Tyrosinase and Tyrosinase-Related Protein 1 Require Rab7 for Their Intracellular Transport
2002; Elsevier BV; Volume: 119; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2002.01832.x
ISSN1523-1747
AutoresKuninori Hirosaki, Toshiharu Yamashita, Haiying Jin, Kowichi Jimbow, Ikuo Wada,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoWe have recently identified the association of Rab7 in melanosome biogenesis and proposed that Rab7 is involved in the transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes, possibly passing through late-endosome-delineated compartments. In order to further investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and tyrosinase-related protein by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection of the recombinant adenoviruses carrying tyrosinase (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected SK-mel-24 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal lgp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization of tyrosinase and lgp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected SK-mel-24 cells, TRP-1, which is reported to be present exclusively in melanosomes, was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules. Our findings indicate that the dominant-negative mutant of Rab7 impairs vesicular transport of tyrosinase and TRP-1, suggesting that the transport of these melanogenic proteins from the trans-Golgi network to maturing melanosomes requires passage through endosome-delineated compartments. We have recently identified the association of Rab7 in melanosome biogenesis and proposed that Rab7 is involved in the transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes, possibly passing through late-endosome-delineated compartments. In order to further investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and tyrosinase-related protein by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection of the recombinant adenoviruses carrying tyrosinase (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected SK-mel-24 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal lgp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization of tyrosinase and lgp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected SK-mel-24 cells, TRP-1, which is reported to be present exclusively in melanosomes, was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules. Our findings indicate that the dominant-negative mutant of Rab7 impairs vesicular transport of tyrosinase and TRP-1, suggesting that the transport of these melanogenic proteins from the trans-Golgi network to maturing melanosomes requires passage through endosome-delineated compartments. adapter protein dihydroxy-indole-2-carboxylic acid lysosome-associated membrane protein lysosomal acid phosphatase multiplicity of infection plaque-forming unit trans-Golgi network tyrosinase-related protein wild-type Tyrosinase and tyrosinase-related proteins (TRPs) are membrane-bound melanogenic proteins expressed exclusively in the melanocyte and localized in melanosomes (Prota, 1992Prota G. Melanin and Melanogenesis. Academic Press, New York1992: 1-290Google Scholar;Jimbow et al., 1999Jimbow K. Quevedo Jr, W.C. Prota G. Fitzpatrick T.B. Biology of melanocytes.in: Freedberg M. Eisen A.Z. Wolff K. Austin K.F. Goldsmith L.A. Katz S.I. Fitzpatrick T.B. Dermatology in General Medicine. 5th edn. McGraw-Hill, New York1999: 192-222Google Scholar). They share structural similarities such as an N-terminal signal sequence, a cysteine-rich region, and two copper-binding domains. Tyrosinase catalyzes conversion of tyrosine to dopa and subsequently dopa to dopaquinone, which is an initial and rate-limiting step of melanin biosynthesis. TRP-1 was reported to possess dihydroxy-indole-2-carboxylic acid (DHICA) oxidase activity (Kobayashi et al., 1994Kobayashi T. Urabe K. Winder A. et al.Tyrosinase related protein-1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis.EMBO J. 1994; 13: 5818-5825Crossref PubMed Scopus (435) Google Scholar) and to form a stable complex with tyrosinase (Winder et al., 1994Winder A. Kobayashi T. Tsukamoto K. Urabe K. Aroca P. Kameyama K. Hearing V.J. The tyrosinase gene family interactions melanogenic proteins to regulate melanogenesis.Cell Mol Biol Res. 1994; 40: 613-626PubMed Google Scholar;Jimenez-Cervantes et al., 1998Jimenez-Cervantes C. Martinez-Esparza M. Solano F. Lozano J.A. Garcia-Borron J.C. Molecular interactions within the melanogenic complex. Formation of heterodimers of tyrosinase and TRP-1 from B16 mouse melanoma.Biochem Biophys Res Commun. 1998; 253: 761-767Crossref PubMed Scopus (31) Google Scholar;Kobayashi et al., 1998Kobayashi T. Imokawa G. Bennett D.C. Hearing V.J. Tyrosinase stabilization by Trp1 (the brown locus protein).J Biol Chem. 1998; 273: 31801-31805Crossref PubMed Scopus (161) Google Scholar), which may enhance eumelanin production in melanocytes. Similar to other secretory membrane proteins, these melanogenic proteins are transported from the trans-Golgi network (TGN) to melanosomes by a vesicular transport system (Jimbow et al., 2000Jimbow K. Chen H. Gomez P.F. et al.Intracellular vesicular trafficking of tyrosinase gene family protein in eu- and pheo-melanosome biogenesis.Pigment Cell Res. 2000; 13: 110-117Crossref PubMed Scopus (41) Google Scholar). Studies on vesicular transport have indicated that Rab GTPases and SNAREs play essential roles for the intracellular transport in the eukaryotic cell system (Olkkonenn and Stenmark, 1997Olkkonenn V. Stenmark H. Role of Rab GTPases in membrane traffic.Int Rev Cytol. 1997; 176: 1-85Crossref PubMed Google Scholar;Pfeffer, 1999Pfeffer S.R. Transport-vesicle targeting: tethers before SNAREs.Nature Cell Biol. 1999; 1: 17-22Crossref PubMed Scopus (361) Google Scholar). It is suggested that Rab GTPases recruit tethering and docking factors to establish molecular interaction between the membranes, after which SNAREs complete the fusion process. Several Rab proteins such as Rab4 and Rab5 are localized in the early sorting and recycling endosomal compartments (Gorvel et al., 1991Gorvel J.-P. Chavrier P. Zerial M. Gruenberg J. Rab5 controls early endosome fusion in vitro.Cell. 1991; 64: 915-925Abstract Full Text PDF PubMed Scopus (858) Google Scholar;Bucci et al., 1992Bucci C. Parton R.G. Mather I.H. Stunnenberg H. Simons K. Hoflack B. Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytotic pathway.Cell. 1992; 70: 715-728Abstract Full Text PDF PubMed Scopus (1118) Google Scholar;van der Sluijs et al., 1992van der Sluijs P. Hull M. Webster P. Male P. Goud B. Mellman I. Toyofuku K. The small GTP-binding protein rab4 controls an early sorting event on the endocytotic pathway.Cell. 1992; 70: 729-740Abstract Full Text PDF PubMed Scopus (509) Google Scholar). Meanwhile, Rab7 is localized on the late endosomes (Chavrier et al., 1990Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments.Cell. 1990; 62: 317-329Abstract Full Text PDF PubMed Scopus (885) Google Scholar), and implicated in downstream endocytotic traffic either from late endosome to lysosome (Feng et al., 1995Feng Y. Press B. Wandinger-Ness A. Rab 7: an important regulator of late endocytic membrane traffic.J Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (535) Google Scholar;Méresse et al., 1995Méresse S. Gorvel J.-P. Chavrier P. The rab7 GTPase resides on a vesicular compartment connected to lysosomes.J Cell Sci. 1995; 108: 3349-3358Crossref PubMed Google Scholar;Mukhopadhyay et al., 1997Mukhopadhyay A. Funato K. Stahl P.D. Rab7 regulates transport from early to late endocytic compartments in xenopus oocytes.J Biol Chem. 1997; 272: 13055-13059Crossref PubMed Scopus (88) Google Scholar;Vitelli et al., 1997Vitelli R. Santillo M. Lattero D. Chiariello M. Bifulco M. Bruni C.B. Bucci C. Role of the small GTPase Rab7 in the late endocytic pathway.J Biol Chem. 1997; 272: 4391-4397Crossref PubMed Scopus (251) Google Scholar) or possibly from early to late endosome (Press et al., 1998Press B. Feng Y. Hoflack G. Wandinger-Ness A. Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment.J Cell Biol. 1998; 140: 1075-1089Crossref PubMed Scopus (225) Google Scholar). Earlier we have proposed that the maturation and three-dimensional assembly of tyrosinase gene family proteins in the endoplasmic reticulum are regulated by calnexin (Jimbow et al., 1994Jimbow K. Hara H. Vinayagamoorthy T. et al.Molecular control of melanogenesis in malignant melanoma. Functional assessment of tyrosinase and lamp gene families by UV exposure and gene co-transfection, and cloning of a cDNA encoding calnexin, a possible melanogenesis 'chaperone'.J Dermatol. 1994; 21: 894-906Crossref PubMed Scopus (24) Google Scholar;Toyofuku et al., 1999Toyofuku K. Wada I. Hirosaki K. Park J.S. Hori Y. Jimbow K. Promotion of tyrosinase folding in COS 7 cells by calnexin.J Biochem. 1999; 125: 82-89Crossref PubMed Scopus (63) Google Scholar). Indeed, albino-associated mutants of tyrosinase were shown to have defects in appropriate folding and interaction with calnexin and calreticulin, which result in their retention in the endoplasmic reticulum (Halaban et al., 1997Halaban R. Cheng E. Zhang Y. et al.Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells.Proc Natl Acad Sci USA. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar;Halaban et al., 2000Halaban R. Svedine S. Cheng E. Smicun Y. Aron R. Hebert D.N. Endoplasmic reticulum retention is a common defect associated with tyrosinase-negative albinism.Proc Natl Acad Sci USA. 2000; 97: 5889-5894Crossref PubMed Scopus (155) Google Scholar). The molecular mechanism of vesicular transport of tyrosinase and TRPs between the TGN and melanosomes has remained unclear, however. Recently, we have found that late-endosome-delineated compartments and Rab7 are involved in the transport of tyrosinase and TRP-1 between the TGN and melanosomes (Jimbow et al., 1997Jimbow K. Gomez P.F. Toyofuku K. Chang D. Miura S. Tsujiya H. Park J.S. Biological role of tyrosinase related protein and its biosynthesis and transport from TGN to stage I melanosome, late endosome, through gene transfection study.Pigment Cell Res. 1997; 10: 206-213Crossref PubMed Scopus (39) Google Scholar;Jimbow et al., 2000Jimbow K. Chen H. Gomez P.F. et al.Intracellular vesicular trafficking of tyrosinase gene family protein in eu- and pheo-melanosome biogenesis.Pigment Cell Res. 2000; 13: 110-117Crossref PubMed Scopus (41) Google Scholar;Gomez et al., 2001Gomez P.F. Luo D. Hirosaki K. et al.Identification of rab7 as a melanosome-associated protein involved in the intracellular transport of tyrosinase-related protein-1.J Invest Dermatol. 2001; 117: 81-90Crossref PubMed Google Scholar). In order to further investigate whether Rab7 GTPase is involved in the vesicular transport of tyrosinase family proteins, we expressed tyrosinase and TRP-1 by adenovirus vector and examined the effect of a dominant-negative mutant of Rab7 on the transport of melanogenic proteins. We show here that vesicular transport of tyrosinase and TRP-1 requires a transient passage through Rab7-associated endosomal compartments to be transported to melanosomes. Untransformed human fibroblasts, MRC5, SV40-transformed monkey kidney cell line, COS7, and adenovirus type 5 DNA-transformed human kidney cell line, 293, were purchased from American Type Culture Collection (Rockville, MD). Amelanotic SK-mel-24 and melanotic SK-mel-23 human melanoma cell lines were provided by Dr. Houghton of Sloan Kettering Cancer Center, New York. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco BRL, Paisley, U.K.) supplemented with 5% fetal bovine serum (ICN Flow, Basingstoke, U.K.) and antibiotics. Ad-LacZ, β-galactosidase-expressing recombinant adenovirus, was kindly provided by Dr. M Imperiale of Michigan University. Construction of recombinant adenoviruses carrying cDNA 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., 1996Takahashi M. Ilan Y. Chowdhury N.R. Guida J. Horwitz M. Chowdhury J.R. Long-term correction of bilirubin-UDP-glucuronosyl transferase deficiency in Gunn rats by administration of a recombinant adenovirus during the neonatal period.J Biol Chem. 1996; 271: 26536-26542Crossref PubMed Scopus (108) Google Scholar;Yamano et al., 1999Yamano S. Tokino T. Yasuda M. et al.Induction of transformation and p53-dependent apoptosis by adenovirus type 5, E4orf6/7 cDNA.J Virol. 1999; 73: 10095-10103Crossref PubMed Google Scholar). Briefly, human tyrosinase and TRP-1 cDNAs were prepared from pcDNA-HT (Singh and Jimbow, 1998Singh M.V. Jimbow K. Tyrosinase transfection produces melanin synthesis and growth retardation in glioma cells.Melanoma Res. 1998; 8: 493-498Crossref PubMed Scopus (12) Google Scholar) and pRHOHTαπ (Takimoto et al., 1995Takimoto H. Suzuki S. Masui S. Shibata K. Tomita Y. Shibahara S. Nakano H. MAT-1, a monoclonal antibody that specifically recognizes human tyrosinase.J Invest Dermatol. 1995; 105: 764-768Crossref PubMed Scopus (15) Google Scholar) by cleaving them with EcoRI and SalI +XbaI, respectively. cDNAs of wtRab7 and its dominant-negative mutant Rab7N125I (Feng et al., 1995Feng Y. Press B. Wandinger-Ness A. Rab 7: an important regulator of late endocytic membrane traffic.J Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (535) Google Scholar;Vitelli et al., 1997Vitelli R. Santillo M. Lattero D. Chiariello M. Bifulco M. Bruni C.B. Bucci C. Role of the small GTPase Rab7 in the late endocytic pathway.J Biol Chem. 1997; 272: 4391-4397Crossref PubMed Scopus (251) Google Scholar;Press et al., 1998Press B. Feng Y. Hoflack G. Wandinger-Ness A. Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment.J Cell Biol. 1998; 140: 1075-1089Crossref PubMed Scopus (225) Google Scholar) were cleaved off from pGEM-Rab7 and pGEM-Rab7N125I by BamHI. After the cDNA fragments were blunted by T4 polymerase, they were inserted into the HindIII site, which had also been blunted by T4 polymerase, of pAd-BglII at the downstream of human cytomegalovirus early promoter (Takahashi et al., 1996Takahashi M. Ilan Y. Chowdhury N.R. Guida J. Horwitz M. Chowdhury J.R. Long-term correction of bilirubin-UDP-glucuronosyl transferase deficiency in Gunn rats by administration of a recombinant adenovirus during the neonatal period.J Biol Chem. 1996; 271: 26536-26542Crossref PubMed Scopus (108) Google Scholar). Parts of the inserted cDNAs in pAd-BglII were verified by nucleotide sequencing by the dideoxy termination method using the Applied Biosystems Model 373S DNA Sequencing system. Then, pAd-BglII plasmids were separately cotransfected with pJM17 (McGrory et al., 1988McGrory W.J. Bautista D.S. Graham F.L. A simple technique for the rescue of early region 1 mutants into infectious adenovirus type 5.Virology. 1988; 163: 614-617Crossref PubMed Scopus (550) Google Scholar) into 293 cells. Resulting recombinant adenoviruses obtained from transfected 293 cell culture, which showed cytopathic effect, were cloned and propagated in 293 cells, and infected cell lysate was aliquoted and frozen at -80°C until use. Cells were infected with the virus at the multiplicity of infection (moi) of more than 20 plaque-forming units (pfu) per cell, as nearly maximum numbers of cells were found to express β-galactosidase after Ad-LacZ infection at the moi of 20 pfu per cell in various types of cells (Yamashita et al., 2001Yamashita T. Tokino T. Tonoki H. Moriuchi T. Jin H.-Y. Orori F. Jimbow K. Induction of apoptosis in melanoma cell lines by p53-family members.J Invest Dermatol. 2001; 117: 914-919Crossref PubMed Google Scholar). Expression of tyrosinase and TRP-1 was detected by Western blotting as described previously (Yamashita et al., 1999Yamashita T. Tonoki H. Nakata D. Yamano S. Segawa K. Moriuchi T. Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53.Micro Immunol. 1999; 43: 1037-1044Crossref PubMed Scopus (8) Google Scholar). Four × 105 cells were infected with Ad-HT or Ad-TRP-1 at the moi of 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 fluoride, they were sonicated for 10 s with Branson's sonicator. Protein concentration was determinated by using a BCA protein assay kit (Pierce, Rockford, IL). Samples containing 5.0 µg protein were separated by 5%-20% gradient SDS polyacrylamide gel electrophoresis (PAGE) (Bio-Rad, Tokyo, Japan) at 100 V for 80 min. The proteins in the polyacrylamide gel were transferred onto a nitrocellulose membrane (Protran, Schleicher & Schuell, Dassel, Germany) by electroblotting at 100 V for 80 min. The membrane was probed with the primary antibodies as follows: 1 µg per ml of mouse monoclonal antibody for tyrosinase (Novocastra, Newcastle upon Tyne, U.K.), 5 µl of anti-TRP-1 rabbit serum, or 1 µg per ml of antihuman α-actin monoclonal antibody (AL-40, Sigma, MI) in 5 ml of PBS-T (0.1% Tween-20 in phosphate-buffered saline) at room temperature for 1 h. The blot was then washed with PBS-T and incubated with horseradish peroxidase conjugated to a secondary antibody. The specific complexes were detected by the ECL chemiluminescence reagent (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Five × 105 cells were infected with recombinant adenovirus at the moi of 20 pfu per cell and cultured for 48 h. After cells were lyzed in 300 µl of 1% Triton X-100 in 0.1 M phosphate buffer (pH 6.8), samples were sonicated and centrifuged at 8000 rpm for 10 min. 100 µl of 0.15% dopa solution was added to the 100 µl of obtained supernatant and incubated at 37°C for 10 min. The tyrosinase activity was measured with a spectrophotometer at 475 nm. After 4 × 105 cells were seeded in 6 cm dishes and cultured for 24 h, they were infected with recombinant adenovirus at the moi of total 20 pfu per cell and cultured for 48 h. Cells were detached and collected in Eppendorf tubes. Then, cells were dissolved in 1 ml of alkaline solution (1 N NaOH in 10% dimethylsulfoxide) and incubated at 80°C for 2 h. After centrifugation at 1000 × g for 10 min, the supernatant was taken to measure the protein concentration and melanin. The amount of melanin was measured with a spectrophotometer at 420 nm by using synthetic melanin (Sigma M-8631, St. Louis, MI) as a standard. Anti-lgp85, a rabbit polyclonal antiserum used for a lysosomal marker, was a generous gift of Dr. H. Fujita of Kyusyu University, Fukuoka, Japan. Anti-EEA-1 (a marker for early endosome) and antisyntaxin 8 (a marker for late endosome) mouse monoclonal antibodies are products of Transduction Laboratories (Los Angeles, CA). Antihuman tyrosinase mouse monoclonal antibody was purchased from Novocastra. Antihuman TRP-1 antibody was raised in rabbits by injecting C-terminal amino acid residues, CNQPLLTD QYQSYAEE, of human TRP-1 into domestic rabbits. Secondary antibodies conjugated to Alexa 488 and Alexa 594 were purchased from Molecular Probes (Eugene, OR). For immunofluorescence experiments, the cells were grown on poly L-lysine (10 mg per ml) coated round coverslides in 12-well plates (Coster, NY). Cells cultured on the coverslides were fixed and permeabilized with methanol at -20°C for 4 min. Fixed cells were washed with blocking washing buffer [PBS containing 0.5% (wt/vol) bovine serum albumin] twice and with 0.025% Tween-20 diluted in the washing buffer once, and were incubated with various antibodies for 30 min at room temperature. Coverslips were washed once with the washing buffer for 5 min, rinsed with water, and mounted in Vectashield (Vector Laboratories, CA). Fluorescent images were obtained with immunofluorescent microscopes: (i) Olympus upright microscope with an argon laser (Mitsubishi Optiplex GXM 5133, Tokyo) and Fluoview software, and (ii) Nikon Fluoview confocal laser scanning microscope and Bio-RAD MRC 1024 software (Bio-Rad, Tokyo, Japan). First, SK-mel-24 cells were infected with Ad-HT or Ad-TRP-1 at the moi of 20 pfu per cell, cultured for 48 h, and collected to examine the expression of tyrosinase and TRP-1 by Western blotting (Figure 1a, b). A tyrosinase band became detectable by 12 h and reached a maximum level at 24 h after viral inoculation. Tyrosinase was detected as an approximately 70 kDa broad band, and TRP-1 was also about 70 kDa (Figure 1a, b). Infected SK-mel-24 cells contained a larger amount of tyrosinase than SK-mel-23 cells did (Figure 1a). The immunostaining against tyrosinase- or TRP-1-specific antibodies also became detectable under the confocal microscope in the perinuclear region 4 h after viral infection, and then the immunostains were dispersed to the entire cytoplasm showing fine granules at 24 h (data not shown). In order to confirm that tyrosinase and TRP-1 expressed by the recombinant adenovirus are biologically and biochemically functional, we measured dopa oxidase activity and intracellular melanin in Ad-HT- and/or Ad-TRP-1-infected SK-mel-24 cells. After cells were infected with Ad-LacZ, Ad-HT, Ad-TRP-1, or Ad-HT plus Ad-TRP-1 and cultured for 48 h, cellular lysates were collected and processed to measure tyrosinase activity and melanin content as described in Materials and Methods. Even with a single introduction of tyrosinase gene, dopa oxidase activity and melanin content were significantly increased (Figure 2a, b). When tyrosinase and TRP-1 were coexpressed, tyrosinase activity and melanin content were increased approximately 3.0-fold and 1.3-fold, respectively, compared to those in the case of tyrosinase expression alone (Figure 2a, b). When SK-mel-24 cells were infected with Ad-HT for 5 h and doubly stained with antityrosinase and anti-Lamp-1 (a marker for lysosome and melanosome) antibodies (Orlow, 1995Orlow S.J. Melanosomes are specialized members of the lysosomal lineage of organelles.J Invest Dermatol. 1995; 105: 3-7Crossref PubMed Scopus (236) Google Scholar), the majority of tyrosinase immunostains were found to be colocalized in the Lamp-1-positive vesicles (Figure 3a–c). This suggests that the newly synthesized tyrosinase accumulates in lysosomes as well as melanosomes when overexpressed in SK-mel-24 cells. In order to further test if Rab7 function is required for transport of tyrosinase family proteins from the TGN to melanosomes passing through the endosomal compartments (e.g., early endosomes, late endosomes, or both), effects of Rab7N125I expression on the localization of tyrosinase or TRP-1 were examined. The mutant Rab7N125I exists preferentially in the nucleotide-free form and impairs Rab7 function in vesicular transport of various proteins (Feng et al., 1995Feng Y. Press B. Wandinger-Ness A. Rab 7: an important regulator of late endocytic membrane traffic.J Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (535) Google Scholar;Vitelli et al., 1997Vitelli R. Santillo M. Lattero D. Chiariello M. Bifulco M. Bruni C.B. Bucci C. Role of the small GTPase Rab7 in the late endocytic pathway.J Biol Chem. 1997; 272: 4391-4397Crossref PubMed Scopus (251) Google Scholar). SK-mel-24 cells were infected first with Ad-LacZ, Ad-Rab7wt, or Ad-Rab7N125I at the moi of 50 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 infection were cultured for another 24 h, and then puromycin (40 µM) was added to inhibit the following protein synthesis. Coexpression of β-galactosidase (Figure 4a–c) or wtRab7 (data not shown) did not alter the localization of tyrosinase, which was observed in the lgp85 (another lysosomal marker)-positive vesicles. In contrast, in Rab7N125I-expressing SK-mel-24 cells, the majority of tyrosinase was localized in the cytoplasm located differently from lgp85, and it was accumulated in enlarged vesicular structures at the peripheral area of cells (Figure 4d–f). The rate of colocalization of lgp85 and tyrosinase was 49.4% in Ad-LacZ-infected cells, whereas it was 17.9% in Ad-Rab7N125I-infected cells at the distal region of the cytoplasm (Figure 5a). This result suggests that the over-expression of Rab7N125I impairs the transfer of tyrosinase to lysosomes, melanosomes, or both.Figure 5Rates of colocalization of melanogenic proteins and lysosomal or endosomal marker proteins.(A)Colocalization of tyrosinase and lgp85 in Ad-HT-infected SK-mel-24 cells. Determined from the experiment shown in Figure 4. (B)Colocalization of TRP-1 and EEA-1 in Ad-TRP-1-infected SK-mel-24 cells. Determined from the experiment of Figure 6. (C)Colocalization of TRP-1 and syntaxin 8 in Ad-TRP-1-infected SK-mel-24 cells. Percentages were determined from several fields of photographs of confocal microscopy.View Large Image Figure ViewerDownload (PPT) We had no polyclonal antityrosinase antibody, which was necessary to obtain doubly stained images with anti-EEA-1 (an early endosomal marker) or syntaxin 8 (a late endosomal marker) monoclonal antibodies. Recently, another melanosome-targeted protein TRP-1 was shown to reach almost exclusively to melanosomes (Raposo et al., 2001Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.J Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (357) Google Scholar). Thus, we investigated the localization of another melanosome-targeted protein TRP-1, which was detectable by a polyclonal rabbit serum against human TRP-1, as well as by monoclonal antibodies against EEA-1 and syntaxin 8 as site markers. SK-mel-24 cells were infected with Ad-LacZ or Ad-Rab7N125I and then Ad-TRP-1 followed by culture in the presence of puromycin as described above. As expected, in the mutant Rab7-expressing cells, the enlarged peripheral vacuoles in which TRP-1 was confined were intensely stained by an anti-EEA-1, early endosomal antibody (Figure 6d–f). Little or no colocalization of TRP-1 with EEA-1 was observed in the cells expressing β-galactosidase (Figure 6a–c). Rates of colocalization of these proteins were 26.4% in Ad-LacZ-infected cells, and 91.3% in Ad-Rab7N125I-infected cells (Figure 5b). In contrast, immunostaining with the antibody against syntaxin 8 of a late endosomal marker showed little or no colocalization with TRP-1 in either the Ad-LacZ- or Ad-Rab7N125I-infected cells (Figure 5c). These results suggest that intracellular transport of TRP-1 requires functional Rab7, probably passing through early to late endosomal compartments. Taken together, we conclude that the transport of tyrosinase family proteins to lysosomes (tyrosinase), and/or melanosomes (tyrosinase and TRP-1) transits Rab7-associated vesicular transport from endosome-delineated compartments in SK-mel-24 cells. There are two major classes of melanin pigment in humans and animals, 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 common vacuoles, hence being called stage I melanosomes (Jimbow et al., 2000Jimbow K. Chen H. Gomez P.F. et al.Intracellular vesicular trafficking of tyrosinase gene family protein in eu- and pheo-melanosome biogenesis.Pigment Cell Res. 2000; 13: 110-117Crossref PubMed Scopus (41) Google Scholar). Our recent studies have indicated that the previously defined stage I melanosomes, which are spherical and morphologically resemble multivesicular bodies, are related to late-endosomal compartments (Jimbow et al., 2000Jimbow K. Chen H. Gomez P.F. et al.Intracellular vesicular trafficking of tyrosinase gene family protein in eu- and pheo-melanosome biogenesis.Pigment Cell Res. 2000; 13: 110-117Crossref PubMed Scopus (41) Google Scholar). Recently, it was also reported that tyrosinase and dopachrome tautomerase are present in stage II melanosomes associated with their enzymatic activities, but they, as well as TRP-1, MART1, and gp100, are cleaved and inactivated in stage I melanosomes (Kushimoto et al., 2001Kushimoto T. Basrur V. Valencia J. et al.A model for melanosome biogenesis based on the purification and analysis of early melanosomes.Proc Natl Acad Sci USA. 2001; 98: 10698-10703Crossref PubMed Scopus (193) Google Scholar). It has been suggested that the trafficking of tyrosinase family proteins to melanosomes is mediated by a number of membrane-bound carrier intermediates (Jimbow et al., 2000Jimbow K. Chen H. Gomez P.F. et al.Intracellular vesicular trafficking of tyrosinase gene family p
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