From the Department of Biochemistry and Molecular
Biology, Program in Molecular and Cellular Biology, University of
Massachusetts, Amherst, Massachusetts 01003 and the ¶ Department
of Dermatology, Yale University School of Medicine,
New Haven, Connecticut 06520
Received for publication, October 9, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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Tyrosinase is a type I membrane glycoprotein
essential for melanin synthesis. Mutations in tyrosinase lead to
albinism due, at least in part, to aberrant retention of the protein in
the endoplasmic reticulum and subsequent degradation by the
cytosolic ubiquitin-proteasomal pathway. A similar premature
degradative fate for wild type tyrosinase also occurs in amelanotic
melanoma cells. To understand critical cotranslational events, the
glycosylation and rate of translation of tyrosinase was studied in
normal melanocytes, melanoma cells, an in vitro cell-free
system, and semi-permeabilized cells. Site-directed mutagenesis
revealed that all seven N-linked consensus sites are
utilized in human tyrosinase. However, glycosylation at Asn-290
(Asn-Gly-Thr-Pro) was suppressed, particularly when translation
proceeded rapidly, producing a protein doublet with six or seven
N-linked core glycans. The inefficient glycosylation of
Asn-290, due to the presence of a proximal Pro, was enhanced in
melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino
acids/s). Slowing the translation rate with the protein synthesis
inhibitor cycloheximide increased the glycosylation efficiency in live
cells and in the cell-free system. Therefore, the rate of
protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential
cotranslational maturation events.
Whereas prokaryotic proteins fold posttranslationally due to their
rapid rate of translation, the maturation of nascent proteins in
eukaryotic cells often begins cotranslationally as a vectorial process
and continues posttranslationally after the release of the protein from
the ribosome (1-4). The slower rate of protein translation observed in
eukaryotic cells has been proposed to play an important role in the
proper folding of proteins in the cell by permitting the sequential
folding of individual domains during the translation process (5).
Understanding how proteins acquire their native structure in the cell
is of fundamental significance. Because key protein maturation events
for eukaryotic cells occur cotranslationally and have a large impact on
the fidelity of the overall maturation process, it is important to
fully understand these cotranslational processes.
For proteins that traverse the secretory pathway, the cotranslational
processes include the translocation of the protein across the
endoplasmic reticulum (ER)1
membrane, the site of entry into the secretory pathway. In this case,
protein folding commences upon emergence of the polypeptide chain into
the lumen of the ER. The ER is an organelle that specializes in the
efficient folding, modification, and assembly of proteins to their
native structures prior to their packaging into transport vesicles. The
milieu of the ER is topologically equivalent to the extracellular
space, with oxidizing conditions permitting the cotranslational
and posttranslational formation of disulfide bonds (1, 6-9).
Additional covalent modifications in the ER include the transfer of
N-linked core glycans to Asn residues found in the consensus sequence Asn-X-(Thr/Ser) (10). This transfer can occur
cotranslationally after the Asn is 12-14 amino acids into the ER
lumen, positioning it proximal to the active site of the oligosaccharyl
transferase (OST) (1, 11). Immediately after its transfer, the glycan side chains are trimmed by glucosidases I and II, generating
glycoproteins possessing monoglucosylated glycans that are substrates
for the lectin chaperones calnexin and calreticulin (12-15). Release
from the chaperones is then initiated after the cleavage of the third glucose by glucosidase II (14, 16). The binding of these lectin chaperones to their substrates promotes correct folding and oligomeric assembly (17-19). Thus, oligosaccharides play a central role in the
quality control system of the ER that determines the fate of the
maturing cargo glycoproteins (20-22).
Melanocytes are specialized cells dedicated to the production of
melanin. Tyrosinase (monophenol, L-dopa:oxygen
oxidoreductase, EC 1.14.18.1), is the key melanocyte-specific enzyme
that catalyzes the oxidation of tyrosine and DOPA to DOPAquinone, and
5,6-dihydroxyindole to indole-5,6-quinone (23-25). The biosynthesis of
melanin takes place in post-Golgi endomembranous compartments called
melanosomes or pigmented granules. Mutational analysis of
tyrosinase-positive albinism has identified AP-3 as an important
protein involved in the sorting of tyrosinase in the
trans-Golgi to melanosomes (26-28, reviewed in Ref. 29).
However, additional key sorting decisions for tyrosinase are made in
the early secretory pathway that are critical for pigmentation
(30-32).
Tyrosinase is a membrane glycoprotein with an N-terminal signal
sequence that targets the protein to the ER (Fig. 1A)
(33-36). The human protein possesses 7 putative N-linked
glycosylation sites and 15 lumenal Cys residues that can participate in
disulfide bond formation. Mutations in tyrosinase are the cause of
tyrosinase-negative oculocutaneous albinism 1, an autosomal recessive
genetic disorder characterized by the absence of melanin (reviewed in
Refs. 37 and 38). The mutant protein in several tyrosinase-negative
albino melanocytes of human and mouse origin is retained in the ER (31, 39). The essential role of the ER in the regulation of tyrosinase has
also been demonstrated in amelanotic melanoma cells in which ER
retention of wild type tyrosinase leads to subsequent degradation by
the 26 S proteasome (30).
Here, we demonstrate that the faster rate of translation of tyrosinase
in melanoma cells than that of normal melanocytes hampered glycosylation at the inefficient Asn-290 site. This was determined by
studying tyrosinase glycosylation and maturation under conditions that
altered the rate of translation in normal melanocytes and melanoma
cells, as well as in a cell-free system that recapitulated the ER processes.
Materials--
Rabbit reticulocyte lysate (RRL), wheat germ
(WG), dithiothreitol, and RNasin were from Promega Corp. (Madison, WI).
Canine pancreas microsomes were a generous gift from Dr. R. Gilmore
(Worcester, MA). [35S]Methionine/cysteine (EasyTag) and
CHAPS were from PerkinElmer Life Sciences and Pierce,
respectively. Restriction endonucleases and ribonucleotide
triphosphates were from New England Biolabs, Inc. (Beverly, MA).
mMessage mMachine and T7 transcription kits were from Ambion (Austin,
TX), and the QuikChange site-directed mutagenesis kit was from
Stratagene (La Jolla, CA). Zysorbin (fixed and killed Staphylococcus aureus) was obtained from Zymed
Laboratories Inc. (San Francisco, CA). All other reagents,
including the anti-FLAG M2 monoclonal antibody, were from Sigma.
Construction of Plasmids Encoding Wild Type and Mutant Tyrosinase
Proteins--
The plasmid pcTYR carrying the human tyrosinase gene
(GenBankTM accession number Y00819) was a gift from
Dr. R. Spritz (Denver, CO). The EcoRI tyrosinase fragment
excised from pcTYR was subcloned into pGEM 7Zf (Promega). To improve
in vitro translation/translocation, the original signal
sequence of tyrosinase was exchanged with the murine major
histocompatibility complex class I molecule Kb
signal sequence as follows. A XbaI restriction site was
introduced at the end of the 18-amino acid signal sequence in pGEM
7Zf-TYR plasmid by site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene). The tyrosinase XbaI fragment
(encoding the full sequence of tyrosinase minus the signal peptide) was inserted into the XbaI restriction site of
pSP72/KbSS-CD3
To generate single-site glycosylation deletion mutant proteins, the
consensus N-linked glycosylation sites Asn-X-Thr/Ser in pSP72/KbSS-TYR were eliminated or modified in the cDNA
by changing threonine or serine to an alanine, except for Thr-373,
which was changed to the albino mutation T373K. The site at Asn-290 was
modified by exchanging the proline at position 293 to alanine (P293A). The P293A and T292A mutations were also introduced to tyrosinase cDNA in the enhanced green fluorescent protein (enhanced
GFP) vector (31). In addition, the wild type and mutant
cDNAs were subcloned from the enhanced GFP plasmids into the
KpnI/HindII cloning sites of p3XFLAG-CMV-14
expression vector (Sigma) to generate FLAG-tagged tyrosinase proteins.
In all cases, DNA sequencing of the entire tyrosinase gene verified the
inserted mutations. Transfection of plasmids into mouse melanocytes was
done as described (31).
Transcription, Translation, and Translocation--
Messenger RNA
was prepared by in vitro run-off transcription of the
cDNA that was linearized with NdeI or HaeII,
according to the manufacturer's instructions. Radioactive
35S-labeled tyrosinase was translated for 1 h at
27 °C in RRL (14) or in WG and translocated cotranslationally into
canine pancreas microsomes. In the latter case, WG lysate (40 µl) was
mixed with ribonuclease-treated rough ER (RER) microsomes (6 µl), an
amino acid mixture lacking methionine and cysteine (6 µl of a 1 mM solution of each), [35S]Met/Cys (63 µCi), RNase-free water (18.4 µl), RNase inhibitor (1.6 µl), and
mRNA (1 µg/µl, 3.5 µl). Samples were alkylated with N-ethylmaleimide (20 mM) to block free
sulfhydryls (7). Alkylated samples were either analyzed directly by
SDS-PAGE or immunoprecipitated with anti-tyrosinase antibodies
(
Semipermeabilized cells were prepared from subconfluent mouse B10BR
melanocytes (41) permeabilized with 20 µg/ml digitonin using a method
described previously by Wilson et al. (42). Radioactive 35S-labeled tyrosinase was translated for 1 h at
27 °C in RRL with semipermeabilized cells (1.3 × 104 cells/µl) replacing the RER microsomes.
Identification of Translocated Protein--
To separate
glycosylated (TYR) from nonglycosylated (untranslocated
(UTYR)) proteins, 10 µl of the translation mixture was
solubilized in 200 µl of 2% CHAPS buffer (2% CHAPS, 50 mM HEPES, 200 mM sodium chloride, pH 7.5), and
glycoproteins were captured on wheat germ agglutinin-bound beads at
4 °C for 2 h under constant rotation. The beads were pelleted
by centrifugation at 2500 × g and washed once with
0.5% CHAPS buffer. The bound proteins were then eluted in SDS-sample
buffer at 95 °C for 5 min. Alternatively, untranslocated tyrosinase
was separated from the translocated protein by centrifugation of the
translation products (10 µl) through a sucrose cushion (100 µl, 0.5 M sucrose, 50 mM triethanolamine, 1 mM dithiothreitol, pH 7.4) in a Beckman Airfuge
ultracentrifuge for 10 min. For protease protection, translation
products were digested with proteinase K (0.35 µg/µl) in the
absence or presence of 1% Triton X-100 for 1 h on ice. Protease
digestion was stopped with 10 mM phenylmethylsulfonyl fluoride. Samples were added to a 100-µl solution of 0.1 M Tris, 1% SDS (pH 8) and heated at 95 °C for 10 min.
Deglycosylation, Immunoprecipitation, and Trichloroacetic
Acid Precipitation--
Translation mixtures were centrifuged
through a sucrose cushion, and pellets were solubilized in a solution
containing 0.2% SDS, 100 mM sodium phosphate, 25 mM EDTA (pH 6.9) at 95 °C for 5 min. The samples were
cooled to room temperature, diluted with 2% Triton X-100 in 100 mM sodium phosphate, 25 mM EDTA (pH 6.9), and
digested with 0.1-1 µl of 1 units/µl PNGase F at 37 °C for the
indicated times. 35S-Labeled tyrosinase was
immunoprecipitated with anti-tyrosinase antibodies (30). To determine
radioactivity in total proteins, lysates were blotted on filter paper
and treated with 10% trichloroacetic acid on ice for 30 min. Samples
were washed with ice-cold 100% ethanol. The radioactivity of the blots
was determined in a liquid scintillation counter.
Calculation of TYR Concentration--
The approximate
concentration of TYR in the in vitro translation reactions
was determined as described below. Radioactive samples labeled with
[35S]Met/Cys were fully resolved by SDS-PAGE to quantify
the intensity of the translocated tyrosinase band by phosphorimaging.
Equivalent samples were also loaded on the same gel after the run was
70% completed. Therefore, the samples were not resolved and the
intensity of the total free label in the reaction could be determined.
Because tyrosinase has a total of 28 Met and Cys, its approximate
concentration is the following: [Tyr] = (T/F) × [Met/Cys]/28, where T
is the intensity of the tyrosinase band and F is the
intensity of the total free label. [Met/Cys] is the sum of the
concentrations of endogenous reticulocyte lysate (3.9 µM) and the radioactive [35S]Met/Cys (0.9 µM).
Metabolic Labeling of Tyrosinase--
Normal human melanocytes
were cultured from newborn foreskins in Ham's F-10 medium supplemented
with 7% fetal bovine serum and several ingredients required for their
proliferation, including 12-O-tetradecanoylphorbol-13-acetate,
3-isobutyl-1-methylxanthine, dbcAMP (N6,
2'-O-dibutyryladenosine 3, 5-cyclic
monophosphate) and cholera toxin (43). Melanoma cells strains 501 mel
and YUSIT1 were grown in Ham's F-10 medium plus serum (30). Cells were
starved for 2 h in Met/Cys free-RPMI 1640 medium (supplemented
with 3% dialyzed calf serum, 1% glutamine, 0.1 mM
3-isobutyl-1-methylxanthine, and 50 µg/ml
12-O-tetradecanoylphorbol-13-acetate), were then incubated
with [35S]Met/Cys (0.7 mCi/ml) for 10 min, scraped into
phosphate-buffered saline supplemented with 20 mM
N-ethylmaleimide, and lysed in 2% CHAPS buffer. Cell
extracts were immunoprecipitated with anti-tyrosinase antibodies
(30).
Translation and Translocation of Tyrosinase--
Newly synthesized
tyrosinase from normal melanocytes migrated as a 70-kDa doublet band
separated by ~3 kDa (Fig. 1B,
lane 1) (30). An [35S]TYR doublet of similar
mobility also accumulated in melanoma cells, but the intensities of the
individual doublet bands varied. The ratio of the larger TYR to the
smaller TYR in melanoma cells was half of that in normal melanocytes
(0.4-0.5 compared with 0.8, respectively), indicating that the faster
migrating form (TYRS) accumulated more predominantly in the
melanoma cells.
To identify the cause of the TYR doublet, TYR was expressed in a
cell-free system that facilitated experimental manipulation. The
translation system consisted of rabbit reticulocyte lysate and mRNA
encoding human TYR in the presence and absence of isolated RER
membranes (Fig. 2). The native tyrosinase
mRNA or a modified message termed KbSS-TYR, in which
the TYR signal sequence was exchanged with the corresponding signal
sequence from murine major histocompatibility complex class I molecule
Kb (KbSS) (40, 44), was used. A 60-kDa protein,
recognized by anti-tyrosinase antibodies, was generated in the absence
of microsomes that corresponded to untranslocated and unglycosylated
TYR (Fig. 2, A, lanes 1 and 5, UTYR). However, a 70-kDa doublet identical to that
observed previously in intact melanocytes (30) (Fig. 1B)
accumulated in the presence of RER microsomes. This band represented
translocated tyrosinase that had received multiple N-linked
carbohydrates (Fig. 2A, lanes 3 and 7). The
KbSS-TYR chimera was translated and translocated 4-6-fold
more efficiently than native tyrosinase (Fig. 2A, compare
lane 3 to lane 4). The untranslocated
KbSS-TYR migrated slightly slower than wild type TYR (Fig.
2A, compare lane 1 to lane 2 and
lane 5 to lane 6, UTYR) due to
the different sizes of signal sequence (24 amino acids in
KbSS and 18 amino acids in TYR). However, the mobility of
the translocated products was identical (Fig. 2A, compare
lane 3 to lane 4 and lane 7 to
lane 8, TYR) and was similar to newly synthesized
tyrosinase produced in intact melanocytes, justifying the use of the
KbSS-TYR in all subsequent cell-free experiments.
The protein doublet constituted the translocated and glycosylated
tyrosinase because it was precipitated with wheat germ agglutinin-bound beads (Fig. 2B, lane 4, TYR), and it sedimented
through a sucrose cushion designed to isolate only membranes decorated
with ribosomes (Fig. 2B, lane 6, TYR). Digestion
with proteinase K degraded UTYR but not the TYR form,
unless the microsomes were first solubilized by detergent (Fig.
2B, lanes 7-9). The downward mobility shift caused by
proteinase K cleavage of the 29-amino acid cytosolic tail of the
translocated form is consistent with correct insertion of the type I
membrane protein into the microsomal membrane bilayer (Fig.
2B, compare lane 6 to lane 8, TYR).
Taken together, the data supported the conclusion that the in
vitro translated 70-kDa doublet represented ER translocated and
glycosylated forms of tyrosinase.
Tyrosinase Exists as Two Alternatively Processed N-Linked Core
Glycans, TYR7 and TYR6--
The two
translocated ER isoforms of tyrosinase could be generated by
heterogeneous glucose or mannose trimming of N-linked glycans in the ER or through differential recognition by the OST generating differences in the total number of attached
N-linked glycans. Differential trimming of glucose residues
as the source for this variability was ruled out by the persistent
production of doublet protein in the presence of DNJ, although the
translated and processed products displayed slower mobility due to
inhibition of glucosidases I and II activities (Fig.
3A, lanes 3 and 5). Likewise, differential ER carbohydrate trimming was excluded by the
persistence of the doublet in the presence of the mannosidase inhibitor
deoxymannojirimycin (data not shown).
Heterogeneity in the carbohydrate side chains as the source of TYR
doublet was indicated after digestion with the bacterial endoglycosidase PNGase F. This endoglycosidase removed all
N-linked side chains coalescing the doublet into a single
band that migrated slightly faster than the untranslocated form (Fig.
3A, lanes 6 and 7). The faster mobility of the
PNGase F digested over the untranslocated form of tyrosinase (Fig.
3A, compare lanes 1 and 2 to
lanes 6 and 7) was due to the cleavage of the
signal sequence during RER processing. Therefore, we concluded that the
doublet represented tyrosinase glycoforms with different numbers of
N-linked glycans.
The number of glycans was then resolved by creating a ladder of
partially cleaved PNGase F glycosidase products (Fig. 3B). Tyrosinase was produced in the presence of DNJ to eliminate variation due to glucose trimming. Complete digestion with high concentration of
PNGase F generated an unglycosylated protein (Fig. 3B, lanes 4-6, band 0). However, PNGase F at lower concentrations produced a discrete ladder of eight bands corresponding to proteins with seven
to zero glycans (Fig. 3B, lane 3; top and
bottom bands, respectively). This analysis revealed that the
doublet represented tyrosinase with seven (TYR7) and six
(TYR6) N-linked glycans.
Glycosylation of Asn-290 at the Consensus site Asn-Gly-Thr-Pro Is
Suppressed by the Presence of Pro at Position 293--
The partial
digestion by PNGase F suggested that the doublet was generated by
inefficient glycosylation of one of the consensus sites. The smaller
glycoform (TYRS) could be the product of site-specific
inefficient glycosylation or random vacancy of any one of the sites.
Glycosylation of a consensus site could be suppressed by inefficient
transfer of truncated dolichol pyrophosphate precursors, competition
with disulfide bond formation, or the presence of inefficient sites (45-48). The involvement of truncated dolichol pyrophosphate sugar precursor was excluded because complete glycosylation of a variety of
multiglycosylated substrates has been reported with this cell-free system (14, 49, 50), and the doublet appeared in cells. Furthermore, we
ruled out competition by disulfide bond formation, because the doublet
persisted even when tyrosinase was synthesized in the presence of the
reducing agent dithiothreitol (Fig. 2 and data not shown).
A clue that a proline residue had a role in the hypoglycosylation came
from experiments using the proline analogue azetidine-2-carboxylic acid
(Azc). Although the doublet persisted when tyrosinase was synthesized
in the presence of equal molar Azc and proline (Fig. 4A, lane 6), hypoglycosylation
was arrested, and complete glycosylation to TYR7 was
observed with an excess of Azc (Fig. 4A, lane 4). These
results suggested that a proline located proximal to the
hypoglycosylated site was involved in the hypoglycosylation of
TYR6.
A scan of the human tyrosinase amino acid sequence revealed a Pro
immediately downstream from the Asn-290 consensus glycosylation sequence (Asn-Gly-Thr-Pro-293). To determine whether hypoglycosylation of Asn-290 was the source of the doublet, this glycosylation site was
removed by substituting Thr with an Ala at position 292 (TYR-T292A). In vitro translation of TYR-T292A produced a single band
with mobility equivalent to TYR6 (Fig. 4B, lane
3). In contrast, a single band with mobility of TYR7
was produced when Pro-293 was exchanged for an Ala (TYR-P293A) (Fig.
4B, lane 4). Likewise, in vivo synthesis of
FLAG-tagged wild type and mutant tyrosinase proteins transiently
expressed in mouse melanocytes produced similar results (Fig.
4C). In the latter case, the differences in the migration
pattern was restricted to the ER form, as all ectopically expressed
proteins possessed Golgi processed complex carbohydrates and matured
normally (Fig. 4C, arrow). Because the mutant TYR-P293A was
completely glycosylated, we concluded that the proximal proline at
position 293 was the source of tyrosinase hypoglycosylation.
The utilization of the other six consensus N-linked
glycosylation sites was confirmed by site directed mutagenesis. Here, in vitro translated proteins were translocated into the ER
of semi-permeabilized melanocytes. As observed with the RER microsomes, the wild type protein migrated as a protein doublet (Fig.
5, lane 1). Consistent with
the results obtained by PNGase F partial digestion (Fig.
3B), exchanging the Thr or Ser of each of the seven
consensus sites with Ala (or Lys in the case of Thr-373) induced an
identical downward mobility shift of TYR (Fig. 5, lanes
2-8). The T373K mutation was utilized here, because it is a
common human mutation that causes albinism due to the retention of the
protein in the ER (31). It should also be noted that a doublet was
produced with all mutants excluding T292A.
Glycosylation of Asn-290 Is More Efficient at Slow Rates of
Translation--
The intensity of the individual bands of the doublet
was dependent on the translation system employed, as the ratio of
TYR7/TYR6 was 0.77 in the wheat germ
translation system and 0.50 or smaller in the RRL (Fig.
6A, lanes 1 and 2).
These two systems also displayed differences in translation times. The
translation time for tyrosinase was 15 min with the WG and 10 min with
the RRL system (Fig. 6B, as determined by the x
intercept). These translation times at 27 °C corresponded to
translation rates of 0.6 and 0.9 amino acids/s in WG and RRL,
respectively. Therefore, we explored the possibility that rapid
translation rates contributed to hypoglycosylation of tyrosinase at the
Asn-290 site.
Toward this aim, the protein synthesis inhibitor cycloheximide (CHX)
was employed. Although CHX blocks protein synthesis at concentrations
above 100 µM, it only slows translation at lower concentrations (7). Indeed, slowing the translation time of tyrosinase
in the RRL system to 20 min with 0.3 µM CHX increased the
ratio of TYR7/TYR6 to 0.71 (Fig. 6A, lane
3, and Fig. 6B, RRL + CHX), indicating an increase in
the level of glycosylation. Higher concentration of CHX resulted in
additional decrease in translation rate, further increasing
glycosylation (Fig. 6A, lane 4). Therefore, we concluded that the suppressive effect of Pro on glycosylation at the
Asn-Gly-Thr-Pro consensus site was alleviated when the rate of
translation was reduced.
Cellular Translation Rates--
We went on to determine whether
the difference in glycosylation levels of tyrosinase in normal
melanocytes versus melanoma cells was due to differences in
translation rates. Translation rates were determined by performing
short radioactive pulses with [35S]Met/Cys, followed by
the quantification of TYR levels. Half-times of synthesis were
determined as the x intercept of the plot of TYR level
versus pulse time (Fig. 7),
minus the time for general radiolabel incorporation into the cells
determined by trichloroacetic acid precipitation (data not shown) (7,
51, 52).
The synthesis time of TYR in normal melanocytes was 2.53 min (3.5 amino
acids/s), compared with 1.15 (7.7 amino acids/s) and 0.89 min (10.0 amino acids/s) (Fig. 7B) in the amelanotic melanoma cell
lines 501 mel and YUSIT1, respectively. As seen earlier in the
cell-free system, the slower rate of TYR translation corresponded to a
higher level of TYR7 (Fig. 7A, compare
lanes 1-5 to lanes 6-16). Moreover, slowing the
rate of translation in melanoma cells with 1 or 10 µM CHX to 3.4 and 8.1 min, respectively, permitted more complete glycosylation to TYR7 (Fig. 8), as seen
before in the cell-free system. This was accompanied by a dramatic
increase in the glycosylation of TYR, as reflected by an increase in
the ratio of TYR7/TYR6 from 0.4 to 1.7 in the
presence of 10 µM CHX. Therefore, the rate of TYR
translation influenced the glycosylation profiles of TYR synthesized in
the cell-free system and in live cells. However, slowing the
translation rate by decreasing the temperature had no effect on the
TYR7/TYR6 ratio, likely due to the glycan
transfer reaction also being slowed (data not shown).
Similar analysis was performed with two other proteins to determine
whether the difference in the rate of TYR synthesis between normal
melanocytes and melanoma cells was general or protein specific (Table
I). The results show similar synthesis
times for calnexin and tyrosinase-related protein 1 (TRP1) in the two
melanoma cells and normal melanocytes. This indicated that the
decreased translation rate observed in normal melanocytes was specific
to tyrosinase.
The important role of the ER quality control system in the
maturation of tyrosinase has been observed in normal and malignant melanocytes (30). In normal human melanocytes, tyrosinase slowly reaches the Golgi, with a half-time of ~1.5 h,
while most of the wild type protein in amelanotic melanoma cells
is retained in the ER and is then targeted for degradation by the
ubiquitin-dependent cytosolic proteasomal system. To
further understand this critical quality control process, we have
investigated the maturation of human tyrosinase in normal melanocytes,
melanoma cells, semipermeabilized cells, and a cell-free system. The
cell-free system reproduced the early stages of maturation in the ER
and permitted the detailed analysis of tyrosinase translocation and
glycosylation. Furthermore, it helped resolve the nature of human
tyrosinase early glycoforms that appeared as a doublet in melanocytes.
Upon translocation into the lumen of the ER, tyrosinase
cotranslationally received multiple N-linked glycans,
resulting in a 12-kDa gain in molecular mass. Limited PNGase F
digestion and site-directed mutagenesis showed that all consensus sites
are being utilized and that the doublet ER translocated form represents a protein with six or seven glycans. We found that the source of the
heterogeneity was the inefficient recognition by the OST of Asn-290
(Asn-Gly-Thr-Pro). Interestingly, mouse tyrosinase lacks the
inefficient glycosylation site at Asn-290 but retains the remaining six
observed in the human protein. However, unlike human tyrosinase, the
second and third sites in the mouse counterpart (Asn-111 and Asn-161)
were reported to be unrecognized, producing a protein with only four
glycans (53).
Hypoglycosylation at Asn-290 of human tyrosinase occurred during
in vivo and cell-free processing based on the following
observations. 1) Partial digestion of tyrosinase with PNGase F showed
that the upper and lower bands of the doublet corresponded to
tyrosinase with seven and six processed glycans, termed
TYR7 and TYR6, respectively, indicating
inefficient glycosylation of a single site. 2) Substituting Pro with
its analogue Azc during translation of tyrosinase produced more
efficient glycosylation, identifying a role for Pro in the
hypoglycosylation. Indeed, a Pro is located immediately carboxyl to the
glycosylation site of Asn-290 (Pro-293). 3) Mouse tyrosinase has six
consensus glycosylation sites, lacking the one corresponding to Asn-290
in human tyrosinase (33, 34, 36). Consistent with this difference, the
early glycoform of mouse tyrosinase migrates in SDS-PAGE as a protein
singlet (54), whereas early glycoforms of human tyrosinase produced in
normal melanocytes appear as a doublet (Fig. 1B and Ref.
30). 4) The substitution of Pro-293 with an Ala in human TYR produced a
single protein band that migrated with mobility corresponding to
TYR7. 5) Likewise, abolishing the glycosylation site at
Asn-290 with an Ala at position 292 produced a single protein band that
corresponded to TYR6. Together, our results support the
conclusion that the source for tyrosinase hypoglycosylation is the
inefficient recognition of Asn-290 by the OST due to suppression by
Pro-293.
Proline suppresses glycan transfer to the consensus site
Asn-X-(Thr/Ser)-Y when found in the X
position (55). However, its effect on glycosylation when in other
proximal locations is poorly understood. Sites that have a Pro in the
Y position often go unrecognized by the OST (46), as well as
by many of the protein analysis software programs used to identify
potential glycosylation sites in a primary amino acid sequence. Protein
heterogeneity caused by Pro suppression of glycosylation is likely to
be found for a large range of glycoproteins.
The efficiency of glycosylation of Asn-Gly-Thr-Pro correlated inversely
with the rate of translation. The fully glycosylated TYR7
species was produced more efficiently when tyrosinase was translated in
the slower translation systems (cell-free WG system or normal melanocytes), as well as in the RRL translation system and melanoma cells, both of which were slowed by CHX. A tight window for
cotranslational glycosylation exists shortly after the consensus
sequence enters the ER lumen, determined by the position of the OST
active site 30-40 Å, or 12-14 residues from the membrane into the
lumen of the ER (11). Therefore, slowing down the rate of translation may maintain the inefficient Asn-290-Gly-Thr-Pro glycosylation site in
human tyrosinase in proximity to the OST active site for sufficient
time to allow glycosylation.
In addition to the precise distance from the membrane, recognition by
the OST may also have conformational requirements. It appears that
transfer entails roughly a parallel configuration of the
Asn-X-(Thr/Ser) sequence in relation to the ER membrane (46). Therefore, it is likely that in the context of the full-length protein, interference by Pro proximal to the Asn-290 consensus site
decreases the probability that the consensus sequence attains the
correct orientation that permits transfer of the glycan to the
inefficient site.
The level of glycosylation did not appear to influence the ability of
tyrosinase to exit the ER as both TYR6-FLAG and
TYR7-FLAG obtained complex glycans in the Golgi as
indicated by the appearance of 90-kDa forms. These results were
confirmed by direct visualization of TYR and GFP chimeras
(TYR6-GFP and TYR7-GFP) in the cell periphery,
indicative of melanosomal localization (data not shown). It remains to
be determined whether having the additional glycan confers any
maturational or functional advantage.
The translation rate of tyrosinase synthesized in normal melanocytes
was slower than that found in the melanoma cell lines. However, the
rates of translation of TRP1, a protein homologous to tyrosinase, and
the ER molecular chaperone calnexin were similar in both cell types.
Why the translation of TYR in normal melanocytes is slowed compared
with other proteins in both normal melanocytes and melanoma cells
remains to be determined. Because the protein is wild type in both cell
systems (30), the differential regulation of translation cannot involve
differences in the TYR gene. Furthermore, RT-PCR analysis demonstrated
that TYR mRNA was 2-3-fold more abundant in normal melanocytes
compared with melanoma cells, excluding the possibility that high
message levels caused the faster translation rate in melanoma cells
(data not shown).
The folding state of a protein in the ER can be communicated to the
translation machinery through the unfolded protein response. Here, during times of stress when large concentrations of
unfolded proteins have accumulated in the ER and the unfolded protein
response is turned on, the ER membrane kinase PERK
phosphorylates eIF2- Ribosomal or translational pausing has been implicated in signal
recognition particle-dependent targeting to the ER
membranes, helping to ensure efficient targeting (59). Translational
control has also been demonstrated to occur during the translocation of apolipoprotein B100 across the ER membrane, dependent upon the availability of newly synthesized lipids (60), and during the synthesis
of the membrane-bound chloroplast reaction center protein D1, which is
hypothesized to facilitate the cotranslational binding of its cofactors
(61). Translational control could also play an important role in the
vectorial folding of sequential folding domains and other
cotranslational maturation events including glycosylation. Revealing
the components in the translation system that regulate translation and
influence tyrosinase maturation may provide important insights into our
understanding of why wild type tyrosinase is targeted for degradation
in melanoma cells, and may offer a more general explanation for other
phenotypes associated with cancerous cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plasmid (from Drs. J. Huppa and H. Ploegh, Boston, MA) (40). This created a hybrid molecule comprising the
class I heavy chain H2-Kb signal peptide in frame with the
tyrosinase protein downstream of the T7 promoter, termed
pSP72/KbSS-TYR.
-TYR) prior to electrophoresis. Deoxynojirimycin (DNJ) (0.5 mM) was used to inhibit ER glucosidases when indicated.
Half of each sample was subjected to nonreducing SDS-PAGE, and
the other half was reduced by the addition of dithiothreitol (100 mM) and resolved by reducing SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
Topological map of human tyrosinase.
A, the 58-kDa tyrosinase includes an 18-amino acid signal
sequence that directs the protein to the ER and a 29-amino acid
cytosolic tail. Tyrosinase has seven putative glycosylation sites
(branched structures) that increase its size to 70 kDa in
the ER. It also contains two copper binding domains (CuA and
CuB) that form a binuclear copper binding site.
B, normal human melanocytes (NM) or melanoma
cells (501 mel and YUSIT1) were pulsed with
[35S]Met/Cys for 5 min. Tyrosinase was immunoprecipitated
and resolved by SDS-PAGE and autoradiography. High and low
molecular weight forms of tyrosinase are designated
TYRL (large molecular weight) and
TYRS (small molecular weight) respectively.
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[in a new window]
Fig. 2.
In vitro translation and
translocation produces normally processed tyrosinase.
A, replacing the native 18-amino acid signal sequence
(SS) of tyrosinase (MLLAVLYCLLWSFQTSAG) with the 24-amino
acid murine major histocompatibility complex class I KbSS
(MNSMVPCTLLLLLAAALAPTQTRG) increases efficiency of translation and
translocation. Radiolabeled proteins synthesized in the presence or
absence of RER microsomes (Ms) in the RRL translation system
were resolved by 7.5% SDS-PAGE directly (lanes 1-4) or
after immunoprecipitation with anti-tyrosinase antibodies (lanes
5-8). Molecular mass markers in kDa are indicated on the
right, and translocated (TYR) and untranslocated
(UTYR) bands are indicated on the left. *
denotes a RRL background band. B, the translocated doublet
form of tyrosinase is a glycosylated protein. 35S-Labeled
tyrosinase was synthesized in the absence (lanes 1, 3, 5, and 7) or in the presence (lanes 2, 4, 6, 8, and
9) of microsomes with the RRL translation system. Samples
were resolved directly, by SDS-PAGE (lanes 1 and
2), or after affinity precipitation with wheat germ
agglutinin (WGA) (lanes 3 and 4).
Alternatively, to assess translocation, the lysates were spun through a
sucrose cushion, and the resuspended pellets were fractionated
(lanes 5 and 6). Finally, digestion with
proteinase K, which has access to untranslocated tyrosinase and
to the cytoplasmic tail of the translocated protein, further
demonstrated that TYR is correctly inserted into the ER microsomes
(lanes 7-9).
View larger version (56K):
[in a new window]
Fig. 3.
Tyrosinase doublet is due to differential
glycosylation of tyrosinase by the OST. A,
[35S]TYR was translated under reducing conditions in the
presence and absence of DNJ and microsomes. Translocated protein
(TYR) was isolated from untranslocated
(UTYR) by ultracentrifugation and treated with
PNGase F prior to resolution by SDS-PAGE. B,
[35S]TYR was synthesized in the presence of DNJ using the
RRL cell-free system. Limited digestion with varying concentrations of
PNGase F produces a ladder of differentially glycosylated proteins. The
numbers next to each band indicate the total number of glycans per
tyrosinase molecule.
View larger version (37K):
[in a new window]
Fig. 4.
Pro-293 at the consensus glycosylation site
(Asn-Gly-Thr-Pro) suppresses glycosylation. A,
[35S]TYR was translated under reducing conditions in the
presence of varying concentrations of proline or the proline analogue
Azc. B, mutant forms of tyrosinase, T292A and P293A,
were translated along with wild type in the presence of RER microsomes
under reducing conditions and subjected to SDS-PAGE and
autoradiography. [35S]TYR in normal human melanocytes
(cell) was synthesized with a 10-min pulse of
[35S]Met/Cys, and the protein was immunoprecipitated with
anti-tyrosinase antibodies prior to electrophoresis. C,
in vivo processing of FLAG-tagged wild type (WT) and
mutant tyrosinase proteins. Mouse melanocytes were harvested 1 week
after transfection with plasmids encoding TYRwt-FLAG, TYR-T292A-FLAG,
or TYR-P293A-FLAG. Cell lysates were subjected to immunoprecipitation
with anti-tyrosinase antibodies and Western blotting with mAb against
FLAG. The arrow designates the fully glycosylated forms of
the chimeric proteins. Size markers are in kDa.
View larger version (43K):
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Fig. 5.
All seven glycans in tyrosinase are
processed. All seven putative N-linked glycosylation
sites were eliminated individually by mutation of the consensus site
(Thr/Ser to an Ala, or in the case of Thr-373 to a Lys).
[35S]TYR was translated with the RRL system in the
presence of semi-permeabilized melanocyte prior to resolution by
reducing SDS-PAGE.
View larger version (28K):
[in a new window]
Fig. 6.
Translation rate affects glycosylation
efficiency of Asn-Gly-Thr-Pro. A,
[35S]TYR was translated under reducing conditions with
either a WG or RRL translation system in the presence of varying
concentrations of CHX and resolved by SDS-PAGE. The
TYR7/TYR6 ratio was calculated after the
quantification of the radioactive protein bands by phosphorimaging.
B, amount of translocated tyrosinase (TYR6 + TYR7) versus the time of translation in RRL
(filled squares), in RRL with 0.3 µM CHX
(open squares), or in WG (circles). The
x intercept of the curves approximates the total synthesis
time of full-length tyrosinase under the different conditions.
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Fig. 7.
Synthesis time of tyrosinase.
A, normal melanocytes and melanoma cells (501 mel
and YUSIT1) were pulsed with [35S]Met/Cys for
the times indicated. The lysates were immunoprecipitated with -TYR,
subjected to SDS-PAGE, and quantified by phosphorimager
analysis. B, [35S]TYR levels were plotted
versus the time of the radioactive pulse. Radioactivity in
trichloroacetic acid precipitates of each sample was determined with a
liquid scintillation counter (data not shown). The x
intercepts of the [35S]TYR and the trichloroacetic acid
precipitation plots fitted by a straight line were subtracted from each
other. This value is equal to the half-time of synthesis (7, 51). For
calculated values, see Table I.
View larger version (47K):
[in a new window]
Fig. 8.
Translation rate determines glycosylation
efficiency in melanocytes. Normal melanocytes (NM) and
melanoma cells (YUSIT1 and 501 mel) were pulsed
with [35S]Met/Cys in the absence of CHX (-) for 5 min or
in the presence of 1 and 10 µM CHX for 10 and 20 min,
respectively. The lysates were immunoprecipitated with -TYR and
resolved by SDS-PAGE and autoradiography.
TYR7/TYR6 ratios were determined by
phosphorimaging.
Synthesis times for proteins in normal melanocytes and melanoma
cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, causing the inhibition of translation
initiation (56, 57). Another known mode of translation regulation
involves the phosphorylation of elongation factor eEF2 by
cAMP-dependent kinase that decreases the rate of protein
elongation (58). Although differences in the initiation rates in normal
melanocytes versus melanoma cells cannot be discounted, the
root of the variation is likely to be the elongation step, because TYR
mRNA levels were higher in normal melanocytes (data not shown), and
treatment of melanoma cells with low concentrations of cycloheximide
recapitulated the normal melanocyte glycosylation pattern. The
involvement of a nonspecific mechanism of translational regulation
known to affect all proteins in a similar manner was also ruled out by
the observations that synthesis times of calnexin and TRP1 were the
same in normal melanocytes and melanoma cells.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. J. Huppa and H. Ploegh (Boston,
MA) for the pSP72/KbSS-CD3 plasmid, Dr. R. Spritz
(Denver, CO) for the human tyrosinase cDNA pcTYR, and Dr. R. Gilmore (Worcester, MA) for RER microsomes. We acknowledge Robert
Daniels for help with the construction of glycosylation mutations,
Sherri Svedine with the confocal microscopy, and Dr. Sergio Trombetta
and members of the Hebert laboratory for their thoughtful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Medical Foundation, Edward Mallinckrodt, Jr. Foundation, and United States Public Health Service Grant CA79864 (to D. N. H.) and by United States Public Health Service Grants AR39848 (to R. H.) and AR41942 (Yale Skin Diseases Research Center; R. E. Tigelaar, Program Investigator).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail:
dhebert@biochem.umass.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M009203200
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ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; Azc, azetidine-2-carboxylic acid; CHX, cycloheximide; DNJ, deoxynojirimycin; GFP, green fluorescent protein; KbSS, murine major histocompatibility complex class I molecule Kb signal sequence; OST, oligosaccharyl transferase; PAGE, polyacrylamide gel electrophoresis; RER, rough ER; RRL, rabbit reticulocyte lysate; TYR, human tyrosinase; TYR6, tyrosinase with 6 glycans; TYR7, tyrosinase with 7 glycans; UTYR, untranslocated tyrosinase; WG, wheat germ; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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