From the
Department of Biochemistry and Molecular
Biology, Program in Molecular and Cellular Biology, University of
Massachusetts, Amherst, Massachusetts 01003 and
Department of Dermatology, Yale University
School of Medicine, New Haven, Connecticut 06520
Received for publication, April 2, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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Proteins that traverse the eukaryotic secretory pathway, such as tyrosinase, are generally co-translationally inserted through a translocon located within the ER membrane (9). Upon emergence of the polypeptide chain into the ER lumen, the protein is immersed into a specialized maturation environment. The oxidizing conditions of the ER support protein-assisted disulfide bond formation (10). Additional ER protein-mediated maturation processes include signal sequence cleavage, protein folding, glycan transfer and trimming, chaperone binding, and oligomerization (11). Understanding these maturation steps has implications for the etiology of albinism, the production of antigenic peptides associated with melanoma, and the wide array of other ER retention diseases.
Human tyrosinase (TYR) contains seven N-linked glycosylation sites. Although all seven sites are utilized, one site (Asn290) is inefficiently processed due to an adjacent Pro residue (Asn-Gly-Thr-Pro) (12). The presence of this inefficient glycosylation site, which is absent in mouse tyrosinase (Tyr), creates a heterogeneous population of TYR possessing six or seven glycans. Glycan trimming by glucosidases I and II generates monoglucosylated side chains, which are, in turn, substrates for calnexin (CNX) and calreticulin (CRT), ER lectin chaperones that specifically bind monoglucosylated glycans (13, 14). The interaction of CNX and CRT with TYR appears to be essential for its proper maturation and the acquisition of its activity within the cell. Inhibition of glucose trimming in human melanoma cells decreased TYR activity and stability (15). In addition, inactive tyrosinase was produced in B16 mouse melanoma cells treated with glucosidase inhibitors (16, 17). The removal of the final glucose by glucosidase II triggers the release of TYR from CNX and CRT. Reglucosylation by the UDP-glucose:glycoprotein glucosyltransferase, which reglucosylates proteins containing non-native regions (18), can initiate rebinding of the substrates to the lectin chaperones (19).
In this study, a semipermeabilized melanocyte system coupled with in vitro translations that supported the proper oxidation, chaperone binding, and oligomerization of TYR was developed. This system provides a high degree of experimental versatility, as well as the ability to isolate and identify the ER components required for TYR maturation. We found that TYR matured to a newly defined homodimer after oxidation and release from CNX. This oligomerization required melanocyte-specific factors.
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MATERIALS AND METHODS |
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Cell LinesWild type black and c-albino mutant (Tyr(C85S))
mouse melanocytes were maintained in culture as described previously
(4). Mouse melanocytes
expressing the null mutant pink eye dilution protein
pcp/p25H (melan-p)
(20) or mutant
tyrosinase-related protein 1 (Tyrp1(C86Y)) (melan-b)
(21,
22) were established in
culture by Dr. D. C. Bennett (Department of Anatomy, University of London,
London, United Kingdom) and obtained from Dr. V. Hearing (Laboratory of Cell
Biology, National Institutes of Health, Bethesda, MD). The melanoma cells 501
mel and YUGEN8 were grown in Ham's F-10 medium as described previously
(8). Chinese hamster ovary
(CHO) cells were grown in -MEM in 10% fetal bovine serum.
Construction of Plasmids Encoding Wild Type and Mutant TYR
The construction of the plasmid encoding WT TYR, termed
pSP72-KbSS/TYR, has been described previously
(12). To generate the mutant
TYR forms C89R and C500S, the corresponding Cys in pSP72-KbSS/TYR
were changed to Arg and Ser, respectively, using QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). The truncated tail-less TYR
(Tail) was created by the insertion of a stop codon in
pSP72-KbSS/TYR at position 499 of the protein. All mutations were
verified by DNA sequencing.
Transcription, Translation, and TranslocationMessenger RNA was prepared by in vitro run-off transcription after linearizing the TYR-encoding plasmid with NdeI restriction enzyme. Radioactive 35S-labeled TYR was translated for 1 h at 27 °C with RRL or WG in the presence of canine pancreas microsomes or semipermeabilized cells. Translation reactions were carried out as described previously (12). In some experiments, the samples were alkylated either with N-ethylmaleimide (20 mM) (23) or with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS; 14 mM) in 80 mM Tris, pH 6.8, 1% SDS to block free sulfhydryls. Lysates of alkylated samples were analyzed directly on SDS-PAGE or after immunoprecipitation with various antibodies before electrophoresis. In most experiments, one half of the sample was subjected to nonreducing SDS-PAGE, and the other half was reduced by the addition of dithiothreitol (100 mM) before electrophoresis.
Preparation of Semipermeabilized CellsSemipermeabilized
cells were prepared from confluent cultures as described previously
(24). In brief, confluent
cells (5 x 107 cells) were detached from the culture
dish (75 cm2) with 4 ml of a solution of 0.25% trypsin/EDTA and
collected in 8 ml of KHM buffer (110 mM KOAc, 2 mM
MgOAc, 20 mM HEPES, pH 7.2). Cells were harvested by centrifugation
at 1,200 rpm for 7 min at 4 °C. Cell pellets were resuspended in KHM
buffer (6 ml) and permeabilized with digitonin (20 µg/ml) on ice for 5 min.
Permeabilization was stopped by the addition of KHM buffer (8 ml) followed by
centrifugation. Cell pellets were resuspended in resuspension buffer (14 ml;
50 mM KOAc, 90 mM HEPES, pH 7.2), incubated on ice for
10 min, and centrifuged at 1,200 rpm for 7 min at 0 °C. Pellets were
resuspended in KHM buffer (
4 x 106 cells/100 µl KHM)
and treated with a calcium-dependent nuclease at 25 °C for 12 min to
remove the endogenous mRNAs. Nuclease treatment was stopped by EGTA (final
concentration, 4 mM). The cells were centrifuged at 8,000 rpm for 5
min at 4 °C, and cell pellets were resuspended in KHM buffer to a final
concentration of 1 x 105 cells/µl. Radioactively labeled
TYR was translated for 1 h at 27 °C in RRL with semipermeabilized cells
(1.3 x 104 cells/µl) substituting for the rough ER-derived
microsomes.
Sucrose Density Gradient CentrifugationTo determine the
size of the monomeric and oligomeric TYR generated with semipermeabilized cell
system, 35S-labeled samples were lysed in 2% CHAPS in 90
mM HEPES and 50 mM NaCl (HBS), pH 7.5. Cell debris were
removed by centrifugation at 14,000 rpm in a microfuge at 4 °C for 5 min,
and the supernatant (150 µl) was layered on top of a 525% linear
sucrose gradient (4 ml) in HBS supplemented with 0.5% CHAPS, pH 7.5. The
gradients were centrifuged at 197,600 x g in a Beckman
(Model L80-M) ultracentrifuge for 15 h at 4 °C; fractions were collected
manually, subjected to immunoprecipitation with anti-TYR antibodies, and
analyzed by SDS-PAGE.
Trypsin DigestionRadioactively labeled in vitro-translated TYR was treated with trypsin to assess its stability. After increasing maturation times, samples were removed, and free thiols were alkylated with N-ethylmaleimide. ER-translocated TYR was isolated by centrifugation at 14,000 rpm for 5 min at 4 °C. Microsome or semipermeabilized cell pellets were solubilized in 1% CHAPS/HBS (25 µl/10 µl translation mixture) and divided into two fractions. Trypsin was added to one set of samples (13 ng/µl), followed by incubation at 27 °C for 10 min. The digestions were stopped with soybean trypsin inhibitor and phenylmethylsulfonyl fluoride (4 mM).
Chemical Cross-linkingTo identify the TYR oligomer, WT Tyr or mutant (TyrC85S) albino mouse melanocytes were suspended in phosphate-buffered saline (13 x 106), and half of the samples were subjected to chemical cross-linking with bismaleimidohexane (BMH; 1 mM) at 25 °C for 30 min followed by blocking with 2-mercaptoethanol (10 mM). After centrifugation, cell pellets were lysed with 2% CHAPS/HBS supplemented with protease inhibitors, cell lysates were cleared from debris by centrifugation, and supernatants were subjected to Western blotting with anti-tyrosinase M-19 goat polyclonal antibodies (Santa Cruz Biotechnology).
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RESULTS |
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To monitor the oxidation of TYR in the ER, WT TYR was translated in RRL or
WG translation systems supplemented with rough ER-derived microsomes. After 1
h of translation, protein synthesis was arrested with cycloheximide, and the
protein was allowed to mature for increasing periods of time
(Fig. 1A). The
oxidative state of TYR was trapped with the alkylating agent
N-ethylmaleimide and analyzed directly (Lysate) or after
immunoprecipitation with anti-tyrosinase antibodies (-TYR) by
nonreducing and reducing SDS-PAGE.
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ER-translocated and N-linked glycosylated TYR (TYR) appeared as a
70-kDa doublet upon reduction (Fig.
1A, bottom panel). The doublet corresponds to
TYR possessing all seven (TYR7) or six
(TYR6) N-linked glycans
(12). As reported previously,
the fraction of TYR6 was increased in the faster-translating RRL
system compared with the slower WG translation, where each form was present in
equal amounts. In the nonreduced (NR) samples, TYR displayed a slight
increase in mobility indicative of intramolecular disulfide formation creating
a more compact structure (Fig.
1A, top panel). TYR aggregates were also
observed at the top of the nonreduced gel. These aggregates disappeared upon
reduction, demonstrating that they involved intermolecular disulfide bonds. An
artifact of the cell-free maturation system is the generation of
untranslocated protein (UTYR) that is not properly
targeted to the ER microsomes. Therefore, translated TYR also accumulates as
unglycosylated protein possessing an intact N-terminal signal sequence with an
electrophoretic mobility of 60 kDa
(Fig. 1A, bottom
panel). The untranslocated protein accounts for a portion of the
aggregates observed upon oxidation.
TYR has 15 lumenal Cys that can potentially form multiple intra- and/or intermolecular disulfide bonds. Because the mobility shift observed under nonreduced conditions was slight, the status of the Cys thiols upon oxidation was determined by modification of free thiols with a bulky membrane-impermeable alkylating agent, AMS (536 Da). Radioactively labeled TYR was translated in RRL or in WG in the presence of ER-derived microsomes under reducing or oxidizing conditions, followed by alkylation with N-ethylmaleimide or AMS before SDS-PAGE analysis under reducing conditions. A large decrease in mobility of translocated and untranslocated TYR was observed when the protein was translated under reducing conditions, in which Cys residues are accessible to AMS (Fig. 1B, lanes 14). However, no mobility shift in TYR was seen under oxidizing conditions (Fig. 1B, lanes 58), indicating that most of the Cys residues were protected from alkylation, probably due to their involvement in disulfide bonds. Similar results were observed regardless of the cell-free translation system used (RRL or WG), indicating that TYR formed disulfide bonds in both conditions.
Calnexin Binding to TYR Persists in the RER Microsomes
Chaperones bind transiently to substrates until they acquire correct tertiary
or quaternary structure. Similarly, TYR associates with two lectin chaperones,
CNX and CRT, before exit from the ER
(7). We therefore determined
whether TYR was maturing correctly in ER-derived microsomes by monitoring
complex formation with CNX. Immunoprecipitation of radioactively labeled TYR
with anti-TYR (-TYR) or anti-CNX (
-CNX)
antibodies showed that TYR interaction with CNX persisted even after 4 h of
processing, regardless of the cell-free system used
(Fig. 1C, lanes
46 and 1012). Persistent interactions were
similarly observed for CRT, the soluble lumenal paralogue of CNX (data not
shown). Therefore, unlike other membrane glycoproteins such as influenza
hemagglutinin and vesicular stomatitis virus G protein
(14,
25), the canine pancreatic
microsomes cell-free system does not support the proper maturation of the type
I membrane glycoprotein TYR.
TYR Matures Efficiently in Semipermeabilized MelanocytesSince TYR is a melanocyte-specific protein, it is possible that a factor specific to melanocytes is required for its proper maturation. To address this concern, we used a semipermeabilized cell system that can be coupled to the in vitro translation of a substrate (26). Treatment of mouse melanocytes with digitonin produced semipermeabilized cells with a leaky plasma membrane but an intact ER as confirmed by confocal immunofluorescence microscopy (data not shown). We have previously found that this semipermeabilized melanocyte system supports the efficient ER translocation and glycosylation of TYR (12).
In vitro-translated TYR translocated into the ER of wild type semipermeabilized melanocytes was analyzed directly or after immunoprecipitation with anti-TYR or anti-CNX antibodies. As previously observed in microsomes, TYR translocated into the ER from semipermeabilized cells migrated as a doublet (Fig. 2A, lane 1). TYR migrated faster in SDS-PAGE under nonreduced (NR) compared with reduced (RD) conditions after 2 and 4 h of maturation, indicative of disulfide bond formation (Fig. 2, compare A with B). Interestingly, a disulfide-linked oligomer band of 140 kDa appeared after 2 h of maturation, increasing in intensity with time (Fig. 2A, arrow). This oligomer disappeared after reduction, suggesting that it was created by intermolecular disulfide bonds (Fig. 2B).
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Evidence for proper maturation in semipermeabilized melanocytes was provided by the transient binding of CNX to TYR. The CNX·TYR complex observed during the first 2 h of processing was greatly diminished at the end of 4 h of maturation (Fig. 2A, lane 9). Furthermore, the 140-kDa TYR oligomer was not bound by CNX. The temporal relationship indicated that TYR oligomerization occurred after CNX release and is a properly folded form because CNX is released from monoglucosylated substrates once the substrate is trimmed of its final glucose and is no longer reglucosylated by UDP-glucose:glycoprotein glucosyltransferase (13, 14, 19). The time-dependent accumulation of trimmed or unglucosylated TYR side chains was supported by the persistence of an increase in TYR mobility observed after reduction with dithiothreitol (Fig. 2B, lanes 1015).
Resistance to limited proteolysis was used as an additional measure for TYR maturation because folded proteins acquire a more stable conformation that is often resistant to digestion by proteases. The vast majority of TYR produced in the RRL microsome system under oxidizing conditions that permit the formation of disulfide bonds remained protease-sensitive, even after 8 h of maturation (Fig. 2C, bottom panel, lanes 58). In contrast, a substantial level of trypsin-resistant TYR was already present after 4 h of maturation, a time at which CNX no longer bound to TYR. Therefore, semipermeabilized melanocytes appear to support the proper folding of TYR in the ER.
Folding and disulfide bond formation commence co-translationally in the ER (27). To determine whether TYR could form oligomers when proper oxidizing conditions were initiated post-translationally, 35S-TYR was translated for 1 h under reducing condition, and the oxidizing agent FAD was added after inhibition of further protein synthesis with cycloheximide (Fig. 2D). Oligomerization of TYR was observed after 4 h of post-translational oxidation, indicating that the physiological co-translational maturation process was not obligatory for oligomerization in semipermeabilized melanocytes (Fig. 2D, top panel, lane 4).
Time-dependent Oligomerization of TYRTo further
characterize the oligomeric state, 35S-TYR generated in the
presence of WT semipermeabilized mouse melanocytes was solubilized with the
nondenaturing detergent CHAPS and subjected to centrifugation through a
525% linear sucrose gradient. After 1 h of processing, TYR was
predominantly in the 5S form (Fig.
3, lanes 6 and 7, 1hr) corresponding to
monomeric TYR. An additional smaller peak in lanes 810 was
indicative of oligomeric TYR of
8S. The quaternary structure of the 8S
form of TYR was disrupted by an ionic detergent because it migrated as a
monomer of 70 kDa on SDS-PAGE. The 8S isoform likely represented TYR
heterocomplexes with CNX and/or CRT because the interaction is stable in CHAPS
but not in SDS-containing buffer
(28).
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After further maturation for 2 and 4 h, the monomeric peak moved from
lane 7 to lane 6, indicative of a decline in CNX and CRT
binding to TYR. In addition, a 7.5 S peak that was resolved as 150 kDa by
SDS-PAGE emerged (Fig. 3,
2hr and 4hr, arrow). The intensity of this peak increased
with time, as did the aggregated form of TYR, which accumulated at the top of
the gel. Both velocity centrifugation and SDS-PAGE analyses indicated that the
150-kDa oligomeric form of TYR entails the addition of 7080 kDa of
molecular mass.
TYR Oligomerization Does Not Require Glycan TrimmingTo determine the roles of glycosidase trimming and subsequent CNX and CRT binding in TYR oligomerization, the maturation of TYR in semipermeabilized melanocytes was monitored in the presence of n-butyl deoxynojirimycin and deoxymannojirimycin, inhibitors of ER glucosidases and mannosidases, respectively. Inhibition of mannose trimming had no effect on CNX binding or TYR oligomerization (Fig. 4, lanes 79 and 1618). As expected, inhibition of glucose trimming resulted in a slower-migrating protein due to the persistence of triglucosylated side chains (Fig. 4, compare lane 4 with lane 1, NR and RD) that were not bound by CNX (Fig. 4, lanes 1315). However, TYR did oligomerize in the presence of the glucosidase inhibitor n-butyl deoxynojirimycin, although with less efficiency than in the absence of inhibitors (35% versus 50%, respectively). Therefore, CNX binding enhanced the efficiency of oligomerization but was not obligatory for this process.
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Inactive Mutant TYR Does Not Form OligomersMutations in
tyrosinase are the cause of oculocutaneous albinism type 1, a recessive
genetic disorder manifested by the absence of melanin in melanocytes
(3). We therefore analyzed
several mutant forms of TYR (described in
Fig. 5A) to determine
whether proper folding is required for oligomerization and to identify the
amino acid residue responsible for intermolecular bonding. The inactive mouse
mutant Tyr(C85S) in the albino Balb c mouse is retained in the ER as an
immature 70-kDa glycoform bound to the ER chaperones CNX and CRT
(4). The mutant protein is
unstable, with a half-life of 3 h, and is rapidly degraded by the ER
protein-associated degradation
pathway.2 The
homologous mutation in humans, TYR(C89R), is also associated with albinism
(Fig. 5A, Cys
mutants). TYR(C89R) was therefore used to determine whether inactive
mutant TYR could also form oligomers. Time course maturation analysis failed
to detect oligomeric forms of TYR(C89R) even at the end of the 4-h reaction
time (Fig. 5B,
lanes 46). However, a larger amount of TYR(C89R) aggregated at
the top of the nonreducing gel when compared with WT TYR. Furthermore, CNX
binding to the mutant TYR(C89R) was transient in a fashion similar to that of
WT protein (Fig. 5B,
lanes 1618). In contrast to what had been observed in intact
albino mouse
melanocytes,2
TYR(C89R) was eventually released from CNX, but it was not degraded because
the proteasome is inhibited in the semipermeabilized melanocyte system by the
hemin from the reticulocyte lysate.
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Residue Cys500 in the Cytosolic Tail Is Required for TYR
OligomerizationAn intermolecular disulfide bond appears to be
involved in the formation of oligomeric TYR because it was disrupted under
reducing conditions. To identify the Cys residues engaged in this process, we
first determined whether the disulfide bond resided in TYR ectodomain or
cytosolic tail by analyzing oligomerization of a tailless TYR possessing an
intact transmembrane domain (Fig.
5A, Tail). Time course maturation
analysis implicated the TYR cytosolic tail in the oligomerization process
because TYR
Tail was unable to form oligomers
(Fig. 5B, lanes
1012), although it achieved proper folding as indicated by the
timely release from CNX (lanes 2224).
The cytosolic tail of TYR has a single Cys residue at position 500 immediately at the interface between the predicted membrane and transmembrane regions (Fig. 5A, Cys mutants). To determine whether this Cys residue was required for oligomerization, we monitored the maturation of TYR(C500S), a mutant in which Cys500 was changed to a Ser. Similar to the TYR tail truncation, TYR(C500S) was released from CNX but did not oligomerize (Fig. 5B, lanes 1921 and 79), indicating that this Cys was involved in TYR oligomer formation.
WT TYR Oligomerization Does Not Occur in Tyr(C85S) Albino Melanocytes or CHO CellsMutations in tyrosinase can cause the retention of an inactive protein in the ER that is eventually degraded by the cytosolic proteasome (4).2 In addition, human amelanotic melanoma cells have been shown to possess a hostile acidic environment that does not support proper maturation of wild type TYR (7, 8, 29). To establish the requirements for TYR oligomer formation, the maturation of WT TYR was monitored in semipermeabilized mouse Tyr(C85S) albino melanocytes (4) and nonmelanocytic CHO cells. As before, oligomeric WT TYR appeared after 2 h of maturation in semipermeabilized wild type mouse melanocytes (Fig. 6, lanes 2 and 3, top panel), whereas the 150-kDa TYR oligomer was not observed when maturation proceeded in mouse Tyr(C85S) albino melanocytes or CHO semipermeabilized cells (Fig. 6, lanes 49).
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Interaction of WT TYR with CNX during maturation in the different cell types was also examined. Co-immunoprecipitation experiments showed that CNX binding/release profiles in albino Tyr(C85S) melanocytes were similar to those in wild type melanocytes, even though TYR oligomerization was not observed (Fig. 6, lanes 1315). In contrast, the interaction of CNX with WT TYR persisted when processed in the CHO semipermeabilized cells, indicating a failure to fold properly in this heterologous cell type. TYR also failed to oligomerize in semipermeabilized human amelanotic melanoma cells (501 mel and YUGEN8), although its binding to CNX was transient (7) (data not shown). Altogether, these results indicated that correct folding and maturation of TYR require factors associated with the ER that are not present or are nonfunctional in semipermeabilized cells lacking tyrosinase activity, such as the albino Tyr(C85S) melanocytes, amelanotic melanoma cells, or CHO cells.
Mouse Tyrp1 Is Required for Tyrosinase Oligomerization Genetic analysis of inherited hypopigmentation disorders has demonstrated that mutations in other genes can modify the stability of tyrosinase and its subcellular localization and function (3034). To determine whether defects in other melanocyte-specific proteins can also disrupt TYR oligomerization, TYR maturation was monitored in melanocytes expressing null p-protein mutant pcp/p25H (melan-p1) or Tyrp1(C86Y) (melan-b) (Fig. 7).
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WT TYR oligomerization and CNX binding were normal when in vitro maturation was performed in semipermeabilized melan-p1 melanocytes, indicating that the p-protein was dispensable for these two processes (Fig. 7A, lanes 46 and 1315). In contrast, WT TYR did not oligomerize when processed in semipermeabilized melan-b melanocytes (Fig. 7B, lanes 46); however, it was released from CNX (Fig. 7B, lanes 1012).
Oligomerization Can Occur in Intact MelanocytesEukaryotic cells maintain redox conditions where the ER lumen provides an oxidizing environment, and the cytosol provides a reducing environment. Therefore, disulfide bond formation, for the most part, occurs in the ER, with the help of dedicated folding catalysts (10). Whereas disulfide bonds can be found in the cytosol, they are uncommon. The partitioning between oxidizing and reducing environments is not fully recapitulated in the in vitro translation and semipermeabilized cell systems. Here, the redox conditions are controlled by the reticulocyte lysate, in which FAD facilitates an oxidizing environment not only in the ER but also on the cis side of the ER membrane, where the Cys500 residue of TYR is localized. Therefore, to ensure that the oligomers observed with semipermeabilized melanocytes also occur in live cells under physiological conditions, we used the membrane-permeable chemical cross-linker BMH, a reagent that reacts with free Cys residues, to trap potential oligomers.
Wild type and Tyr(C85S) albino melanocytes were treated with BMH, and the
cross-linker was then quenched with 2-mercaptoethanol before cell lysis. A
tyrosinase oligomer of 150 kDa was formed after cross-linking in wild
type but not albino Tyr(C85S) melanocytes
(Fig. 8, lanes 2 and
4). The endogenous mouse tyrosinase band corresponded in size to the
human TYR oligomer translated in the semipermeabilized cell system using wild
type melanocytes. The cross-linking efficiency of endogenous tyrosinase in
wild type mouse melanocyte was low because a large fraction remained monomeric
after BMH treatment (Fig. 8,
compare lane 1 with lane 2). This result suggested that
proper folding of tyrosinase involves the formation of a number of
intramolecular disulfide bonds and that only a small number of free thiols are
available for intermolecular cross-linking. In contrast, the cross-linking
efficiency for Tyr(C85S) from albino melanocytes approached 100%
(Fig. 8, compare lane
3 with lane 4), suggestive of multiple Cys residues accessible
for intermolecular bonding by BMH on the malfolded protein. Noticeably, the
Tyr(C85S) intermolecular bonding produced complexes of ≥175 kDa, not the
150-kDa conformation observed in wild type melanocytes, suggesting the
involvement of different protein(s) in the complex. Altogether, these results
confirmed that tyrosinase oligomerizes to a
150-kDa conformation in cells
harboring wild type but not mutant inactive tyrosinase.
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DISCUSSION |
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We provide for the first time the evidence that TYR oligomerizes as it becomes properly folded in the ER. The maturation steps resolved by the in vitro semipermeabilized melanocyte system included binding of nascent TYR to the lectin chaperones CNX and CRT in a monoglucosylated-dependent manner. This binding lasted until intramolecular disulfides were in place and a protease-resistant conformation was reached. Proper folding was followed by oligomerization before TYR exit from the ER. The inability of albino inactive mutant TYR(C89R) to oligomerize supports the conclusion that the normal productive pathway of TYR maturation involves oligomerization in the ER. This oligomer is likely formed by an intermolecular disulfide bond because it was disrupted under reduced conditions. Furthermore, we identified Cys500 in the cytoplasmic tail of TYR as the key residue for dimerization because the protein failed to oligomerize in its absence, as shown for tailless TYR and for a point mutant in which Cys500 was substituted by Ser (TYR(C500S)).
In eukaryotic cells, the lumen of the ER maintains an oxidizing environment, whereas the cytosol provides reducing conditions. Disulfide bond formation, in general, occurs in the ER lumen but has also been observed in the cytosol (10). Our initial detection of TYR oligomers in vitro was facilitated by the creation of a nonphysiological oxidizing environment outside the ER of semipermeabilized melanocytes with the oxidizing agent FAD. We postulate that under normal cellular redox conditions, TYR forms a noncovalent oligomer by the alignment of the transmembrane or cytoplasmic tail regions, bringing the juxtamembrane regions of the cytoplasmic tail of TYR (Cys500) together. Therefore, exposing Cys500 to an oxidizing environment supported the formation of an intermolecular disulfide bond that covalently trapped the oligomer. Cross-linking studies with intact melanocytes possessing wild type but not misfold mutant tyrosinase verified that this oligomer also occurred under physiological redox conditions in cells.
The mobility of the oligomer on nonreducing SDS-PAGE as a 150-kDa
protein and results from hydrodynamic studies demonstrating a 7.5 S complex
were consistent with TYR forming a homodimer within the ER. The conclusion
that this was a TYR homodimer was also supported by our inability to
immunoprecipitate the oligomer with antibodies against a homologous protein of
similar size, Tyrp1 (data not shown), known to exist in a melanogenic complex
with TYR (35). Furthermore,
the cytosolic tail of Tyrp1 lacks Cys residues, and thus failure to form
heterodimers with TYR is consistent with our results demonstrating a
requirement for Cys500 in TYR for intermolecular disulfide bonding.
The same argument can also be applied to another homologous protein,
DOPAchrome tautomerase (DCT; previously named Tyrp2), because this protein
also lacks Cys residues in its cytosolic tail. Our conclusion is in agreement
with the observations that purified tyrosinase behaves as a homodimer by
size-exclusion chromatography in nonionic detergent
(36). However, we cannot
exclude the possibility that the complex contained additional proteins before
ER exit that were not stabilized under the experimental conditions used
here.
Detailed analysis using several semipermeabilized cellular systems as the source for in vitro translation-maturation processes demonstrated requirement for melanocyte-specific factors (Table I). TYR did not mature properly using heterologous sources. The translocation of TYR into rough ER-derived microsomes from pancreatic acinar cells generated a protein that remained associated with CNX, was trypsin-sensitive, and was unable to oligomerize. Similarly, release from CNX and oligomerization were not observed when CHO cells were used as the source for semipermeabilized cells for TYR maturation. The persistent binding to CNX indicated that TYR remained misfolded in these heterologous cellular systems because it was continually reglucosylated by UDP-glucose:glycoprotein glucosyltransferase, an enzyme that recognizes non-native structures (18).
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According to the results described here, two main functions were required for TYR oligomerization, i.e. its own enzymatic activity and Tyrp1 (Table I). We ruled out the involvement of the p-protein. In p-melanocytes with null mutations in the pink-eyed dilution gene, tyrosinase is mislocalized to the extracellular compartment, and a fraction is retained in the ER (34, 37). However, in our hands, CNX binding, glycan trimming, and oligomerization of TYR produced in semipermeabilized melan-p melanocytes were indistinguishable from those produced in semipermeabilized wild type melanocytes.
Several studies demonstrated that TYR is stabilized by its own enzymatic activity (29, 38). Stimulation of TYR in intact human melanoma cells by DOPA and tyrosine enhanced TYR maturation in the ER and its transport to the Golgi (29). Likewise, co-expression of wild type protein with temperature-sensitive TYR mutants corrected the mutant conformation defect in an activity-dependent manner (38). The results from the in vitro translation-maturation system described here are consistent with these observations because WT TYR failed to oligomerize when allowed to mature in the semipermeabilized c-albino melanocytes with null Tyr(C85S) mutant protein and in melanoma cells with extremely low TYR activity.
In vivo, tyrosinase degradation is enhanced in mouse melan-b melanocytes defective for Tyrp1 (32). Here, a role for Tyrp1 in the proper maturation of TYR within the ER was demonstrated by the failure of TYR to dimerize in the ER of semipermeabilized melan-b melanocytes after its proper maturation and release from CNX. Tyrp1 is the most abundant of the melanocyte-specific proteins, but its activity remains controversial. Immunoprecipitated human Tyrp1 was shown to possess catalase activity, but the studies did not rule out that the activity was derived from an associated protein (39). In murine melanocytes, Tyrp1 was reported to function as a 5,6-dihydroxyindole-2-carboxylic acid oxidase, the downstream substrate produced by the tyrosinase reaction (40). However, the same function could not be demonstrated for human Tyrp1 by some of the same investigators (41). Furthermore, another group demonstrated that ectopic mouse Tyrp1 expressed in transfected fibroblasts possessed DOPAchrome tautomerase activity (42). Therefore, at this point, it remains to be determined how Tyrp1 supports TYR oligomerization.
The significance of TYR oligomerization in in vivo maturation processes and transport to the Golgi is not yet clear. Mouse tyrosinase is in large part normally processed in melan-b melanocytes, and the cells (as well as the mutant mouse) suffer only minor reduction in pigmentation (brown instead of black). In addition, TYR can induce pigmentation in nonmelanocytic cells (43). However, the overall efficiency of pigmentation in nonmelanocytic cells expressing tyrosinase is much lower than that in melanocytes. This is due not only to the targeting of TYR to lysosomes in the absence of melanosomes, the subcellular organelles for melanin formation, but also to sluggish maturation, as demonstrated in parallel experiments using melanocytes and CHO cells (38). Perhaps oligomerization is not obligatory for TYR activity or exit from the ER but enhances this process.
Time course analysis showed that TYR dimerization occurred after TYR was released from CNX. This sequence of events is consistent with previous results obtained with the viral glycoprotein hemagglutinin, where its folding and oligomerization occurred after release from chaperones (44). In addition, CNX/CRT binding was not obligatory for TYR oligomerization because dimers were observed after inhibition of glucose trimming. However, the overall efficiency of oligomerization was greatly decreased under these circumstances, suggestive of a role for the lectin chaperones in enhancing the fidelity of the maturation process. The inhibition of glucose trimming and subsequent CNX/CRT binding in mouse melanoma cells by glucosidase inhibition has been found previously to greatly decrease melanin production (16). In that report, inactive tyrosinase was transported to melanosomes. Evidently, CNX/CRT binding to tyrosinase aided in the efficient folding of the protein and served the quality control function of retaining non-native protein in the ER. Abolishing the interaction not only created inactive protein but also disrupted its retention by the quality control machinery of the ER.
CNX binding is mediated by monoglucosylated glycans. Glycoproteins can
persist in the monoglucosylated state through repeated deglucosylation and
reglucosylation cycles. UDP-glucose:glycoprotein glucosyltransferase
reglucosylates proteins containing misfolded or immature structures
(18). The general notion is
that reglucosylation and subsequent CNX binding persist until a protein is
correctly folded or assembled. Under certain conditions in our system, TYR
binding to CNX ceased indicating proper folding, but the protein did not
oligomerize. This pattern was observed with the folding-defective TYR(C89R),
with TYR mutants lacking the critical residue required for oligomerization
(C500S and Tail) expressed in semipermeabilized wild type or melan-p1
melanocytes, and with wild type protein expressed in semipermeabilized albino
and Tyrp1-defective mouse melanocytes
(Table I). How could a
defective protein be released from CNX and remain stable within the ER?
Defective proteins are generally retained in the ER and subsequently
targeted for degradation by the proteasome through the ER protein-associated
degradation pathway (45).
EDEM, a mammalian ER type II membrane glycoprotein, has recently been
implicated as a quality control receptor that extracts proteins from the CNX
binding cycle and sorts them for destruction
(46,
47). Like CNX, EDEM is also a
lectin, but its binding is mediated by the slow trimming of a mannose residue
by ER mannosidase I to the mannose 8 form. Therefore, the release of the
malfolded TYR(C89R) from CNX was likely due to its accumulation on EDEM.
Generally, binding to EDEM is associated with substrate dislocation to the
cytosol and rapid proteasomal degradation. However, to optimize translation,
the in vitro system used in our studies contains hemin, a suppressor
of an inhibitor of the initiation factor eIF2. Hemin also inhibits the
proteasome, thus allowing TYR(C89R) stabilization within the ER.
The use of reduced biological systems that support mechanistic studies can help in elucidating the details of how the cellular machinery operates. Through the use of semipermeabilized melanocytes, we have been able to draw a detailed picture of the ER maturation steps for TYR. Future studies should explain how active tyrosinase and Tyrp1 help with the maturation of TYR. In addition, this in vitro system will permit examination of the quality control components participating in ER retention and sorting of defective proteins to the ER protein-associated degradation pathway reported to operate in albino melanocytes and melanoma cells.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 413-545-0079; Fax: 413-545-3291; E-mail: dhebert{at}biochem.umass.edu.
1 The abbreviations used are: ER, endoplasmic reticulum; BMH,
bismaleimidohexane; Tyrp, tyrosinase-related protein; TYR, human tyrosinase;
Tyr, mouse tyrosinase; WT, wild type; CNX, calnexin; CRT, calreticulin; RRL,
rabbit reticulocyte lysate; WG, wheat germ; CHO, Chinese hamster ovary; AMS,
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2 S. Svedine, T. Wang, R. Halaban, and D. N. Hebert, manuscript in
preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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