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Originally published In Press as doi:10.1074/jbc.M303167200 on April 28, 2003

J. Biol. Chem., Vol. 278, Issue 29, 27035-27042, July 18, 2003
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The Inhibition of Early N-Glycan Processing Targets TRP-2 to Degradation in B16 Melanoma Cells*

Gabriela Negroiu {ddagger}, Raymond A. Dwek § and Stefana M. Petrescu {ddagger} 

From the {ddagger}Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 77700 Bucharest, Romania and the §Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

Received for publication, March 27, 2003 , and in revised form, April 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosinase-related protein-2 (TRP-2) is a DOPAchrome tautomerase catalyzing a distal step in the melanin synthesis pathway. Similar to the other two melanogenic enzymes belonging to the TRP gene family, tyrosinase and TRP-1, TRP-2 is expressed in melanocytes and melanoma cells. Despite the increasing evidence of its efficiency as a melanoma antigen, little is known about the maturation and intracellular trafficking of TRP-2. Here we show that TRP-2 is mainly distributed in the TGN of melanoma cells instead of being confined solely to melanosomes. This, together with the plasma membrane occasional localization observed by immunofluorescence, suggest the TRP-2 participation in a recycling pathway, which could include or not the melanosomes. Using pulse-chase experiments we show that the TRP-2 polypeptide folds in the endoplasmic reticulum (ER) in the presence of calnexin, until it reaches a dithiothreitol-resistant conformation enabling its ER exit to the Golgi. If N-glycosylation inhibitors prevent the association with calnexin, the TRP-2 nascent chain undergoes an accelerated degradation process. This process is delayed in the presence of proteasomal inhibitors, indicating that the misfolded chain is retro-translocated from the ER into the cytosol and degraded in proteasomes. This is a rare example in which calnexin although indispensable for the nascent chain folding is not required for its targeting to degradation. Therefore TRP-2 may prove to be a good model to document the calnexin-independent retro-translocation process of proteasomally degraded proteins. Clearly, TRP-2 has a distinct maturation pathway from tyrosinase and TRP-1 and possibly a second regulatory function within the cell.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRP-2 is a member of the tyrosinase-related protein family (TRP)1 controlling the major steps of the pigmentation process in mammals (1). The analysis of the TRPs sequences revealed a high degree of homology between the three proteins of this family, tyrosinase, TRP-1, and TRP-2 (2, 3). TRPs are type I membrane proteins having an N-glycosylated luminal domain and a short cytoplasmic tail at the COOH-terminal end. The cytoplasmic domain facing the cytosol contains the signals required for the intracellular trafficking of TRPs (4). The luminal domain includes 15 cysteines in conserved positions and two metal binding domains co-ordinating the two metal ions involved in the structure of the catalytic site. While tyrosinase incorporates Cu2+ in its active site (5), TRP-2 was shown to be a Zn2+-binding protein (6). TRP-2 shares the conserved potential N-glycosylation sites with TRP-1 and tyrosinase (7). Although only four out of the six N-glycosylation sequons are occupied in murine tyrosinase, the N-glycans were shown to play important roles in its maturation and folding process (8). In tyrosinase the interaction of the immature glucosylated N-glycans with the ER chaperone CNX was shown to promote the productive folding of both tyrosinase and TRP-1 (9). CNX binding as a lectin to the monoglucosylated high mannose glycans of the nascent chains is known to be a component of the ER quality control (10). CNX acts by driving the incompletely folded nascent chains into glucosylation/deglucosylation cycles (11, 12). Although it does not act directly on the chains, the chaperone is thought to facilitate their interaction with ER folding factors (13). It is therefore likely that CNX association may help the stabilization of the folded structure by disulfide formation or rearrangements. On the other hand CNX binding re-directs the polypeptides into the calnexin cycle where they are again presented to glucosidase II for deglucosylation (14). Once folded, the polypeptide chains lacking the glucosyl tag are able to leave CNX cycle and exit the ER. We and others (15, 16) have previously shown that in the absence of CNX interaction, abolished by treatment with iminosugars, both tyrosinase and TRP-1 are unable to acquire a native conformation, indicating an important role for CNX of the two TRPs.

The TRPs are synthesized in melanocytes, cells specialized in the production of melanin. Melanin synthesis is initiated by tyrosinase, which catalyzes the oxidation of tyrosine to DOPAchrome in a two-step reaction (3). TRP-2 or DOPAchrome tautomerase catalyzes the conversion of DOPAchrome to 5,6,dihydroxyindole-2-carboxylic acid, a precursor of brown melanins with low molecular weight and poor solubility (6, 17, 18). One should note, however, that melanin may form even in the absence of TRP-2, since DOPAchrome can be non-enzymatically converted to an intermediate, 5,6-dihydroxyindole, able to polymerize to black, insoluble melanins. Despite the common opinion that the qualities of melanins should be modified in the absence of TRP-2, there is still not clear evidence whether TRP-2 enzymatic activity is the only factor controlling this process. Therefore, it has been proposed that TRP-2 function may be to maintain cell viability by controlling the concentration of 5,6-dihydroxyindole, a very toxic intermediate in vivo, which in the absence of the enzyme would accumulate in melanocytes (19). Cell-biological evidence suggests that TRP-2 is involved in cellular events related to growth and morphology (20) and that its expression depends of cell-cell contacts (21). However, the postulated melanosomal localization of TRP-2 cannot explain this role which would require TRP-2 trafficking to other organelles including the plasma membrane.

Melanoma cells that represent transformed melanocytes contain also TRP-2 and the other TRPs (22). Investigation of tyrosinases expression in experimentally induced melanomas of transgenic mice revealed that although tyrosinase and TRP-1 expression level fluctuated in these cells, TRP-2 expression was relatively constant (23). Interestingly, TRP-2 is highly expressed in most melanoma cells. Many studies have reported TRP-2 as an important melanoma differentiation antigen and showed that certain antigenic epitopes in TRP-2 can trigger a specific cytotoxic T lymphocytes response (24–26). TRP-2-derived peptides are extensively studied for enhancing antitumor immunity (27) or in different immunotherapeutical strategies against melanoma (28, 29). TRP-2 has also an autoantigenic potential confirmed by significant anti-TRP-2 antibody titer in patients with vitiligo (30, 31). Despite the increasing interest in understanding the differences between normal and transformed melanocytes little is known about the synthesis, folding, and maturation of this melanogenic protein in melanoma cells.

In an attempt to have a better understanding of the actual role of TRP-2, we performed studies related to its intracellular localization, ER folding, and maturation of the polypeptide chain in B16 mouse melanoma cells. Here we show that the polypeptide matures in the ER until it reaches a stable conformation enabling it to avoid the accelerated degradation that occurs in the absence of CNX association. The mature polypeptide exits the ER, traffics through the Golgi, and is found localized mainly in the TGN, which indicates that TRP-2 may be involved in other regulatory functions independent of the melanogenesis pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents, Antibodies, and Enzymes—NB-DNJ was from Sigma. N-Ethylmaleimide, DTT, HEPES, CHAPS, L-methionine, chloroquine, anti-rabbit IgG (whole molecule), and peroxidase conjugate were from Sigma. Protein A-Sepharose was from Amersham Biosciences (Uppsala, Sweden) and protease inhibitor mixture (CompleteTM) was from Roche Diagnostics. [35S]Methionine/cysteine (Tran35S-label 1100 Ci/mmol) was from ICN Biomedicals (Asse-Relegern, Belgium). Lactacystin synthetic was purchased from Calbiochem. The rabbit anti-TRP-2 ({alpha}-Pep8), anti-TRP-1 ({alpha}-Pep1), and anti-tyrosinase ({alpha}-Pep7) antisera generated against synthetic peptides corresponding to the carboxyl termini of TRP-2, TRP-1, and tyrosinase, respectively, were a generous gift from Dr. V. J. Hearing (National Institutes of Health, Bethesda, MD). Rabbit anti-calnexin 3 antibody ({alpha}-CNX3) raised against the peptide 487–505 of canine calnexin conjugated with BSA was a kind gift from Dr. J. J. Bergeron (McGill University). Mouse anti-syntaxin 6 monoclonal antibody was purchased from Stressgen (North Yorkshire, UK). Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 594 goat anti-mouse IgG (H+L) were from Molecular Probes Europe BV (Leiden, The Netherlands). Anti-rabbit Ig biotinylated from donkey and streptavidin-Texas Red were from Amersham Biosciences. cDNA encoding for GFP-Rab27a was subcloned in pEGFP-C2 vector (Clontech), resulting a plasmid encoding a fusion protein called EGFP-Rab27a, which contains Rab27a attached to the carboxyl terminus of the EGFP. This was a gift from Dr. Peter van der Sluijs, Department of Cell Biology, Utrecht University, Utrecht, The Netherlands. Endoglycosidase H (Endo H) and peptide-N-glycosidase F (PNGase F) were from New England BioLabs (Beverly, MA). All other chemicals were from Sigma.

Cell Culture—B16 F1 mouse melanoma cells (European Collection of Animal Cell Cultures, Porton Down, UK) were cultured in RPMI 1640 medium (Paisley, Scotland, UK) containing 10% (v/v) fetal calf serum (Sigma), 50 units/ml penicillin, and 50 mg/ml streptomycin (Invitrogen). The cells were maintained at 37 °C in an atmosphere of air/CO2 (19:1).

Pulse-Chase Experiments—B16 mouse melanoma cells were harvested with EDTA, washed three times with PBS, and resuspended in methionine- and cysteine-free RPMI 1640 medium (Invitrogen). Cells (107 cells/ml) were preincubated for 1 h at 37 °C before the addition of [35S]methionine/[35S]cysteine, 200 µCi/ml. Following the 5-min pulse, RPMI medium containing 5 mM cold methionine was added. In NB-DNJ experiments cells were cultured for 2 h before pulse in medium containing 5 mM NB-DNJ, the concentration being maintained during the chase periods. 5 mM DTT, 20 µM lactacystin, and 50 µM chloroquine were added to the cells during the chase periods as described in the legend to Fig. 5. At the indicated times, the chase medium was removed and the cells were harvested by scrapping into ice-cold PBS. Samples analyzed for the S–S bond formation were incubated before lysis for 1 h at 4 °C with 20 mM N-ethylmaleimide to block the free SH groups and to prevent the nonspecific formation of S–S bonds. Cells were lysed in 0.5 ml of lysis buffer (50 mM HEPES, pH 7.5, containing 2% (w/v) CHAPS, 200 mM NaCl, and proteinase inhibitors) for 1 h on ice.



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  		FIG. 5.
TRP-2 processing in B16 cells in the presence of degradation inhibitors. A, to B16 cells treated with 5 mM NB-DNJ (+), 20 µM lactacystin or 50 µM cloroquine were added 1 h before pulse. Cells were pulsed for 5 min with 35S and chased in the presence of NB-DNJ (+) and lactacystin (L) or cloroquine (CQ). For each chase point a control sample without any inhibitor (–) was also processed. B, untreated cells were pulsed and chased as in A in the absence (–) or presence (+) of 20 µM lactacystin. In both A and B, cell lysates were immunoprecipitated with {alpha}-Pep8 antibody, and samples were run on SDS-PAGE in reducing conditions and visualized by autoradiography.

 

Immunoprecipitation and SDS-PAGE—35S-Labeled cell lysates were precleared with 20 µl of protein A-Sepharose for 1 h at 4 °C and incubated with {alpha}-Pep8 (1:50) or {alpha}-CNX3 (1:150) for 2 h at 4 °C. The immunocomplexes were separated by incubation with 20 µl of Protein A-Sepharose for 2 h at 4 °C. The slurry was washed with 50 mM HEPES, pH 7.5, containing 0.5% CHAPS and 200 mM NaCl. TRP-2 was further eluted in: non-denaturing conditions (N) (sample mixed with SDS-PAGE sample buffer without {beta}-mercaptoethanol), in non-reducing conditions (NR) (sample incubated with SDS-PAGE sample buffer without {beta}-mercaptoethanol for 5 min at 100 °C), and in reducing conditions (R) (sample incubated with SDS-PAGE sample buffer with {beta}-mercaptoethanol for 5 min at 100 °C). When TRP-2 bound to calnexin was analyzed, samples were eluted for 1 h at room temperature in lysis buffer containing 1% SDS. SDS concentration was decreased in eluates at 0.1%, and TRP-2 was immunoprecipitated with {alpha}-Pep8. Samples were run in 7.5% polyacrylamide SDS-PAGE and analyzed by autoradiography. Western Blot and Ultracentrifugation—B16 lysates (~50 µg of total protein) were separated by SDS-PAGE in reducing conditions, transferred to Immobilon membranes (Millipore, Bedford, MA), and reacted with {alpha}-Pep8, {alpha}-Pep1, and {alpha}-Pep7 antisera (1:1,000), followed by peroxidase-goat anti rabbit IgG (1:10,000) and visualized with ECL system (Amersham Biosciences). The melanosomal fraction was obtained by ultracentrifugation in Percoll gradient. B16 cells were harvested in a solution including 1 mM EDTA and 0.25 M sucrose, pelleted for 5 min at 2,000 x g, and homogenized with 25 strokes in a Potter homogenizator in5mM HEPES buffer, pH 7.0, containing 0.25 M sucrose and a mixture of proteinase inhibitors. The postnuclear supernatant, obtained by centrifugation at 700 x g for 30 min at 4 °C, was layered on the top of a discontinuous gradient of Percoll of densities 1.05, 1.04, 1.03, and 1.02 g/ml and separated by ultracentifugation for 90 min, at 20,000 rpm in a Beckman SW 41 Ti rotor at 4 °C. Fractions of 0.5 ml were collected, Percoll was 10x diluted with the homogenization buffer, and the pellet obtained at 13,000 rpm was lysed in 10 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 2 mM EDTA, and 0.5% Triton X-100. Distribution of tyrosinase, TRP-1, and TRP-2 was analyzed in various layers of the gradient by Western blot with {alpha}-Pep7, {alpha}-Pep1, and {alpha}-Pep8 antibodies.

Immunofluorescence—For immunofluorescence analysis, B16 cells or B16 cells in which EGFP-Rab27a was transiently expressed using the liposomal transfection kit LipofectAMINE PLUS Reagent (Invitrogen) were grown on coverslips for 24 h. Cells were rinsed with PBS and fixed with 4% p-formaldehyde for 20 min at room temperature and permeabilized with methanol for 20 min at 4 °C. After blocking with 0.5% BSA in PBS for 30 min at room temperature, cells were incubated with {alpha}-Pep8 (1:300), {alpha}-Pep1 (1:300), anti-syntaxin 6 (1:100) diluted in 0.5% BSA in PBS, for 30 min at room temperature, washed three times with PBS, and further incubated with the appropriate Alexa 488 or Alexa 594 conjugated secondary antibodies (1:400) in 0.5% BSA in PBS, for 30 min at room temperature. B16 cells transfected with EGFP-Rab27a were incubated with {alpha}-Pep8 or {alpha}-Pep1 (1:300) and then consecutively with anti-rabbit Ig biotinylated (1:50) for 30 min at room temperature, washed three times with PBS, and next with streptavidin-Texas Red (1:50) to detect endogenous TRP-2 or TRP-1. Finally cells washed three times with PBS were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and observed with a Nikon Eclipse E 600 fluorescent microscope. Images were processed using Adobe Photoshop 5.0 software.

Endo H and PNGase F Digestion—35S-Labeled samples were digested with Endo H or PNGase F as described previously (32). Briefly, TRP-2 samples were eluted from the protein A-Sepharose in Endo H denaturing buffer, by incubation for 5 min at 100 °C. The samples resulted from pulse-chase experiments or samples about to be analyzed in Western blot were digested in the appropriate reaction buffer with 500 units of Endo H or PNGase F for 18 h at 37 °C, run in SDS-PAGE, and further analyzed by autoradiography or by Western blot.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subcellular Localization of TRP-2 in B16 Cells—To determine the localization of TRP-2, we analyzed its distribution by immunofluorescence microscopy. As shown in Fig. 1A, B16 cells showed an intense, polarized perinuclear staining, which became diffuse toward cell periphery. Only occasionally some staining at the plasma membrane could be observed. To identify the perinuclear substructures we performed co-localization experiments with syntaxin 6, a protein primarily localized in the TGN (Fig. 1B) (33). A significant amount of TRP-2 co-localized with syntaxin 6, indicating that TRP-2 was mainly enriched in the TGN area. To identify the punctate structures in the cell periphery also positive for TRP-2, we analyzed the co-localization of TRP-2 and TRP-1 with Rab27a. TRP-1 was previously suggested as a marker for late stage melanosomes (34). Rab27a, highly expressed in melanocytes and melanoma-derived cells (35), was involved in transport and retention of mature melanosomes to the cell periphery, being co-localized with late stage melanosome-resident proteins and with motor protein myosin V (36, 37). Therefore, we transiently expressed in B16 cells Rab27a as a fusion protein, EGFP-Rab27a, and cells were further stained for either TRP-2 with {alpha}PEP8 or for TRP-1 with {alpha}PEP1. Rab27a was distributed mainly on linear or in granular structures extending from the cell body into the periphery or close to the plasma membrane of B16 cells (Fig. 1C, panels b and e). Most of these cellular substructures appeared in yellow on merged images when they were labeled for TRP-1 (Fig. 1C, panel f). In contrast the TRP-2 punctate structures were mainly concentrated in the perinuclear area and very few on the limiting cell membrane (Fig. 1C, panel a), showing weak overlapping with the peripheral substructures positive for Rab27a (panel c).



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  		FIG. 1.
TRP-2 subcellular distribution in B16 melanoma cells. A, cells grown on coverslips were fixed, stained for TRP-2 with {alpha}-Pep8 antibody, and detected using Alexa 488 goat anti-rabbit secondary antibodies. B, cells grown on coverslips were fixed and stained for TRP-2 (green fluorescence) and syntaxin 6 (red flourescence). The yellow color observed on merged images indicates overlapping of TRP-2 with the TGN marker, syntaxin 6. C, cells were transiently transfected with EGFP-Rab27a (panels b and e), fixed after 24 h, and stained for either TRP-2 or TRP-1 with {alpha}-Pep8 and {alpha}-Pep1, respectively. TRPs were detected with anti-rabbit IgG biotinylated followed by streptavidin Texas Red (a and d). The merged images (c and f) show that unlike TRP-1 (f), TRP-2 poorly co-localizes with Rab27a in periferal punctate or filamentous structures (panel c).

 

The finding that most of TRP-2 was not present in substructures identical with late stage melanosomes prompted us to further investigate its presence in purified melanosomes. We used ultracentrifugation on Percoll gradient under conditions that separate high-density melanosomes from other cellular components (36, 38). B16 cell homogenate was layered on a discontinuous Percoll gradient and ultracentrifuged as described under "Materials and Methods." Melanin-laden melanosomes recovered as a black pellet in the bottom fraction of the gradient (1.05 g/ml) were further analyzed for the presence of tyrosinase, TRP-1, and TRP-2 by Western blot with specific antibodies using a total lysate of B16 cells as a control. The data presented in Fig. 2 show that all TRPs, including TRP-2, are found in the late stage melanosomes. Moreover the melanosomal TRP-2 is a glycosylated form migrating as TRP-2 polypeptide following PNGase F digestion.



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  		FIG. 2.
Distribution of TRP-1, tyrosinase, and TRP-2 in the density gradient purified melanosomes from B16 cells. Melaninladen melanosomes separated from cell homogenate by ultracentrifugation in the high density fraction (as described under "Materials and Methods") were lysed (M) and immunoprobed for TRP-1, tyrosinase, and TRP-2 by Western blot. A total lysate of B16 cells (TL) was used as a control. TRP-2 from melanosomal lysate was digested with PNGase F (+).

 

Significant amounts of all TRPs were detected in the upper fractions of the gradient which contained the ER and TGN markers CNX and syntaxin 6, respectively (data not shown). Taken together these data demonstrated that at steady state TRP-2 was mainly localized in the perinuclear/Golgi area and occasionally on plasma membrane and only a very small amount in late stage melanosomes.

Biosynthesis and N-Glycan Maturation of TRP-2 in B16 Cells—We next analyzed TRP-2 expression in B16 mouse melanoma cells by Western blotting. At steady state TRP-2 ran as two bands at 69 kDa and at ~80 kDa, respectively (Fig. 3A). Digestion of the lysate with PNGase F, which removed the attached N-glycans from the polypeptide chains, resulted in TRP-2 migrating as a single band at 55 kDa, which coincided with the predicted molecular mass of the polypeptide. This indicated that the two bands were glycoforms of the same TRP-2 polypeptide (Fig. 3A, lane 3). Endo H digestion revealed that the resistant upper band possessed complex structures (Fig. 3A, lane 2), whereas the digested lower band had only high mannose glycans. These experiments may indicate that although TRP-2 was exported from the ER in the Golgi compartment, a significant amount was retained at the ER/cis Golgi level.



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  		FIG. 3.
Glycosylation and time course of post-translational processing of TRP-2 in B16 cells. A, B16 cell lysate was treated with the glycolytic enzymes, Endo H (+) and PNGase F (+), and TRP-2 was immunodected by Western blot with {alpha}-Pep8 and compared with a non-digested control (–). B, B16 cells were labeled for 5 min with [35S]methionine, chased with cold methionine for the time period indicated, and immunoprecipitated with {alpha}-Pep8 antiserum. Half of each immunoprecipitated sample was digested with Endo H. Digested (+) and non-digested (–) samples were run in 7.5% SDS-PAGE, and protein bands were visualized by autoradiography.

 

To further investigate the maturation process efficiency of the TRP-2 polypeptide chain, we have pulse-chased the 35S-labeled cells and immunoprecipitated the lysates with anti-TRP-2 antiserum. Half of each sample was digested with Endo H and run next to a non-digested control, in reducing SDS-PAGE. As shown in Fig. 3B, newly synthesized TRP-2 ran at 69 kDa for 30-min chase showing complete Endo H sensitivity. Within the next 30 min, an Endo H-resistant band running at ~80 kDa was detected, indicating N-glycan processing to complex structures. At this time point, corresponding to 1 h of chase, almost half of the total pool of labeled TRP-2 polypeptides was left in an Endo H-sensitive form. Within the next 2 h of chase N-glycans maturation continued beyond the ER as shown by the gradual increase in Endo H resistant forms. No further change in the electrophoretic pattern or sensitivity to Endo H was observed between 3 and 8 h of chase, suggesting that no further N-glycan maturation continued after 3 h of chase. However some traces of TRP-2 with high mannose oligosaccharides could be still detected even after a chase time of 8 h. The TRP-2 profiles detected by Western blot or at 1-h chase point were highly similar, indicating that the polypeptide had a relatively long ER retention time, and this might be related to the ER maturation of its N-glycans.

TRP-2 Synthesized in the Presence of ER-Glucosidase Inhibitors Is Targeted for Proteasomal Degradation—To assess the role of glycans in the TRP-2 maturation we investigated its biosynthesis in the presence of NB-DNJ, an inhibitor of early steps of N-glycan processing. NB-DNJ was added to the cells 2 h before pulse and maintained at the initial concentration during pulse and all chase time points. After s 5-min pulse with 35S, labeled cells were chased for the indicated time periods, and N-glycan maturation was monitored by digesting half of each sample with Endo H and run next to a non-digested control (Fig. 4). A significant decrease in the intensity of the TRP-2 band starting with 30-min chase was observed and this continued up to 1 h chase, indicating that a possible degradation of this protein might have occurred when cells were treated with this inhibitor. The protein did not reach the medial and trans Golgi compartments as the Endo H treatment showed that no complex structures of N-glycans were formed.



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  		FIG. 4.
TRP-2 biosynthesis in NB-DNJ-treated cells. B16 cells were incubated for 2 h in the presence of 5 mM NB-DNJ, pulsed for 5 min with 35S, and chased for the indicated time periods. Cell lysates were immunoprecipitated with {alpha}-Pep8 antibody, and half of each sample was digested with Endo H and run in SDS-PAGE next to the undigested control. Samples were visualized by autoradiography.

 

To rule out the possibility that decreasing in signal intensity was caused by a defective antigen-antibody interaction, we analyzed TRP-2 biosynthesis in a pulse-chase experiment in the presence of lactacystin and chloroquine. Lactacystin is an irreversible proteasomal inhibitor (39), whereas chloroquine impairs lysosomal degradative capacity (40). Cells were incubated as it follows: for 2 h with 5 mM NB-DNJ only, with NB-DNJ 2 h, and after 1 h, 20 µM lactacystin or 50 µM chloroquine were added. Samples were pulsed for 5 min with 35S and chased for 30 min, 1 h, and 3 h. NB-DNJ, lactacystin, or chloroquine concentrations were maintained all over the chase periods. In parallel control samples without inhibitors were run (Fig. 5A, lanes 1, 5, and 10). The NB-DNJ-treated samples showed an accumulation of TRP-2 in the presence of lactacystin (lanes 3, 7, and 12), whereas no difference was observed in the presence (lanes 4, 8, and 13) or absence (lanes 2, 6, and 11) of chloroquine. This result demonstrated that TRP-2 synthesized in the presence of ER-glucosidase inhibitor NB-DNJ was re-translocated in the cytosol and degraded by the proteasomal complex.

To observe whether any TRP-2 degradation occurred along its native biosynthetic pathway, we performed the above-described experiment in non-treated cells using the same proteasomal inhibitor. As shown in Fig. 5B no accumulation of TRP-2 in any of the lactacystin-treated samples was observed, indicating that no proteasomal degradation occurred when TRP-2 was processed in normal conditions (compare lanes 1, 3, and 5 with lanes 2, 4, and 6).

TRP-2 Folding Pathway Is Assisted by Calnexin in Normal, but Not in NB-DNJ-treated, Cells—It is known that the proteasomal complex is the site for degradation of proteins which fail to fold in a proper conformation. The observed TRP-2 proteasomal degradation in the presence of the iminosugar NB-DNJ suggested a severe perturbation of TRP-2 folding pathway in the presence of this inhibitor. Our previous data showed that both tyrosinase and TRP-1 folding pathways were controlled by the interaction with the ER-lectin chaperone, CNX (16, 41). To determine the role of CNX in TRP-2 folding, co-immunoprecipitation experiments of TRP-2 with calnexin have been performed. Cells were labeled for 5 min and chased for the indicated time points (Fig. 6). TRP-2 bound to calnexin was retrieved from cell lysates by co-immunoprecipitation with an anti-calnexin antiserum and subsequently with {alpha}-Pep8 as described elsewhere (16). In Fig. 6 it is shown that the amount of TRP-2 bound to calnexin, detected as a band of ~69 kDa, gradually decreased within1hof chase. This result was in good correlation with the kinetics of TRP-2 maturation presented in Fig. 3. TRP-2 associated to calnexin was still detected after ~1-h chase, confirming that TRP-2 was gradually folded in the ER compartment. Preventing N-glycan trimming in the presence of NB-DNJ resulted in the abolishment of TRP-2 calnexin binding (data not shown), suggesting that TRP-2 interacted with calnexin via the monoglucosylated precursor only. No interaction of TRP-2 with calreticulin, the soluble lectin counterpart of calnexin, was detected.



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  		FIG. 6.
TRP-2 interaction with calnexin in B16 cells. Cells were pulsed for 5 min with 35S and chased for the indicated time periods. Proteins interacting with calnexin were immunoprecipitated from cell lysates with anti-calnexin antibody. TRP-2 was subsequently recovered from its immunocomplexes with calnexin by immunoprecipitation with {alpha}-Pep8 antibody. Samples were run in reducing SDS-PAGE and visualized by autoradiography.

 

TRP-2 Acquires Post-translationally a Transport-competent Conformation—To further characterize the ER maturation pathway of TRP-2, the kinetics of stabile conformation production was investigated. B16 cells were pulsed for 5 min, and cell lysates were immunoprecipitated with {alpha}-Pep8 antiserum and analyzed in N conditions, NR conditions, and R conditions (see "Materials and Methods"). It is known that a protein in a reduced or open conformation migrates slower than its compact or oxidized form. As shown in Fig. 7A, TRP-2 bands in N and NR conditions co-migrated, indicating that thermal denaturation did not disrupt TRP-2 conformation. After treatment with the reducing agent TRP-2, polypeptide became fully extended and migrated with a lower electrophoretic mobility than both N or NR samples, and this stood as proof for the presence of disulfide bridges. The difference in the electrophoretic mobility of the NR versus R sample at 0-min chase indicated that disulfides were formed within 5-min pulse. When samples were analyzed in NR conditions along the 10-, 20-, and 30-min chase points no further increase in the electrophoretic mobility to attest the presence of different folding intermediates was observed (data not shown).



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  		FIG. 7.
Analysis of TRP-2 conformation in B16 cells. A, lysates of B16 cells labeled for 5 min with 35S were immunoprecipitated for TRP-2 with {alpha}-Pep8 and analyzed in N, denaturing, and NR and R conditions (as described under "Materials and Methods"). B, cells were labeled for 5 min with 35S and chased for the indicated time periods without DTT (–) and then for additional 10 min in the presence of 5 mM DTT (+). Cell lysates were immunoprecipitated with {alpha}-Pep8. Samples were analyzed in NR or R. C, cells labeled for 5 min with 35S were chased for the indicated time periods without DTT (–) and for additional 40 min with DTT (+). After immunoprecipitation with {alpha}-Pep8 antibody, samples were run in reducing SDS-PAGE. All samples were vizualized by autoradiography.

 

However, still we could not rule out the possibility that TRP-2 native conformation was the result of a sequential process of oxidative folding which retained the chain for 30–60 min in the ER. To verify this working hypothesis, we have performed an additional experiment in which conformational stability was tested using DTT. When added in vivo, DTT can quickly penetrate across the cell membrane and prevents the formation of disulfides in nascent polypeptide or it can reduce S–S bonds, which are exposed in transient, unstable conformation (42). Therefore, immature polypeptides with exposed S–S bonds were readily reduced reduced by DTT added in vivo and migrated as the fully reduced polypeptide in SDS-PAGE, in NR conditions. Mature polypeptides with disulfides resistant to DTT treatment due to a more compact conformation would migrate faster than the fully reduced polypeptide.

Cells were labeled for 5 min and chased for 0, 5, 10, and 20 min. At the end of each chase period, 5 mM DTT was added to each sample, and the chase was continued for additional 10 min, in the presence of the reducing agent. Cell lysates were immunoprecipitated with {alpha}-Pep8, and the immunocomplexes were eluted from protein A-Sepharose in either NR or R conditions and analyzed by SDS-PAGE. As shown in Fig. 7B, all samples up to 20-min chase co-migrated (lanes 1–6), whereas the first shift in the electrophoretic mobility of NR versus R samples appeared at 20-min chase (lanes 7 and 8). These results showed that for the first 20 min following synthesis, the TRP-2 chain was highly unstable, being able to adopt a more compact conformation only after this period of time.

To assess whether the disulfides post-translationally acquired during 20-min chase conferred the TRP-2 polypeptide a transport-competent conformation, we extended the chase periods in the presence of DTT as follows. After 5-min labeling, the cells were chased for 20, 30, 40, and 60 min in the absence of DTT and then for an additional 40 min in the presence of 5 mM DTT, reaching a total chase time of 60, 70, 80, and 100 min, respectively. Samples were analyzed in R conditions to monitor the presence of complex glycans. As shown in Fig. 7C, after 20–30 min in the absence of DTT, most of TRP-2 migrated at 69 kDa (lanes 1 and 2), similar to the immature TRP-2 caring high mannose glycan chains. In contrast, after 60-min chase, in the total absence of DTT, significantly more TRP-2 became Endo H-resistant and was detected as an ~80-kDa band, representing the fully glycosylated protein (lane 4). Disulfides formed after 10 min of polypeptide synthesis were sensitive to DTT treatment in vivo. Within additional 10 min, disulfides were further rearranged and buried in a more protected conformation, resistant to DTT. Approximately 60 min were necessary for the amount of TRP-2 synthesized within 5 min of pulse to acquire the transport-competent conformation and to pass the Golgi compartments where the maturation of its N-linked glycans was completed.

TRP-2 Folding in NB-DNJ-treated Cells—Next we followed the folding pathways of TRP-2 in the presence of NB-DNJ when the interaction with calnexin was prevented. Cells were incubated for 2 h with 5 mM NB-DNJ, pulsed for 5 min, and chased for 0, 5, 10, and 20 min in the presence of the same concentration of the inhibitor. Cell lysates were immunoprecipitated with {alpha}-Pep8, and immunocomplexes were eluted in NR or R conditions and analyzed in SDS-PAGE. The data presented in Fig. 8A show that for all tested chase points, reduced TRP-2 (lanes 2, 4, 6, and 8) migrated in a higher position than TRP-2 in NR conditions (lanes 1, 3, 5, and 7), demonstrating the presence of disulfides. Further we tested DTT resistance of S–S formed in TRP-2 in NB-DNJ-treated B16 cells in an experiment similar with the one presented in Fig. 7B. After a 5-min pulse with 35S, cells were chased in the absence of DTT for 0, 5, 10, and 20 min. 5 mM DTT was added, and chase was continued in the presence of the reducing agent for 10 min. After immunoprecipitation of cell lysates with {alpha}-Pep8 and elution of the immunocomplexes from protein A-Sepharose, half of each sample was treated in NR conditions (lanes 1, 3, 5, and 7) and to the other half the reducing agent, DTT, was added in vitro to obtain the fully reduced conformation of the polypeptide (lanes 2, 4, 6, and 8). Samples were run next to each other to compare their electrophoretic mobilities. As shown in Fig. 8B in cells treated with NB-DNJ, no shift could be observed within 20 min between samples eluted in NR or R conditions. This indicated that S–S formed in NB-DNJ-treated TRP-2 within 20 min were exposed and easily reducible by DTT added at different chase points. In both experiments described in Fig. 8, a significant decrease in the intensity of the TRP-2 band starting with 20-min chase was observed, confirming its degradation in the presence of NB-DNJ.



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  		FIG. 8.
TRP-2 folding in NB-DNJ-treated cells. A, B16 cells incubated with 5 mM NB-DNJ and pulsed for 5 min with 35S were chased in the presence of the inhibitor for the indicated time periods. Samples were immunoprecipitated with {alpha}-Pep8 antibody and analyzed in NR and R conditions (see "Materials and Methods"). B, same as A, except that after the indicated chase periods, 5 mM DTT was added, and chase was continued for additional 10 min, in the presence of DTT. The immunoprecipitated samples were analyzed as for A in NR or R conditions.

 

The above-presented data suggested that in NB-DNJ-treated cells, when interaction of TRP-2 polypeptide with calnexin was prevented, although disulfides were formed they could not be further rearranged and paired in the right position. The result of this unassisted folding process was a grossly misfolded polypeptide, rapidly targeted for degradation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tyrosinase and its related proteins, TRP-1 and TRP-2, are thought to control the melanin synthesis pathways and the architecture of melanosomes. Melanosomes are highly specialized organelles within the melanocytes hosting the melanogenesis pathway. Despite the numerous studies pursued, it is still unclear if all TRPs are required for the melanogenesis process or some are rather involved in the melanosome stability. Several reports have documented a structural role for TRP-1 in stabilization of tyrosinase and of late stage fully melanized melanosomes (43, 44), despite its potential role as a melanogenesis enzyme. On the other hand, tyrosinase has been clearly demonstrated to be the key enzyme of melanogenesis, while TRP-2 was shown to catalyze later steps in the pathway. However, tyrosinase activity was only evidenced in early, and not in late, stage melanosomes where TRP-1 is mainly localized (45). This raises the intriguing question as to whether the three TRPs can participate in melanogenesis even if they are not present simultaneously in the same organelle.

In an attempt to clarify the role of TRP-2 in melanoma, we investigated its subcellular distribution in B16 cells. In immunofluorescence experiments TRP-2 appears concentrated in the perinuclear area, whereas TRP-1 shows a different distribution. TRP-1 overlaps with Rab27a mainly on filamentous structures running toward the cell periphery or occasionally in punctate structures in the cellular dendritic tips. These structures should be melanosomes, since Rab27a is recruited to mature melanosomes for the final stage of their movement from the microtubule across the actin-rich region (46). In contrast, TRP-2 shows a weak co-localization with Rab 27a in peripheral structures, suggesting a scarce presence of TRP-2 in late melanosomes. This is in agreement with data reported by Kushimoto et al. (47) revealing DOPAchrome tautomerase and tyrosinase enzymatic activities in stage II rather than stage IV melanosomes. Surprisingly, the most intense fluorescence for TRP-2 encompasses the Golgi area co-localizing with the TGN-marker syntaxin 6. Previous studies have shown that tyrosinase and TRP-1 are present in punctate structures identified as melanosomes, with TRP-1 being found mainly in melanosomes of late stages (34, 48). The presence of TRP-2 in melanosomes is relatively reduced, since the highly concentrated melanosomal fraction obtained by the ultracentrifugation of B16 cells was required to detect it.

Altogether the data presented here point toward a main TGN localization of TRP-2 at steady state.

That TRP-2 is mainly concentrated in the Golgi area can be due to specific sorting machinery different from for tyrosinase or TRP-1. Thus, the di-leucine sorting signal absent from TRP-2 cytosolic tail, and present in both tyrosinase and TRP-1, is recognized by the AP-3 complex only in tyrosinase, whereas TRP-1 can be trafficked to melanosomes by interacting with the AP-1 complex or with a PDZ-domain protein, which binds to TRP-cytosolic motifs different from the di-leucine sequence (45, 49). Interestingly, the pattern of TRP-2 subcellular distribution is similar to another melanosomal protein, Melan-A/MART-1 (50), known to participate in the early stages of melanosome biogenesis and also known as a melanoma differentiation antigen. It has been noted that a tyrosine-based signal present in cytosolic tails of both TRP-2 and LAMP-1 is similar in sequence and position with a motif in Melan A (50, 51). We showed here that TRP-2 co-localized with TGN marker syntaxin 6, which has been reported to partially co-localize with TGN adapter protein AP-1 on clathrin-coated membranes. In the absence of a di-leucine cytoplasmic sequence, the tyrosine motif in interaction with the AP-1 complex could represent a sorting signal for TRP-2. However, further experiments are required to uncover the sorting machinery that regulates TRP-2 intracellular pathways from the TGN to the melanosomes. At this stage we can only hypothesize that TRP-2 is released from the TGN in minute amounts required for melanin production in early melanosomes probably via an endosomal compartment. This could also explain the presence of TRP-2 only in early melanosomes reported by other authors (48). On the other hand, the TGN localization may be related to a recycling mechanism of TRP-2 from melanosomes, which may imply or not other organelles. The predicted transmembrane domain of TRP-2 has a similar hydrophobicity with TRP-1, which has been shown to reach the plasma membrane before reaching the melanosomes (52). It may be that TRP-2 is kept in the TGN to protect it from degradation in lysosomes, and the pathway to melanosomes goes through the plasma membrane. This would explain the previously postulated role of TRP-2 in cell-cell contacts, which may be particularly important in melanoma where the intercellular contacts are highly favored.

The atypical subcellular localization of TRP-2 in the Golgi prompted us to investigate the maturation pathway of the polypeptide chain in relation to the other two tyrosinases, which have been previously characterized. All the members of tyrosinase family are synthesized in the ER and transported through the secretory pathway toward the melanosome, where melanin synthesis occurs. Tyrosinase is known to require a long processing time for maturation in mouse melanoma cells. We have previously shown that tyrosinase cannot exit the ER for 3 h from synthesis, and this has been correlated with the low folding efficiency of the polypeptide (41). Here we present evidence that the highly homologous protein TRP-2 is synthesized as a precursor acquiring its complex structures in 1-h post-synthesis in melanoma cells. On one hand this demonstrates that TRP-2 is a long resident ER chain as has been shown for tyrosinase and to a certain extent for TRP-1. On the other hand, the analysis of the pulse-chase gel shows that at this point only 40% of the labeled chains are able to exit the ER. The other chains retaining oligomannosidic glycans are unable to reach the Golgi transferases at this point. However, Endo H digestion experiments show that within 3 h of chase all the polypeptide chains are almost completely processed to the mature protein. Therefore, the TRP-2 polypeptide may have a reduced folding efficiency as suggested by the long ER retention time of its polypeptide chains.

To demonstrate that there is a relationship between folding and ER retention, we determined the interaction of TRP-2 with the ER resident chaperones calnexin and calreticulin. Calnexin interacts with TRP-2 for 1 h post-synthesis, as shown by co-immunoprecipitation with anti-calnexin antibodies. This correlates with the time required for the processing of N-glycans to complex structures, indicating that TRP-2 is kept in the calnexin cycle as long as it is necessary for the chain to adopt an ER exit-competent conformation. Even more evidence came from the DTT resistance experiments documenting that S–S formation is a progressive process occurring during TRP-2 transit through the ER. Although the presence of disulfides was detected immediately after 5-min pulse, only at 1-h post-synthesis TRP-2 chain was able to adopt a transport-competent conformation by the formation of its native disulfide bridges. We should note, however, that a more stable conformation of the chain was achieved starting with 20-min chase. This conformation was gradually adopted by an increasing number of chains during the next 40-min of chase. Altogether the data demonstrate that TRP-2 is retained in the ER in the calnexin cycle until the chain adopts its stable conformation stabilized by disulfide bridges. In the absence of calnexin interaction, abolished in the presence of NB-DNJ, TRP-2 acquires nonnative disulfide bridges, which are unable to reshuffle to the native pattern. This leads to the production of a grossly misfolded chain, which is rapidly targeted for degradation. As no interaction with calreticulin could be detected, we can conclude, therefore, that early calnexin association appears to be crucial for the correct folding process of the TRP-2 chain. Similarly, CD1 heavy chain disulfide bond formation was substantially impaired if the chaperone interactions were blocked by NB-DNJ or castanospermine (13).

The degradation process of NB-DNJ treated TRP-2 is greatly reduced in the presence of the proteasome inhibitor lactacystine, indicating that the misfolded chain is retro-translocated from the ER into the cytosol and degraded in proteasomes. Although tyrosinase and TRP-1 have been shown to require calnexin association for productive folding, any of these two related proteins were retained in the ER and degraded. Thus, we have previously shown that TRP-1 folds in a non-native conformation but exits the ER, and its glycans are further processed in the Golgi following the endo-mannosidase removal of the glucosylated arm (16). Similarly, tyrosinase is not grossly misfolded, since in the absence of calnexin it is transported out of the ER. Using N-glycosylation mutants we have shown that tyrosinase folds by domains, with some of the domains folding even in the absence of calnexin and with other domains being completely dependent on calnexin for correct folding (8). Since mutations in the calnexin-dependent domains yield catalytically inactive tyrosinases, we have concluded that the region of the active site folds in a calnexin-dependent manner. The accelerated degradation of TRP-2 in the absence of calnexin supports the notion that calnexin is facilitating the folding of regions required for the overall structure of the molecule. Therefore, while tyrosinase and TRP-1 have indicated a role for calnexin in their biological function, TRP-2 also demonstrates the crucial role of calnexin for the native conformation of a protein from the TRP family. It has been recently documented that the lectin EDEM, in conjunction with calnexin, is driving the chain for retrotranslocation out of the ER (53, 54). This mechanism can explain the behavior of the polypeptide chains that escape the ER associated degradation in the absence of calnexin. ER-glucosidase inhibitors were shown to reduce the degradation of EDEM associated {alpha}1-antitrypsin in cells overexpressing EDEM (53). This protein may be involved in the retro-translocation of tyrosinase and TRP-1, since the abolishment of calnexin interaction results in accelerated ER exit of the chains. However, this model appears not to be valid for TRP-2, since this protein is targeted to accelerated degradation in the absence of calnexin association. Further investigations will be required to demonstrate the EDEM differential binding of tyrosinase/TRP-1 versus TRP-2 and the molecular recognition elements of this interaction. Therefore TRP-2 may prove to be a good model to document the calnexin-independent retro-translocation process of proteasomally degraded proteins.


   FOOTNOTES
 
* This work was supported by the Wellcome Trust Collaborative Research Initiative Grant 064227. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 40-21-223-9069; Fax: 40-21-223-9068; E-mail: Stefana.Petrescu{at}biochim.ro.

1 The abbreviations used are: TRP, tyrosinase-related protein; DTT, dithiothreitol; ER, endoplasmic reticulum; TGN, trans Golgi network; CHAPS, (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), NB-DNJ, N-butyldeoxynojirimycin; Endo H, endoglycosidase H; PNGase F, peptide-N-glycosidase F; CNX, calnexin; N, non-denaturing; NR, non-reducing; R, reducing; BSA, bovine serum albumin; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Dr. V. J. Hearing (National Institutes of Health, Bethesda, MD) for {alpha}-Pep8, {alpha}-Pep1, and {alpha}-Pep7 antisera; Dr. J. J. Bergeron (McGill University, Montreal, Canada) for anti-calnexin antibody; and Dr. P. van der Sluijs (Department of Cell Biology, Utrecht University, Utrecht, The Netherlands) for kindly providing the EGFP-Rab27a construct.



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