Tyrosinase and Tyrosinase-Related Protein 1 Require Rab7for Their Intracellular Transport

Kuninori Hirosaki, Toshiharu Yamashita, Ikuo Wada,* Hai-Ying Jin, and Kowichi JimbowDepartment of Dermatology and *Department of Biochemistry (Section II), Sapporo Medical University School of Medicine, Chuo-ku, Sapporo, Japan

We have recently identi®ed the association of Rab7in melanosome biogenesis and proposed that Rab7 isinvolved in the transport of tyrosinase-related protein1 from the trans-Golgi network to melanosomes,possibly passing through late-endosome-delineatedcompartments. In order to further investigate therequirement of Rab7-containing compartments forvesicular transport of tyrosinase family proteins, weexpressed tyrosinase and tyrosinase-related proteinby recombinant adenovirus and analyzed their local-ization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infectionof the recombinant adenoviruses carrying tyrosinase(Ad-HT) and TRP-1 (Ad-TRP-1) resulted in theenhancement of tyrosinase activity and melanin pro-duction compared to a single infection of Ad-HT. Inthe Ad-HT-infected SK-mel-24 cells many of thenewly synthesized tyrosinase proteins were coloca-

lized in lysosomal lgp85-positive granules of theentire cytoplasm, whereas in the presence ofRab7N125I the colocalization of tyrosinase and lgp85proteins was decreased markedly in the distal area ofthe cytoplasm. In the Ad-TRP-1-infected SK-mel-24cells, TRP-1, which is reported to be present exclu-sively in melanosomes, was detected throughout thecytoplasm, but not colocalized in prelysosomal (earlyendosomal) EEA-1 granules. In the presence ofRab7N125I, however, TRP-1 was retained in theEEA-1-positive granules. Our ®ndings indicate thatthe dominant-negative mutant of Rab7 impairs vesi-cular transport of tyrosinase and TRP-1, suggestingthat the transport of these melanogenic proteinsfrom the trans-Golgi network to maturing melano-somes requires passage through endosome-delineatedcompartments. Key words: melanosome/Rab7/TRP-1/tyrosinase/vesicular transport. J Invest Dermatol 119:475±480, 2002

Tyrosinase and tyrosinase-related proteins (TRPs) aremembrane-bound melanogenic proteins expressedexclusively in the melanocyte and localized inmelanosomes (Prota, 1992; Jimbow et al, 1999).They share structural similarities such as an N-terminal

signal sequence, a cysteine-rich region, and two copper-bindingdomains. Tyrosinase catalyzes conversion of tyrosine to dopa andsubsequently dopa to dopaquinone, which is an initial and rate-limiting step of melanin biosynthesis. TRP-1 was reported topossess dihydroxy-indole-2-carboxylic acid (DHICA) oxidaseactivity (Kobayashi et al, 1994) and to form a stable complex withtyrosinase (Winder et al, 1994; Jimenez-Cervantes et al, 1998;Kobayashi et al, 1998), which may enhance eumelanin productionin melanocytes. Similar to other secretory membrane proteins,these melanogenic proteins are transported from the trans-Golginetwork (TGN) to melanosomes by a vesicular transport system(Jimbow et al, 2000).

Studies on vesicular transport have indicated that Rab GTPasesand SNAREs play essential roles for the intracellular transport inthe eukaryotic cell system (Olkkonen and Stenmark, 1997; Pfeffer,1999). It is suggested that Rab GTPases recruit tethering anddocking factors to establish molecular interaction between themembranes, after which SNAREs complete the fusion process.Several Rab proteins such as Rab4 and Rab5 are localized in theearly sorting and recycling endosomal compartments (Gorvel et al,1991; Bucci et al, 1992; van der Sluijs et al, 1992). Meanwhile,Rab7 is localized on the late endosomes (Chavrier et al, 1990), andimplicated in downstream endocytotic traf®c either from lateendosome to lysosome (Feng et al, 1995; MeÂresse et al, 1995;Mukhopadhyay et al, 1997; Vitelli et al, 1997) or possibly from earlyto late endosome (Press et al, 1998).

Earlier we have proposed that the maturation and three-dimensional assembly of tyrosinase gene family proteins in theendoplasmic reticulum are regulated by calnexin (Jimbow et al,1994; Toyofuku et al, 1999). Indeed, albino-associated mutants oftyrosinase were shown to have defects in appropriate folding andinteraction with calnexin and calreticulin, which result in theirretention in the endoplasmic reticulum (Halaban et al, 1997; 2000).The molecular mechanism of vesicular transport of tyrosinase andTRPs between the TGN and melanosomes has remained unclear,however. Recently, we have found that late-endosome-delineatedcompartments and Rab7 are involved in the transport of tyrosinaseand TRP-1 between the TGN and melanosomes (Jimbow et al,1997; 2000; Gomez et al, 2001). In order to further investigatewhether Rab7 GTPase is involved in the vesicular transport of

Manuscript received December 6, 2001; revised March 25, 2002;accepted for publication April 4, 2002.

Reprint requests to: Dr. Kowichi Jimbow, Department of Dermatology,Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo 060, Japan. Email: jimbow@sapmed.ac.jp

Abbreviations: AP, adapter protein; DHICA, dihydroxy-indole-2-carboxylic acid; Lamp, lysosome-associated membrane protein; LAP,lysosomal acid phosphatase; moi, multiplicity of infection; pfu, plaque-forming unit; TGN, trans-Golgi network; TRP, tyrosinase-related protein;wt, wild-type.

0022-202X/02/$15.00 ´ Copyright # 2002 by The Society for Investigative Dermatology, Inc.

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tyrosinase family proteins, we expressed tyrosinase and TRP-1 byadenovirus vector and examined the effect of a dominant-negativemutant of Rab7 on the transport of melanogenic proteins. Weshow here that vesicular transport of tyrosinase and TRP-1 requiresa transient passage through Rab7-associated endosomal compart-ments to be transported to melanosomes.

MATERIALS AND METHODS

Cells and cell culture Untransformed human ®broblasts, MRC5,SV40-transformed monkey kidney cell line, COS7, and adenovirus type5 DNA-transformed human kidney cell line, 293, were purchased fromAmerican Type Culture Collection (Rockville, MD). Amelanotic SK-mel-24 and melanotic SK-mel-23 human melanoma cell lines wereprovided by Dr. Houghton of Sloan Kettering Cancer Center, NewYork. Cells were cultured in Dulbecco's modi®ed Eagle's medium(Gibco BRL, Paisley, U.K.) supplemented with 5% fetal bovine serum(ICN Flow, Basingstoke, U.K.) and antibiotics.

Recombinant adenovirus Ad-LacZ, b-galactosidase-expressingrecombinant adenovirus, was kindly provided by Dr. M Imperiale ofMichigan University. Construction of recombinant adenoviruses carryingcDNA of human tyrosinase (Ad-HT), human TRP-1 (Ad-TRP-1),wild-type (wt) Rab7 (Ad-wtRab7), or mutant Rab7 (Ad-Rab7N125I)were carried out as described previously (Takahashi et al, 1996; Yamanoet al, 1999). Brie¯y, human tyrosinase and TRP-1 cDNAs were preparedfrom pcDNA-HT (Singh and Jimbow, 1998) and pRHOHTa p(Takimoto et al, 1995) by cleaving them with EcoRI and SalI + XbaI,respectively. cDNAs of wtRab7 and its dominant-negative mutantRab7N125I (Feng et al, 1995; Vitelli et al, 1997; Press et al, 1998) werecleaved off from pGEM-Rab7 and pGEM-Rab7N125I by BamHI. Afterthe cDNA fragments were blunted by T4 polymerase, they were insertedinto the HindIII site, which had also been blunted by T4 polymerase, ofpAd-BglII at the downstream of human cytomegalovirus early promoter(Takahashi et al, 1996). Parts of the inserted cDNAs in pAd-BglII wereveri®ed by nucleotide sequencing by the dideoxy termination methodusing the Applied Biosystems Model 373S DNA Sequencing system.Then, pAd-BglII plasmids were separately cotransfected with pJM17(McGrory et al, 1988) into 293 cells. Resulting recombinant adenovirusesobtained from transfected 293 cell culture, which showed cytopathiceffect, were cloned and propagated in 293 cells, and infected cell lysatewas aliquoted and frozen at ±80°C until use. Cells were infected withthe virus at the multiplicity of infection (moi) of more than 20 plaque-forming units (pfu) per cell, as nearly maximum numbers of cells werefound to express b-galactosidase after Ad-LacZ infection at the moi of20 pfu per cell in various types of cells (Yamashita et al, 2001).

Western blotting Expression of tyrosinase and TRP-1 was detectedby Western blotting as described previously (Yamashita et al, 1999).Four 3 105 cells were infected with Ad-HT or Ad-TRP-1 at the moiof 20 pfu per cell and cultured for 0±48 h. After cells were collected and

lyzed in 10 mM KCl, 1.5 mM MgCl2, 10 mM Tris (pH 7.4), 0.5%sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl ¯uoride,they were sonicated for 10 s with Branson's sonicator. Proteinconcentration was determinated by using a BCA protein assay kit(Pierce, Rockford, IL). Samples containing 5.0 mg protein were separatedby 5%±20% gradient SDS polyacrylamide gel electrophoresis (PAGE)(Bio-Rad, Tokyo, Japan) at 100 V for 80 min. The proteins in thepolyacrylamide gel were transferred onto a nitrocellulose membrane(Protran, Schleicher & Schuell, Dassel, Germany) by electroblotting at100 V for 80 min. The membrane was probed with the primaryantibodies as follows: 1 mg per ml of mouse monoclonal antibody fortyrosinase (Novocastra, Newcastle upon Tyne, U.K.), 5 ml of anti-TRP-1 rabbit serum, or 1 mg per ml of antihuman a-actin monoclonalantibody (AL-40, Sigma, MI) in 5 ml of PBS-T (0.1% Tween-20 inphosphate-buffered saline) at room temperature for 1 h. The blot wasthen washed with PBS-T and incubated with horseradish peroxidaseconjugated to a secondary antibody. The speci®c complexes weredetected by the ECL chemiluminescence reagent (Amersham PharmaciaBiotech, Buckinghamshire, U.K.).

Tyrosinase (dopa oxidase) activity Five 3 105 cells were infectedwith recombinant adenovirus at the moi of 20 pfu per cell and culturedfor 48 h. After cells were lyzed in 300 ml of 1% Triton X-100 in 0.1 Mphosphate buffer (pH 6.8), samples were sonicated and centrifuged at8000 rpm for 10 min. 100 ml of 0.15% dopa solution was added to the100 ml of obtained supernatant and incubated at 37°C for 10 min. Thetyrosinase activity was measured with a spectrophotometer at 475 nm.

Measurement of melanin After 4 3 105 cells were seeded in 6 cmdishes and cultured for 24 h, they were infected with recombinantadenovirus at the moi of total 20 pfu per cell and cultured for 48 h.Cells were detached and collected in Eppendorf tubes. Then, cells weredissolved in 1 ml of alkaline solution (1 N NaOH in 10%dimethylsulfoxide) and incubated at 80°C for 2 h. After centrifugation at1000 3 g for 10 min, the supernatant was taken to measure the proteinconcentration and melanin. The amount of melanin was measured witha spectrophotometer at 420 nm by using synthetic melanin (Sigma M-8631, St. Louis, MI) as a standard.

Antibodies for immuno¯uorescence Anti-lgp85, a rabbit polyclonalantiserum used for a lysosomal marker, was a generous gift of Dr. H.Fujita of Kyusyu University, Fukuoka, Japan. Anti-EEA-1 (a marker forearly endosome) and antisyntaxin 8 (a marker for late endosome) mousemonoclonal antibodies are products of Transduction Laboratories (LosAngeles, CA). Antihuman tyrosinase mouse monoclonal antibody waspurchased from Novocastra. Antihuman TRP-1 antibody was raised inrabbits by injecting C-terminal amino acid residues, CNQPLLTD-QYQSYAEE, of human TRP-1 into domestic rabbits. Secondaryantibodies conjugated to Alexa 488 and Alexa 594 were purchased fromMolecular Probes (Eugene, OR).

Immuno¯uorescence analysis For immuno¯uorescence experiments,the cells were grown on poly L-lysine (10 mg per ml) coated roundcoverslides in 12-well plates (Coster, NY). Cells cultured on thecoverslides were ®xed and permeabilized with methanol at ±20°C for4 min. Fixed cells were washed with blocking washing buffer [PBS

Figure 1. Detection of tyrosinase and TRP-1 by Westernblotting. Cells infected with Ad-HT or Ad-TRP-1 (20 pfu per cell)were lyzed 48 h after virus inoculation, and cellular lysates containing5.0 mg of protein were fractionated by 5%±20% SDS-PAGE. Aftercellular proteins were transferred to a nitrocellulose membrane, theywere reacted with speci®c antibodies against tyrosinase (A1), TRP-1(B1), or a-actin (A2, B2). The speci®c bands were visualized bychemiluminescence as described in Materials and Methods.

Figure 2. Tyrosinase (dopa oxidase) activity and melanin contentof Ad-HT- and/or Ad-TRP-1-infected cells. SK-mel-24 cells wereinfected with recombinant adenoviruses and cultured for 48 h andprocessed to measurement of dopa oxidase activity and melanin content.(A)Relative value of dopa oxidase activity was determined by the valueat 475 nm of infected cell lysate after incubation with 0.15% dopa at37°C for 20 min. (B)Melanin content was determined by the value at420 nm of infected cell lysate using synthetic melanin as a standard.

476 HIROSAKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

containing 0.5% (wt/vol) bovine serum albumin] twice and with 0.025%Tween-20 diluted in the washing buffer once, and were incubated withvarious antibodies for 30 min at room temperature. Coverslips werewashed once with the washing buffer for 5 min, rinsed with water, andmounted in Vectashield (Vector Laboratories, CA). Fluorescent imageswere obtained with immuno¯uorescent microscopes: (i) Olympusupright microscope with an argon laser (Mitsubishi Optiplex GXM5133, Tokyo) and Fluoview software, and (ii) Nikon Fluoview confocallaser scanning microscope and Bio-RAD MRC 1024 software (Bio-Rad,Tokyo, Japan).

RESULTS

Adenovirus-mediated transfer and expression of tyrosinaseand TRP-1 First, SK-mel-24 cells were infected with Ad-HT orAd-TRP-1 at the moi of 20 pfu per cell, cultured for 48 h, andcollected to examine the expression of tyrosinase and TRP-1 byWestern blotting (Fig 1A, B). A tyrosinase band became detectableby 12 h and reached a maximum level at 24 h after viralinoculation. Tyrosinase was detected as an approximately 70 kDabroad band, and TRP-1 was also about 70 kDa (Fig 1A, B).Infected SK-mel-24 cells contained a larger amount of tyrosinasethan SK-mel-23 cells did (Fig 1A). The immunostaining againsttyrosinase- or TRP-1-speci®c antibodies also became detectableunder the confocal microscope in the perinuclear region 4 h afterviral infection, and then the immunostains were dispersed to theentire cytoplasm showing ®ne granules at 24 h (data not shown).

Tyrosinase and TRP-1 collaborate to produce melanin inamelanotic melanoma cells In order to con®rm that tyrosinaseand TRP-1 expressed by the recombinant adenovirus arebiologically and biochemically functional, we measured dopaoxidase activity and intracellular melanin in Ad-HT- and/or Ad-TRP-1-infected SK-mel-24 cells. After cells were infected withAd-LacZ, Ad-HT, Ad-TRP-1, or Ad-HT plus Ad-TRP-1 andcultured for 48 h, cellular lysates were collected and processed tomeasure tyrosinase activity and melanin content as described inMaterials and Methods. Even with a single introduction of tyrosinase

gene, dopa oxidase activity and melanin content were signi®cantlyincreased (Fig 2A, B). When tyrosinase and TRP-1 werecoexpressed, tyrosinase activity and melanin content wereincreased approximately 3.0-fold and 1.3-fold, respectively,compared to those in the case of tyrosinase expression alone(Fig 2A, B).

Effect of dominant-negative expression of mutant Rab7 onthe localization of tyrosinase When SK-mel-24 cells wereinfected with Ad-HT for 5 h and doubly stained with antityrosinaseand anti-Lamp-1 (a marker for lysosome and melanosome)antibodies (Orlow, 1995), the majority of tyrosinaseimmunostains were found to be colocalized in the Lamp-1-positive vesicles (Fig 3A±C). This suggests that the newlysynthesized tyrosinase accumulates in lysosomes as well asmelanosomes when overexpressed in SK-mel-24 cells.

In order to further test if Rab7 function is required for transportof tyrosinase family proteins from the TGN to melanosomes passingthrough the endosomal compartments (e.g., early endosomes, lateendosomes, or both), effects of Rab7N125I expression on thelocalization of tyrosinase or TRP-1 were examined. The mutantRab7N125I exists preferentially in the nucleotide-free form andimpairs Rab7 function in vesicular transport of various proteins(Feng et al, 1995; Vitelli et al, 1997). SK-mel-24 cells were infected®rst with Ad-LacZ, Ad-Rab7wt, or Ad-Rab7N125I at the moi of50 pfu per cell and cultured for 48 h, and then infected with Ad-HT at the moi of 50 pfu per cell. Cells from each group of infectionwere cultured for another 24 h, and then puromycin (40 mM) wasadded to inhibit the following protein synthesis. Coexpression of b-galactosidase (Fig 4A±C) or wtRab7 (data not shown) did not alterthe localization of tyrosinase, which was observed in the lgp85(another lysosomal marker)-positive vesicles. In contrast, inRab7N125I-expressing SK-mel-24 cells, the majority of tyrosinasewas localized in the cytoplasm located differently from lgp85, and itwas accumulated in enlarged vesicular structures at the peripheralarea of cells (Fig 4D±F). The rate of colocalization of lgp85 andtyrosinase was 49.4% in Ad-LacZ-infected cells, whereas it was17.9% in Ad-Rab7N125I-infected cells at the distal region of thecytoplasm (Fig 5A). This result suggests that the over-expression ofRab7N125I impairs the transfer of tyrosinase to lysosomes,melanosomes, or both.

Effect of mutant Rab7 on the localization of TRP-1 Wehad no polyclonal antityrosinase antibody, which was necessary toobtain doubly stained images with anti-EEA-1 (an early endosomalmarker) or syntaxin 8 (a late endosomal marker) monoclonalantibodies. Recently, another melanosome-targeted protein TRP-1 was shown to reach almost exclusively to melanosomes (Raposoet al, 2001). Thus, we investigated the localization of anothermelanosome-targeted protein TRP-1, which was detectable by a

Figure 3. Colocalization of tyrosinase and Lamp-1 in SK-mel-24cells. After cells were infected with Ad-HT for 5 h, they were reactedwith antityrosinase (FITC, A)or anti-Lamp-1 (Texas Red, B). (C) is amerge of (A) and (B). Scale bar: 20 mm.

Figure 4. Effect of Rab7N125I on thelocalization of tyrosinase in SK-mel-24 cells.Cells were infected ®rst with Ad-LacZ (A, B) orAd-Rab7N125I (D, E) and then with Ad-HT (A,B, D, E) and incubated in the puromycin-containing medium. Infected cells were reactedwith anti-lgp85 serum (A, D) or antityrosinaseantibody (B, E). In (C) (a merge of A and B),colocalized signals are con®ned in the lgp85 (alysosomal marker)-reacted vesicles, whereas in (F)(a merge of D and E) immunostains of tyrosinaseare differently located from those of lgp85. Scalebar: 10 mm.

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polyclonal rabbit serum against human TRP-1, as well as bymonoclonal antibodies against EEA-1 and syntaxin 8 as sitemarkers. SK-mel-24 cells were infected with Ad-LacZ or Ad-Rab7N125I and then Ad-TRP-1 followed by culture in thepresence of puromycin as described above.

As expected, in the mutant Rab7-expressing cells, the enlargedperipheral vacuoles in which TRP-1 was con®ned were intenselystained by an anti-EEA-1, early endosomal antibody (Fig 6D±F).Little or no colocalization of TRP-1 with EEA-1 was observed inthe cells expressing b-galactosidase (Fig 6A±C). Rates of coloca-lization of these proteins were 26.4% in Ad-LacZ-infected cells,and 91.3% in Ad-Rab7N125I-infected cells (Fig 5B). In contrast,immunostaining with the antibody against syntaxin 8 of a lateendosomal marker showed little or no colocalization with TRP-1in either the Ad-LacZ- or Ad-Rab7N125I-infected cells (Fig 5C).These results suggest that intracellular transport of TRP-1 requiresfunctional Rab7, probably passing through early to late endosomalcompartments. Taken together, we conclude that the transport oftyrosinase family proteins to lysosomes (tyrosinase), and/ormelanosomes (tyrosinase and TRP-1) transits Rab7-associatedvesicular transport from endosome-delineated compartments inSK-mel-24 cells.

DISCUSSION

There are two major classes of melanin pigment in humans andanimals, i.e., brown black eumelanin and yellow red pheomelanin.The two pigments are synthesized in melanosomal compartments,which follow four stages of maturation starting from the commonvacuoles, hence being called stage I melanosomes (Jimbow et al,2000). Our recent studies have indicated that the previouslyde®ned stage I melanosomes, which are spherical and morpholo-gically resemble multivesicular bodies, are related to late-endosomalcompartments (Jimbow et al, 2000). Recently, it was also reportedthat tyrosinase and dopachrome tautomerase are present in stage IImelanosomes associated with their enzymatic activities, but they, aswell as TRP-1, MART1, and gp100, are cleaved and inactivated instage I melanosomes (Kushimoto et al, 2001). It has been suggested

that the traf®cking of tyrosinase family proteins to melanosomes ismediated by a number of membrane-bound carrier intermediates(Jimbow et al, 2000). Of small GTP-binding proteins, Rab3, Rab7,and Rab8 were found in the melanosomal fraction (Gomez et al,2001). Our sense and antisense oligonucleotide experimentsindicated (i) that Rab7 is a melanogenesis-associated molecule,(ii) that TRP-1 is present in late-endosome-delineated granules,and (iii) that Rab7 is involved in the transport of TRP-1 from late-endosome-delineated granules to melanosomes (Gomez et al,2001). This study has provided additional new information forthe requirement of Rab7 in the intracellular traf®cking of tyrosinaseand TRP-1, and identi®ed the involvement of early-endosome-delineated granules in this melanogenesis cascade.

The amelanotic melanoma cell line SK-mel-24 containsunmelanized melanosomes but does not express melanogenictyrosinase or TRP-1 (Luo et al, 1994). In this study ofadenovirus-mediated tyrosinase gene transfer, we succeeded inexpressing the melanin production in SK-mel-24 cells. SK-mel-24cells with coinfection of Ad-HT and Ad-TRP-1 revealed highertyrosinase activity and a larger amount of melanin pigment thanthose infected with Ad-HT alone (Fig 2). In contrast with murineTRP-1, human TRP-1 seems to possess no DHICA oxidaseactivity (Boissy et al, 1998). It was therefore suggested that theenhancement of tyrosinase-mediated melanin production by TRP-1 results from the formation of a stable complex of TRP-1 withtyrosinase (Winder et al, 1994; Jimenez-Cervantes et al, 1998;Kobayashi et al, 1998).

Tyrosinase and TRP-1 share structural similarities, and theirmelanosomal localization depends on the cytoplasmic, membrane-proximal di-leucine-based targeting signals (Vijayasaradhi et al,1995; Blagoveshchenskaya et al, 1999; Calvo et al, 1999; Simmen etal, 1999; Jimbow et al, 2000). The intracellular traf®cking of theseproteins is not identical, however, as the di-leucine-based signal oftyrosinase interacts with adapter protein 3 (AP-3) in vitro andtyrosinase is mislocalized in AP-3-de®cient melanocytes, whereasTRP-1 seems to be properly targeted in them with the involve-ment of AP-1 (Hoening et al, 1998; Huizing et al, 2001).

In our study, it is likely that exogenously introduced tyrosinase istargeted both to lysosomes and to melanosomes of SK-mel-24 cells,as (i) Lamp-1 is present in the melanosomal and lysosomalcompartments (Orlow, 1995; Jimbow et al, 1997) and (ii) electronmicroscope observation of tyrosinase-gene-transfected SK-mel-24

Figure 5. Rates of colocalization of melanogenic proteins andlysosomal or endosomal marker proteins. (A)Colocalization oftyrosinase and lgp85 in Ad-HT-infected SK-mel-24 cells. Determinedfrom the experiment shown in Fig 4. (B)Colocalization of TRP-1 andEEA-1 in Ad-TRP-1-infected SK-mel-24 cells. Determined from theexperiment of Fig 6. (C)Colocalization of TRP-1 and syntaxin 8 in Ad-TRP-1-infected SK-mel-24 cells. Percentages were determined fromseveral ®elds of photographs of confocal microscopy.

Figure 6. Effect of Rab7N125I on the localization of TRP-1 inSK-mel-24 cells. Cells were successively infected with Ad-LacZ (A, B)or Ad-Rab7N125I (D, E) and with Ad-TRP-1 (A, B, D, E) andincubated in the puromycin-containing medium. Infected cells werereacted with anti-EEA-1 antibody (A, D) or anti-TRP-1 serum (B, E).In (C) (a merge of A and B), TRP-1 and EEA-1 stains are differentlylocalized, whereas in (F) (a merge of D and E) immunostains of TRP-1are colocalized with those of EEA-1. Scale bar: 10 mm.

478 HIROSAKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

cells detected spherical lysosomal compartments with melanindeposition (Luo et al, 1994). In contrast with tyrosinase, however,TRP-1 is transported exclusively to melanosomes, not to lysosomes(Raposo et al, 2001). We have proposed recently that the biogenesiscascades of eumelanosomes and pheomelanosomes may be differ-ent; pheomelanosome biogenesis involves lysosomal compartmen-talization in which only tyrosinase is accumulated to form pheo(false) melanin (yellow red pigment) through the formation of acysteine complex (benzothiazine unit) deriving from lysosomes andits subsequent auto-oxidation, whereas eumelanogenesis involvesthe compartmentalization of a tyrosinase and TRP complex, whichresults in the formation of eu (true) melanin (brown black pigment)through enzymatic reaction (Jimbow et al, 2000).

Recent studies using the mutants have shown that the RabGTPases have essential roles in the speci®c membrane transportpathways (Zerial and Stenmark, 1993; Stow, 1995). Dominant-negative Rab7 mutants do not affect the internalization of vesicularstomatitis virus G protein but impair its degradation in lysosomes(Feng et al, 1995). A similar result was observed in low-densitylipoprotein in BHK21 cells in the presence of Rab7 mutants (Vitelliet al, 1997). A part of the Rab7 activating mutant (Rab7Q67 L) wascolocalized with lysosomal marker lgp120 (MeÂresse et al, 1995).The yeast homolog of Rab7, Ypt7p, has also been shown tofunction at a later step in transport to the vacuole and in homotypicvacuole fusion events (SchimmoÈller and Riezman, 1993; Haas et al,1995). All these suggest that Rab7 has an important role in theprocess of vesicular transport in endosome and lysosome biogenesis.

APs, especially AP-3, have now been identi®ed in melanocytes.AP-3 was shown to play an important role in the vesicular transportof tyrosinase as can be seen in coat pigmentation of mice (Odorizziet al, 1998). The mutation of genes encoding the b3A subunit ofAP-3 causes the conditions relevant to Hermansky±Pudlak syn-drome in mice (Feng et al, 1999), an autosomal recessive diseasecharacterized by abnormal synthesis and/or secretion of threestorage-related organelles, i.e., melanosomes, lysosomes, andplatelet-derived granules. APs for lysosome-associated proteins,e.g., Lamp-1 and lysosomal acid phosphatase (LAP), appear to bedifferent from AP-3. Hoening et al (1998), in their study ofDrosophilia eye pigmentation, found a low af®nity of tyrosinase-associated adapters to a cytoplasmic tail of Lamp-1 or LAP, eventhough the interaction of AP-1 and AP-2 with Lamp-1 or LAP washigh. Recently, the product of ocular albinism type 1 gene, amelanocyte-speci®c integral glycoprotein, was reported to belocalized to late endosomes/lysosomes but not melanosomes andinvolved in reorganization of endolysosomal compartments andtraf®cking of vesicules to developing melanosomes (Samaraweera etal, 2001; Shen et al, 2001).

In an immunoelectron microscope study of heavily pigmentedmelanocytes, Raposo et al (2001) found recently that melanosomesare distinct from conventional endosomal organelles but integratedwith the endocytotic pathway. Speci®cally they indicated that,whereas Lamp-1 and TRP-1 probably cosegregate during post-Golgi sorting, TRP-1 is not detected in lysosomes but in maturingand mature melanosomes. In contrast, Lamp-1-enriched granulesare not melanosomes but lysosomes. It is speculated that there willbe a common cascade between melanosomes and lysosomes at theearly stage of their biogenesis. This study has clearly shown thattyrosinase and TRP-1 require Rab7 in this early stage ofmelanosomal biogenesis before being transported from the TGNto maturing melanosomes in human melanoma cells.

This work was supported in part by Grants-in-Aid from the Ministry of Education

(#12470179 and #13877131), Science, Sports and Culture of Japan. We thank

Dr. Wandinger-Ness of North-western University and Dr. M. Zerial of Max

Planck Institute for Molecular Cell Biology and Genetics for providing recombinant

plasmids containing wild-type and mutant Rab7. We also thank Dr. H. Fujita of

Kyushu University for providing anti-lgp85 rabbit serum.

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