. We thank Dr. J. T. August for kindly providing antimouse LAMP1 monoclonal antibody, Dr. Y. Tanaka for helpful assistance on the subcellular fractionation, M. Ohara for comments on the manuscripts, and Dr. K. Jimbow for helpful comments and discussion. This work was supported in part by a CREST grant from the Sciences and Technology Corporation of Japan, the Kao Foundation For Arts and Sciences, and Grants-in-Aid for Scienti®c Research from the Ministry of Education, Science, Sports, and Culture of Japan.
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Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; *University of Occupational and Environmental Medicine, Kita-Kyushu, Japan; ²Yamanashi Medical University, Yamanashi, Japan; ³Sapporo Medical University, Hokkaido, Japan
stage IV melanosomes. Terminal lysosomes loaded with TR-dextran are also distinct from melanosomes, thus indicating that melanosomes are isolated from the endocytic pathway that is a representative route to lysosomes. Because AP-3 mutation leads to mistargeting of both melanosome and lysosome membrane proteins, we propose that there is a late sorting step for melanosomes and lysosomes in melanocytes after AP-3 sorting. Key words: protein sorting/ targeting signal/AP-3. Journal of Investigative Dermatology Symposium Proceedings 6:19±24, 2001
We report here morphologic and biochemical evidence that melanosomes are distinct from lysosomes. Immuno¯uorescence analysis revealed that TRP-1, a melanosomal membrane protein, did not colocalize with lysosomal membrane proteins LAMP1 and LGP85 in melan-a cells. Wortmannin treatment of melanocytes enhanced the distinct compartmentalization of these melanosomal/lysosomal membrane proteins by the swelling of the endosomal-lysosomal systems. The heavily melanized melanosomes did not have an altered shape, which suggests a lesser degree of membrane dynamics of
L
targeting signals in their cytoplamic tails. Tyrosinase and TRP-1, melanosomal membrane proteins, contain more than two potential targeting motifs (Simmen et al, 1999; Vijayasaradhi et al, 1995), whereas lysosomal membrane proteins have one of each. Currently it is not clear whether melanosomal membrane proteins require two targeting motifs or if one of each is suf®cient for targeting. Genetic analysis revealed that gene products of coat color mutation in mice and eye color mutation in the fruit ¯y, Drosophilla, encoded various membrane transport factors (Lloyd et al, 1998; Odorizzi et al, 1998), mutation in one of the AP-3 subunits was identi®ed as mocha in mice and garnet in the ¯y (Simpson et al, 1997; Kantheti et al, 1998). Moreover, VPS41 (light in ¯y) (Warner et al, 1998), pallidin (syntaxin 13 binding protein; pallid in mice) (Huang et al, 1999), and myosin-V (dilute in mice) (Wu et al, 1998) are possibly implicated in vesicle transport and sorting to lysosomes and melanosomes. AP-3 recognizes the Di-leucine-based motif of membrane proteins (Honing et al, 1998) and myosin-V is required for melanosomes interactions with actin ®laments at the peripheral site of melanocytes (Wu et al, 1998). This genetic and biochemical evidence suggests that both melanosomes and lysosomes utilize the same mechanism for membrane transport; however, there are speci®c features in melanosomes, melaninsynthesizing enzymes, and matrix ®laments or the laminar matrix inside the membrane. Furthermore, melanosomes are transferred to adjacent cells, keratinocytes, by cytophagocytosis (Okazaki et al, 1976). Therefore, it is likely that melanosomes differ from lysosomes and have a speci®c protein-targeting mechanism. Our immunolocalization analysis revealed that melanosomes are distinct from lysosomes and we propose that melanosomes are not just another lysosome but have a speci®c membrane transport route.
ysosomes are general organelles that are expressed in most cells, while melanosomes are speci®cally expressed in melanocytes. There are many common features between lysosomes and melanosomes, such as acidic compartments and with acid hydrolases (Boissy et al, 1987; Diment et al, 1995), and it has been suggested that melanosomes are specialized lysosomes (Orlow, 1995, 1998). Morphologic analyzes suggest the melanosomes biogenesis model, in which the membrane of melanosomes originate from smooth ER, and this compartment receives melanin-synthesizing enzymes from the TGN-derived vesicles (Jimbow et al, 1997; Orlow, 1998). Immature melanosomes gradually mature into heavily melanized melanosomes and are ®nally transferred to adjacent cells, keratinocytes, by cytophagocytosis (Okazaki et al, 1976); however, there is little biochemical evidence to support this hypothesis and the origin of the membrane and how the melanosomes construct the ®lamentous matrix inside of the membrane during maturation are not clear. Other researchers reported that membrane proteins are targeted to the lysosomes and melanosomes using the same targeting motif, socalled tyrosine or di-leucine based motifs (Williams and Fukuda, 1990; Vega et al, 1991a, b; Vijayasaradhi et al, 1995; Calvo et al, 1999). Both lysosomal and melanosomal membrane proteins contain Manuscript received June 14, 2001; accepted for publication June 14, 2001. Reprint requests to: Dr. Masaru Himeno, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan. Email: [email protected] Abbreviations: AP, adaptor protein; GFP, green ¯uorescent protein; LAMP1, lysosome-associated membrane protein-1; LGP, lysosomal glycoprotein; PFA, paraformaldehyde; PLC, prelysosomal compartment; TGN, trans-Golgi network; TRP-1, tyrosinase-related protein-1. 1 These authors contributed equally to this work. 1087-0024/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 19
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MATERIALS AND METHODS Cell culture Murine melanoma, melan-a cells were maintained in DMEM supplemented with 10% fetal bovine serum and phorbol 12myristate 13-acetate (Life Technology, Gaithersburg, MD). For immuno¯uorescence experiments, the cells were grown on the poly lysine (10 mg per ml) coated coverslips. Immuno¯uorescence analysis Cells were ®xed with 4% PFA in phosphate-buffered saline (PBS) and then permeabilized with methanol at 4°C. Fixed cells were blocked with PBS containing 1% (wt/vol) bovine serum albumin and processed for indirect immuno¯uorescence using the following primary antibodies: anti-TRP-1 tail antibody (af®nity puri®ed rabbit polyclonal, 5 mg per ml), anti-LGP85 tail antibody (af®nity puri®ed rabbit polyclonal, 5 mg per ml), antirat cathepsin L antibody (af®nity puri®ed rabbit polyclonal, 25 mg per ml), antimouse LAMP1 antibody (rat monoclonal ascites, 1:100 dilution). All the secondary antibodies (Cy3-goat antirat, Alexa 488-goat antirabbit) were used at 1:200±300 dilution. Antibodies Synthetic peptide corresponding to the middle of the deduced cytoplasmic portion of mouse TRP-1 (509±528: EANQPLLTDHYQRYAEDY EEC) and LGP85 tail (457±477: CRGQGSTDEGTADERAPLIRT, the tail sequences are identical among mouse, rat, and human) were synthesized and coupled to bovine serum albumin as immunogen (Sawady Technology, Tokyo, Japan). Rabbit polyclonal antibodies against each peptide were generated by KBT Oriental (Tosu, Japan) and af®nity puri®cation was performed with the peptide column, respectively. Rabbit polyclonal anti-rat cathepsin L was generated with GST-mature cathepsin L fusion protein as the immunogen and the speci®c antibody was prepared by the sequential af®nity puri®cation with GST and a GST-fusion one coupled to Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden). Rat monoclonal antimouse LAMP1 antibody (1D4B) was a generous gift from Dr. T. August (Johns Hopkins University, Baltimore, MD). Secondary antibodies coupled with Cy3 or Alexa488 were purchased from Jackson (West Grove, PA) and Molecular Probes (Eugene, OR), respectively. Plasmid construction and transfection Plasmid encoding the chimeric protein, human TRP-1, and GFP was generated by the polymerase chain reaction (PCR) with oligonucleotides corresponding to the amino- and carboxyl-terminal of human TRP-1. Primers (Hokkaido System Science, Sapporo, Japan) were designed to obtain an Eco RI site at the 5¢ end (5¢-CTCTGAATTCAAGCAGAATGAGTGCTCC-3¢) and Sal I site at the 3¢ end (5¢-CTGGAGTCGACCATATTCTTTCTTCAGC-3¢). The PCR fragment was directionally subcloned into pHSG399 (TAKARA, Kyoto, Japan), sequenced, and re-cloned into pEGFP-N1 vector (Clontech, Palo Alto, CA). Plasmid transfection was performed with Fugen6 Transfection Reagent (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's protocol. Imaging Immuno¯uorescence images were observed on a Leica DMRB microscope (Wetzlar, Germany), acquired through cooled CCD camera, MicroMAX (Princeton Instruments, Trenton, NJ) and digitally
JID SYMPOSIUM PROCEEDINGS
processed using IPlab Software (Scanalytics, Fairfax, VA). All images from the real time movie, immuno¯uorescence microscopy, and western blotting were subsequently assembled and labeled using Adobe Photoshop (Adobe Systems, Mountain View, CA). Immunoisolation of melanosomes and lysosomes Preparation of heavy granular fractions was done essentially according to Gahl's method from cultured melan-a cells (Gahl et al, 1995). Granular fractions were incubated with af®nity puri®ed tail speci®c antibodies (anti-TRP-1 tail or anti-LGP85 tail) or control IgG (anti-GST) at 4°C for 16 h. Subsequently, bovine serum albumin blocked protein A agarose (Boehringer Mannheim, Indianapolis, IN) was added and followed by a further incubation for 3 h. The sedimented beads were washed four times with 0.25 M sucrose/Hepes/EDTA buffer and bound organelle was solubilized with Laemmli's sample buffer for the western blotting. TR-dextran labeling of melan-a Melan-a cells were labeled with 2 mg per ml of TR-dextran (Molecular Probes) for 15 min at 37°C, chased for the indicated time, and then ®xed for immuno¯uorescence.
RESULTS Wortmannin-induced vacuole formation in melan-a cells Wortmannin, a phosphatidyl inositide-3-kinase inhibitor, causes vacuolation of endosomes and lysosomes because of transport inhibition from prelysosomal compartments (PLC) to lysosomes (Reaves et al, 1996). We examined the effect of wortmannin on melanosomes in mouse melanocyte, melan-a cells. Wortmannin (10 mM)-treated melan-a cells were monitored every 3 min in living cells (Fig 1). Small black granules (thin arrows at 0 min) within the cytoplasm are melanosomes. Spherical small vacuoles began to appear near the nucleus 6 min after wortmannin treatment (thick arrows) and these small vacuoles moved around, increased in size, and sometimes fused; however, the small black granules (melanosomes) never got into the vacuoles. This ®nding suggested that although vacuolation of endosomes and lysosomes had been observed in the melanocytes, the heavily melanized melanosomes (stage IV) did not change in shape and never reached wortmannin-induced vacuoles in melan-a. TRP-1 did not colocalize with either lamp1 or LGP85 in melanocytes The staining of melanosomes with anti-TRP-1 antibody had the cytoplasmic punctuate pattern with strong concentration at the perinuclear in melan-a cells (Fig 2B). Monoclonal antibody against Lamp1, a lysosomal membrane protein, also showed strong perinuclear staining representing both late endosomes and lysosomes, in addition to the peripheral cytoplasmic signals indicating early endosomes (Fig 2A). Although they shared a partial overlap at the perinucleous region (probably at the late endosomes), cytoplasmic and peripheral staining was distinct (Fig 2C). In the presence of wortmannin, both
Figure 1. Wortmannin-induced vacuole formation in melan-a cells. Wortmannin (10 mM)-treated melan-a cells were monitored every 3 min in living cells. Small black granules (thin arrows at 0 min) within the cytoplasm are melanosomes. Spherical small vacuoles begin to appear near the nucleus at 6 min after wortmannin treatment (thick arrows) and these small vacuoles moved around, increased in size, and sometimes fused.
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melanosomes and lysosomes were swollen, probably because of an imbalance of membrane retrieval or due to inhibition of the fusion (Fig 2D, E). There were few vacuoles stained with both TRP-1 and LAMP1 but many vacuoles were positive for one but not the
MEMBRANE TRAFFIC TO MELANOSOMES AND LYSOSOMES
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other (Fig 2F). The wortmannin-induced vacuolation had an exaggerated distinctive compartmentalization in the cytoplasm. LGP85, another lysosomal membrane protein (Fujita et al, 1991), revealed perfect colocalization with LAMP1 (data not shown),
Figure 2. Localization of LAMP1 and TRP-1 in melan-a cells. Cells were treated without (A±C) or with (D±F) 10 mM wortmannin for 1 h at 37°C. TRP-1 did not colocalize with LAMP1. Large arrows indicate solely LAMP1-positive structures and small arrows represent TRP-1-speci®c compartments, respectively. Wortmannin-induced vacuolation exaggerated the distinct compartmentalization of each membrane protein.
Figure 3. Localization of hTRP-1-GFP and LGP85 in melan-a cells. hTRP-1-GFP plasmid was transfected and the cells were treated without (A±C) or with (D±F) 10 mM wortmannin for 3 h at 37°C. Small arrows indicate solely TRP-1-GFP-positive structures and large arrows represent LGP85-speci®c compartments, respectively. Both proteins were distributed into the distinct vacuole in the presence of wortmannin.
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thereby suggesting that the distinct staining pattern between TRP1 and LAMP1 was speci®c and signi®cant. Interestingly, GFPtagged TRP-1 behaved in the same way as endogenous TRP-1 in melanocytes (data not shown), but did not colocalize with LGP85 in the absence or presence of wortmannin (Fig 3A±F). Immunoseparation of lysosomes from melanosomes suggesting the distinct but partial overlap of melanosomes with lysosomes To observe the distinct compartmentalization of melanosomes and lysosomes in melanocytes, we wanted to immunoseparate melanosomes from lysosomes, using tail-speci®c antibodies, rabbit polyclonal antibodies against the cytoplasmic tails of both LGP85 and TRP1, respectively, following protein-A
Figure 4. Immunoisolation of melanosomes and lysosomes using tail-speci®c antibodies. LGP85-positive compartments and TRP-1positive compartments were immunoisolated with cytoplasmic tailspeci®c antibodies, respectively. The isolated fractions were analyzed by western blotting. LGP85-positive compartments are devoid of TRP-1, whereas TRP-1-positive fractions contained signi®cant amounts of LGP85.
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agarose beads sedimentation. The isolated fractions were analyzed by western blotting (Fig 4). The subcellular fraction isolated with the antibody to LGP85 tail was devoid of TRP-1, which suggested that the isolated fraction was highly enriched in lysosomes but not in melanosomes, i.e., melanosomes were distinct from lysosomes. The reciprocal experiment, the separation of melanosomes from lysosomes with the antibody against TRP-1, was not successful. TRP-1 positive fractions contained signi®cant amounts of LGP85, which may mean that lysosomes were more abundant than melanosomes in melan-a. Therefore, the anti-LGP85 antibody was primarily saturated with lysosomes, whereas the TRP-1 antibody fully interacted with both melanosomes and lateendosomes, the sorting place of TRP-1 and LGP85. Melanosomes are distinct from terminal lysosomes We then asked if melanosomes are the terminal of the endocytic pathway as lysosomes. In melan-a cells, TR-dextran was internalized and chased for the indicated time, then the ®xed cells were immunostained with anti-LAMP1, cathepsin L, and TRP-1 antibodies, respectively. Cathepsin L is one of the major acid hydrolases in lysosomes and is well characterized as the marker enzyme of terminal lysosomes with dense and acidic compartments. At an early time point (30 min or 1 h), TR-dextran was localized in the early endocytic compartments and did not colocalize with LAMP1 or TRP-1 (Fig 5A, E). TR-dextran colocalized with cathepsin L at 24 h, which suggested that TRdextran was delivered to the terminal lysosomes in melanocytes at this time point (Fig 5D). LAMP1 colocalized with TR-dextran at later time points, 8 and 24 h after the chase period; however, the extent of colocalization was slightly less than that seen with cathepsin L at 24 h (Fig 5B, C). This may mean that LAMP1 localizes on not only lysosomes but also the late/early endosomes. In contrast, there was no colocalization of TR-dextran with TRP1 positive compartments at any time point (Fig 5E±G). These ®ndings suggested that melanosomes are not the terminal of the endocytic pathway as lysosomes. The endocytic molecules never encountered the biosynthetic route of melanosomes.
Figure 5. Relationship between the endocytic routes and melanosomes. Melan-a cells were labeled with TR-dextran, chased for the indicated times at 37°C, and then processed for the immuno¯uorescence analysis with antibodies against LAMP1 (A±C), cathepsin L (D), and TRP-1 (E±G), respectively. At an early time point, TR-dextran was internalized via ¯uid phase endocytosis and delivered to early endosomes those are devoid of LAMP1 (A). At a later time point, TR-dextran reached late endosomes and lysosomes and colocalized with LAMP1 (B, C) and cathepsin L (D); however, TRP-1 never colocalized with TR-dextran at any time point (E±G).
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Figure 6. Model of the membrane traf®c to melanosomes and lysosomes in melanocytes. In biosynthetic pathways, the traf®c route of melanosome membrane protein (TRP-1) partially overlaps but is distinct from that of the endo/ lysosomal one (LAMP1, LGP85). The melanosome pathway is separated from the lysosome one after the AP-3-dependent sorting steps, probably at late endosomes and/or PLC. Melanosomes gradually maturate into heavily melanized melanosomes (from stage I to stage II±IV) with the accumulation of melanin inside the membrane. In the presence of wortmannin, there is membrane retrieval from the lysosomes or immature melanosomes, but not from the heavily melanized melanosomes. The melanosome pathway is also segregated from the endocytic pathway (TR-dextran).
DISCUSSION Melanosomes and lysosomes are highly related organelles and the protein targeting to these compartments is based on the vesicular transport. Accumulating evidence suggests targeting signals and molecular mechanisms that regulate the vesicle formation, transport, and fusion are common; however, it is not clear to what extent melanosomes are related to lysosomes in melanocytes. To characterize the membrane property of melanosomes we applied wortmannin to melanocytes. Wortmannin treatment (10 mM) caused the appearance of the spherical vacuoles as with other types of cells; however, the black granules (heavily melanized melanosomes) never got into these vacuoles. Ultrastructural analysis also indicated that the heavily melanized melanosomes did not change shape and size in the presence of this drug (data not shown). As they have an organized internal matrix (i.e., ®laments) within a membrane and are rigid, they have little membrane retrieval. When melanosomes have matured, they are separated from any membrane transport pathway and lose the membrane dynamics. We have found that melanosomes and lysosomes are distinct in melanocytes and wortmannin-induced vacuolation exaggerated the distinct compartmentalization of each membrane protein. LAMP1 and LGP85 colocalized either in the presence or in the absence of wortmannin, whereas TRP-1 indicated a little overlap with lysosome markers only at the perinuclear region where TGN or late endosomes are located. The appearance of swollen TRP-1speci®c vacuoles in the presence of wortmannin suggests that TRP-1 is not restricted on the mature melanosomes and has signi®cant distribution on the immature melanosomes, which are wortmannin. Interestingly, TRP-1 redistributed into the distinct vacuoles from that of lysosomes in the presence of wortmannin. A likely explanation for the appearance of swollen TRP-1-speci®c vacuoles is that TRP-1 is not restricted on the mature melanosomes and has signi®cant distribution on the immature melanosomes, where they are wortmannin sensitive but distinct from both heavily melanized melanosomes and lysosomes. This compartment re¯ects the existence of premelanosomal compartments, which are equivalent to PLC in nonmelanocytes. Orlow et al have shown that TRP-1 is limited to a less dense melanosomal compartment in contrast to tyrosinase, which is eventually localized to heavily melanized melanosomes. Their observation also supports the heterogeneity of melanosomes and the existence of the premelanosomal compartments (Orlow et al, 1993); however, they did not distinguish melanosomes from lysosomes and indicated the colocalization of LAMP1 and TRP-1 in melanocytes. The discrepancy between our results and theirs might be explained by the difference of cell types or the degree of melanization. Because
TRP-1 and LAMP-1/LGP85 are already segregated at the premelanosomal compartment, the ®nal step of the sorting could be taking place before this, possibly at late endosomes. Lysosomes are regarded as the terminal compartment of the endocytic pathway, which contain acid hydrolases but have less membrane retrieval in the presence of wortmannin (Reaves et al, 1996; Bright et al, 1997). The PLC is thought to be an intermediate compartment between lysosomes and late endosomes. Transiently, this compartment is formed by direct fusion of lysosomes with late endosomes. Contents are rapidly mixed and the membranes are retrieved and redistributed to their original positions. Therefore PLC, so-called late endosome-lysosome hybrid organelle, is very dynamic and unstable (Mullock et al, 1998; Luzio et al, 2000; Pryor et al, 2000). Wortmannin inhibits antetrograde movement from late endosomes to lysosomes, and consequently enhances formation of hybrid compartments. We assume that in melanocytes there are both PLC and pre-melanosomal compartments, whereas nonmelanocytes contain only PLC. In melanocytes, wortmannin may induce vacuolation of both compartments. Immunoelectron microscopic analysis strongly supported our assumption, as it is clear that heavily melanized melanosomes are devoid of both LGP85 and LAMP1 (data not shown). We also found a colocalization of both TRP-1 and LAMP1 in immature melanosomes and late endosomes (data not shown), which might be the sorting place for the melanosome proteins. A selective isolation of the subset of lysosomes from melanosomes indicates direct evidence that lysosomes are distinct from melanosomes; however, it is controversial as to whether the TRP1-positive compartment contains signi®cant amounts of LGP85. One interpretation of this discrepancy is that the number of lysosomes is more abundant than that of melanosomes in melanocytes. Therefore, the LGP85 tail antibody easily reacts and is saturated with lysosomes, whereas the TRP-1 tail antibody pulls down the mixture of melanosomes and late endosomes, which are the sorting sites of melanosomes and lysosomes. Further careful analysis needs to be carried out to support this. Based on the endocytic analysis in melanocytes, melanosomes are also distinct from the endocytic pathway. When terminal lysosomes were labeled with TR-dextran for 24 h chase, however, TRP-1 never colocalized with endocytic markers at any time point. Therefore, we propose that melanosomes are segregated from the endocytic route. Because heavily melanized melanosomes have little membrane retrieval, it is unlikely that they receive endocytic molecules from the outside of the cells. Interestingly the endocytosed TR-dextran colocalized well with LAMP-1 at an early time point, such as 8 h after the internalization, whereas they took longer to reach the cathepsin L-positive terminal lysosomes.
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JID SYMPOSIUM PROCEEDINGS
This result also represents the existence of the subpopulation and the heterogeneity of lysosomes. LAMP1 is known as a recycling molecule between lysosomes and plasmamembrane, therefore this colocalization is found at early and late endosomes rather than at lysosomes. There is evidence for retrograde traf®c between terminal lysosomes and PLC (Reaves et al, 1996); however, there is no evidence for the presence of premelanosomal compartments. Our results are a demonstration of the presence of the intermediate compartments and the membrane. In summary, we propose the following model of membrane protein transport to the lysosomes and melanosomes in melanocyte (Fig 6). Both melanosome and lysosome membrane proteins follow the AP-3 pathway until the late endosomes and/or PLC, because AP-3 mutation causes mistargeting of both membrane proteins to the cell surface (Le Borgne et al, 1998); however, after leaving the late endosome and/or PLC, they must take different routes to their ®nal destinations. Whereas lysosome membrane proteins are delivered to dense lysosomes, melanosome membrane proteins reach to the immature melanosomes (stage I). At this stage, it is likely that melanosome membrane protein, TRP-1 can be retrieved from there to the former sorting stations (PLC and/or late endosomes). Stage I melanosomes gradually maturate into heavily melanized melanosomes (stages II, III, and IV), lose the membrane dynamics, and then are transferred to the adjacent cells, keratinocytes by the cytophagocytosis. It is reasonable that melanosomes and lysosomes have different contributions to the cell homeostasis; melanosomes are for the pigmentation and lysosomes are for the degradation. To perform these distinct roles, they need to have a speci®c protein-sorting and transport system. Our ®ndings support the idea that the melanocyte has a speci®c sorting place and a mechanism to have melanosomes independent from lysosomes. Furthermore, heavily melanized melanosomes have unique membrane structures and proteins/lipids composition to be speci®cally transferred to the adjacent cells. This is the only example of the intercellular organelle transfer phenomena. Identi®cation the molecular mechanism of this phenomenon is very important to understand the pigmentation in melanocytes. How is the sorting of melanosome and lysosome performed or what kind of molecules are implicated in these steps? The genetic analysis of coat color mutation in mouse (Odorizzi et al, 1998) and eye color mutation in the fruit ¯y Drosophilla (Lloyd et al, 1998) may give us the answer to these questions. We thank Dr. J. T. August for kindly providing antimouse LAMP1 monoclonal antibody, Dr. Y. Tanaka for helpful assistance on the subcellular fractionation, M. Ohara for comments on the manuscripts, and Dr. K. Jimbow for helpful comments and discussion. This work was supported in part by a CREST grant from the Sciences and Technology Corporation of Japan, the Kao Foundation For Arts and Sciences, and Grants-in-Aid for Scienti®c Research from the Ministry of Education, Science, Sports, and Culture of Japan.
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