1 Pigment Cell Biology Section, Laboratory of Cell Biology, National Cancer
Institute, National Institutes of Health, Bethesda, MD 20892, USA
2 Pathology Section, National Heart, Lung and Blood Institute, National
Institutes of Health, Bethesda, MD 20892, USA
3 Department of Veterinary Pathobiology, Texas A and M University, College
Station, TX 77843, USA
* Author for correspondence (e-mail: hearingv{at}nih.gov)
Accepted 3 April 2003
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Summary |
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Key words: OCA4, Underwhite, Tyrosinase, Pigmentation, Albinism
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Introduction |
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OCA1 is an autosomal recessive disorder associated with deficient catalytic
function of tyrosinase, the rate-limiting enzyme in melanin biosynthesis,
which results from mutations of its encoding gene (TYR). Individuals
with OCA1 show a complete absence of pigment in their skin, eyes and hair;
this persists throughout life in the subtype OCA1A
(Spritz et al., 1990), whereas
patients with subtype OCA1B have some residual tyrosinase activity and thus
gradually accumulate minor amounts of pigment in their tissues
(Oetting and King, 1999
). OCA2
is the most common form of albinism; patients are usually born with minimal
pigmentation and become darker during adulthood, finally showing
minimal-to-moderate pigmentation of their skin, hair and eyes
(King, 1998
;
Manga and Orlow, 1999
). OCA3
is a rare form of OCA and patients are born with minimal pigmentation but
increase their pigmentation as they age
(Boissy et al., 1996
;
Manga et al., 1997
).
Studies using immortalized melanocytes derived from mice carrying mutations
in the genes responsible for OCA1 and OCA3 have shown that these disorders are
endoplasmic reticulum (ER) retention diseases
(Halaban et al., 2000;
Toyofuku et al., 2001a
;
Toyofuku et al., 2001b
),
wherein tyrosinase is abnormally targeted for digestion in proteasomes rather
than being transferred to the Golgi apparatus for further glycosylation. By
contrast, studies of immortalized p-mutant melanocytes (the model for OCA2)
have shown that tyrosinase in those cells is correctly processed through the
Golgi, but it is then secreted from the cells rather than being sorted to
melanosomes, thus showing that OCA2 is a hypopigmentary disease resulting from
altered intracellular trafficking of tyrosinase
(Manga et al., 2001
;
Chen et al., 2002
;
Toyofuku et al., 2002
). In
this context, investigations into the molecular basis of OCA4 are necessary to
classify its mechanism into one of the above types of diseases or into yet a
new distinct type.
In this study, we established primary melanocyte cultures from mice carrying the uw/uw mutation at the underwhite locus and from wild-type mice as a control. We show that in uw-mutant melanocytes, tyrosinase processing through the ER and Golgi is normal but that subsequent intracellular trafficking to melanosomes is aberrant and a significant amount of tyrosinase (and other melanogenic enzymes) is secreted from the cells in vesicles and immature melanosomes, rather than melanin being produced intracellularly in mature melanosomes. Thus, the molecular basis of OCA4 shows a mechanism similar to that involved in OCA2 but which occurs later in the melanosomal maturation pathway, and in this context our results provide new and important insights about the involvement of transporters in the normal physiology of melanocytes.
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Materials and Methods |
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Antibodies and enzymes
PEP1,
PEP7 and
PEP8 are rabbit antisera raised in our
laboratory against the carboxyl-terminal peptides of Tyrp1, Tyr and Dct
(DOPAchrome tautomerase), respectively, as previously described
(Jiménez et al., 1989
;
Tsukamoto et al., 1992
).
Anti-rabbit IgG horseradish peroxidase-linked (whole antibody) was from
Amersham Pharmacia Biotech (Piscataway, NJ), KDEL (SPA-827) polyclonal
antibody was purchased from StressGen (Victoria, BC, Canada) and HMB-45
monoclonal antibody (Schaumburg-Lever et
al., 1991
; Kikuchi et al.,
1996
) was purchased from DAKO (Carpinteria, CA). Normal horse
serum, normal goat serum, Texas-red anti-rabbit IgG (H+L) and fluorescein
isothiocyanate anti-mouse IgG (H+L) were all from Vector (Burlingame, CA).
Endoglycosidase H (EndoH) [EC 3.2.1.96,
glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine
1,4-N-acetyl-ß-glucosaminohydrolase] and
peptide:N-glycosidase F (PNGaseF) [EC 3.5.15.2,
N-linked-glycopeptide-(N-acetyl-ß-D-glucosaminyl)-L-asparagine
amidohydrolase] were from New England Biolabs (Beverly, MA).
Cellular fractionation
Melanocytes were harvested with trypsin/EDTA and were washed three times
with cold PBS without CaCl2 and MgCl2. Cells were
solubilized in extraction buffer [1% Nonidet P-40 in PBS containing a protease
inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany)] for 1 hour on ice
with occasional vortexing. The samples were centrifuged at 14,000
g for 30 minutes at 4°C and the supernatants were
recovered and kept at 70°C for further experiments. Protein
concentrations were determined with the BCA assay kit (Pierce, Rockford, IL)
using bovine serum albumin as a standard.
To collect vesicles secreted by melanocytes, medium collected from cells cultured for one week was centrifuged at 14,000 g for 30 minutes at 4°C. The precipitates were solubilized in the extraction buffer and were stored as described above.
To isolate the melanosome-enriched fraction, we used a method previously
described (Valverde et al.,
1992). Briefly, the cells were harvested, washed three times with
PBS without CaCl2 and MgCl2 and lysed with 10 mM
phosphate buffer, pH 7.2, containing 0.25 M sucrose. A postnuclear supernatant
was obtained by centrifugation at 700 g for 15 minutes at
4°C and was then further fractionated by centrifugation at 14,000
g for 30 minutes at 4°C to yield the melanosome-enriched
pellet.
Metabolic labeling
Metabolic labeling and immunoprecipitation experiments were performed as
previously reported (Kobayashi et al.,
1994). Briefly, melanocytes were cultured in 6-well tissue culture
plates for 48 hours before labeling. The cells were then incubated in
methionine- and cysteine-free Dulbecco's Modified Eagle Medium (GIBCO) for 30
minutes at 37°C in a humidified incubator with 5% CO2, and were
then labeled for 1 hour with 0.5 mCi of [35S]-Methionine and
Cysteine Mixture (Redivue Promix, Amersham Biosciences). Following the 1 hour
labeling period, the isotope-supplemented medium was removed and complete
medium containing 1 mM unlabeled methionine was added. Media were then
collected and the cells were harvested by scraping immediately and after 3 or
24 hours of chase. The cells were washed three times with PBS at 4°C and
then solubilized for 1 hour at 4°C in lysis buffer (50 mM Tris-HCl, pH
7.4, 150 mM NaCl containing 1% Nonidet P-40, 0,01% SDS and protease inhibitor
cocktail). [35S]-labeled cell lysates were precleared with 20 µl
Protein-G Sepharose 4 Fast Flow (Amersham) for 2 hours at 4°C with mixing.
The supernatants were collected following centrifugation at 5000
g for 1 minute at 4°C and then incubated with
PEP1
or
PEP7 for 2 hours at 4°C with mixing, as were the media collected
at each of the chase periods. The immune complexes were separated by
incubation with 20 µl Protein-G Sepharose 4 Fast Flow for 2 hours at
4°C and were further washed three times by centrifugation with the lysis
buffer. The final pellets were resuspended in sample buffer, heated at
100°C for 5 minutes and centrifuged. The samples were separated on 8%
SDS-PAGE (sodium dodecyl sulphatepolyacrylamide electrophoresis) gels. The
dried gels were exposed in a storage phosphor screen, scanned with a STORM
Phosphor Imager (Molecular Dynamics, Sunnyvale, CA) and then analyzed with the
Image Quant program.
Western blotting
For western blotting, samples were separated by electrophoresis under
reducing conditions, as previously described
(Negroiu et al., 1999).
Briefly, lysates were mixed 1:2 (v/v) with sample buffer (BioRad, Hercules,
CA) containing 2-mercaptoethanol and were boiled for 10 minutes. Samples were
separated by PAGE in SDS (8% Tris-glycine gels, Invitrogen) and transferred to
polyvinylidene difluoride membranes (Millipore, Bedford, MA). Blots were
blocked in 5% non-fat milk in PBS for 1 hour at room temperature and were then
incubated with the appropriate first antibodies diluted 1:1000 in 1% non-fat
milk in PBS, for 1 hour at room temperature. After six washes (10 minutes
each) with 0.05% Tween 20 (Bio-Rad) in PBS, the blots were incubated in
anti-rabbit horseradish peroxidase-linked whole antibody (1:1500) (Amersham)
in 1% non-fat milk in PBS for 1 hour at room temperature. After three further
washes (10 minutes each) with 0.05% Tween 20 in PBS, the immunoreactivity of
the blots was detected using an Enhanced ChemiLuminescence western blotting
detection kit (Amersham), according to the manufacturer's instructions.
EndoH and PNGaseF digestions
Samples to be digested were denatured in the EndoH- or PNGaseF-denaturing
buffer for 5 minutes at 100°C, cooled and then mixed with 1/10
concentrated EndoH (0.5 M sodium citrate, pH 5.5) or PNGaseF [0.5 M sodium
phosphate, pH 7.5 containing 10% (v/v) Nonidet P40] reaction buffers as
previously described (Negroiu et al.,
1999). Samples were then digested with 500 units of EndoH or
PNGaseF, respectively, for 3 hours at 37°C and analyzed by PAGE.
Melanogenic assays
Tyrosinase enzymatic activity was measured as described previously
(Hearing and Ekel, 1976).
Briefly, 30 µl of each sample to be tested were added to 10 µl phosphate
buffer containing ß-3,4-dihydroxyphenylalanine (DOPA) as cofactor and 10
µl 0.25 mCi/mmol [U-14C]-tyrosine (NEN, Boston, MA). The
reactions (usually performed in duplicate or triplicate) were mixed and
incubated at 37°C for 3 hours. 40 µl of each sample were transferred to
Whatman 3MM filter paper discs and air dried. Each filter was then washed
three times in 0.1 N HCl, twice in 95% ethanol, once in acetone and finally
air-dried. Radioactive melanin retained by the filters was determined in a
Beckmann scintillation counter. Tyrosinase activity is defined as pmol
[14C]-tyrosine incorporated into melanin/µg protein/hour at
37°C.
The melanin content was measured using a modification of a previously
reported method (Siegrist and Eberle,
1986). Briefly, melanocytes were cultured until they became
confluent. They were solubilized in lysis buffer containing the protease
inhibitor cocktail and protein concentrations were measured. Melanin pellets
were dissolved by incubation in 1 N NaOH at 37°C for 18 hours. Aliquots of
each sample were transferred to 96-well plates and were quantitated by
absorbance at 405 nm using an automatic microplate reader (Molecular Devices,
Sunnyvale, CA) and calibrated against a standard curve generated using
synthetic melanin (Sigma).
Immunohistochemical staining
Dual labeling, using immunofluorescence methods and laser scanning confocal
fluorescence microscopy, was used to evaluate the localization of melanogenic
proteins in wild-type and in uw/uw melanocytes, as previously
reported (Kushimoto et al.,
2001). The following antibody dilutions were used:
PEP1,
1:40;
PEP7, 1:40;
PEP8, 1:40; KDEL, 1:10; HMB-45, 1:10.
Melanocytes were plated in 2-well Lab-Tek chamber glass slides (Nalge,
Naperville, IL) 24-72 hours before each experiment and were fixed in 4%
paraformaldehyde for 15 minutes at 4°C. Cells were subsequently
permeabilized with methanol for 20 minutes at 4°C and then with 1% Triton
X-100 for 3 minutes at room temperature (in experiments using the KDEL
antibody); all subsequent processing was done at room temperature. After
washing in PBS, cells were incubated in PBS containing 5% normal serum (normal
horse serum for monoclonal antibodies and normal goat serum for polyclonal
antibodies) for 1 hour as a blocking step. The cells were then incubated
overnight at 4°C with the appropriate first antibody diluted in 2% normal
serum. This step was followed by incubation with the appropriate secondary
antibody (dilution, 1:100) for 1 hour. Nuclei were counterstained with
4',6'-diamidino-2-phenylindole (DAPI) (Vector blue
fluorescence) for 15 minutes at room temperature. Immunoreactive cells were
classified into three categories according to whether they showed green, red
or yellow fluorescence (the latter color indicating colocalization of the red
and green signals). All preparations were examined with a Model TCS4D, DMIRBE
confocal microscope (Leica, Heidelberg, Germany) equipped with argon and
argon-krypton laser sources. Controls included sections stained as detailed
above, but omitting the first antibody.
Electron microscopy
Confluent flasks of wild-type and of uw-mutant melanocytes were fixed with
2% glutaraldehyde for 24 hours at 4°C, collected by centrifugation and
then washed twice with cold PBS. Further, analysis of uw-mutant melanocytes
was performed using the DOPA reaction, an enzyme histochemical method to
detect tyrosinase activity. For DOPA staining, melanocytes were incubated for
3 hours at 37°C in 0.1% L-DOPA in 0.1 M phosphate buffer, pH 7.4, and
fixed in 2% glutaraldehyde as above. All samples were post-fixed in 1% osmium
tetroxide in 0.1 M phosphate buffer, pH 7.2, for 1 hour, dehydrated through a
graded ethanol series and embedded in PolyBed 812 at 60°C for 48 hours.
Ultrathin sections were cut and stained with uranyl acetate and were then
examined in a JEOL JEM-1200 EX electron microscope.
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Results |
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The pattern of melanin present in the extracellular medium was similar to
but distinct from that previously shown for OCA2 p-mutant melanocytes
(Potterf et al., 1998). In
OCA2 p-mutant melanocytes, active tyrosinase was secreted into the medium and
the melanin produced in the medium was of very low molecular weight and could
not be sedimented by centrifugation. By contrast, the pigment present in the
medium of uw-mutant melanocytes was in large particles
(Fig. 1F) that could be readily
sedimented by centrifugation (Fig.
1H). We next characterized the nature of tyrosinase synthesis and
processing to elucidate the intracellular mechanism(s) responsible for the
hypopigmentation of uw-mutant melanocytes.
Subcellular distribution of tyrosinase activity and melanin in
wild-type and uw-mutant melanocytes
Wild-type and uw-mutant melanocytes were cultured until they became
confluent, and they were then fractionated and solubilized in lysis buffer.
Tyrosinase activity was measured in the cell extracts and also in the
melanosome and secreted vesicle fractions
(Table 1). In wild-type
melanocytes, the specific activity of tyrosinase in melanosomes was
approximately ninefold higher than in the cell extract, which is the normal
distribution pattern for tyrosinase trafficking. Tyrosinase activity in the
vesicle fraction secreted by wild-type melanocytes was very low and
represented less than 1% of the activity seen in the total extract. By
contrast, tyrosinase activity in extracts of uw-mutant melanocytes was
significantly lower (about fivefold lower) than that found in wild-type
melanocytes. Tyrosinase activity in melanosomes of uw-mutant melanocytes was
only threefold higher than that found in the total cell extract, and more than
5% of the enzyme activity was present in the secreted vesicle fraction. The
total melanin content in uw-mutant melanocytes is negligible compared with
wild-type melanocytes (Table
1), which is consistent with the report of Lehman et al.
(Lehman et al., 2000).
|
Synthesis and processing of melanogenic proteins in uw-mutant and
wild-type melanocytes
Thus, the melanin content of uw-mutant OCA4 melanocytes is consistent with
their hypopigmented phenotype and also with the reduced levels of tyrosinase
and melanin found in OCA1, OCA2 and OCA3 melanocytes. To further study the
mechanism behind that reduction, we used metabolic labeling and western
immunoblotting to examine the synthesis and processing of tyrosinase in
uw-mutant compared with wild-type melanocytes. As shown in
Fig. 2, the amount of
tyrosinase synthesized (at 0 chase time) by wild-type and by uw-mutant
melanocytes was virtually identical, showing that there was no defect in
tyrosinase gene transcription or translation, as expected. Quantitation of the
64 kDa band representing the newly synthesized tyrosinase revealed that the
amount of tyrosinase synthesized by uw-mutant melanocytes represents 90%
the level produced by wild-type melanocytes (n=4 independent
experiments). Tyrosinase is synthesized as a 55 kDa polypeptide, which almost
instantly undergoes early glycosylation events in the ER and is seen as a 64
kDa band. The fully glycosylated mature form of the enzyme is 68 kDa in size.
In fact, the synthesis and processing of tyrosinase from the immature form to
the mature form was complete within 3 hours in wild-type and in uw-mutant
melanocytes and was strikingly similar at that time. However, the stability of
tyrosinase was markedly different, with virtually 100% of the tyrosinase
remaining in wild-type melanocytes after 24 hours, but very little remaining
in uw-mutant cells. For comparison, the pattern for OCA1 melanocytes is shown,
wherein tyrosinase was synthesized at the same rate as in wild-type and in
uw-mutant melanocytes. However, tyrosinase was not processed to the mature
form correctly and was then quickly degraded, as previously reported
(Toyofuku et al., 2001b
).
|
Similar patterns of Tyrp1 synthesis and processing are shown for comparison, and no significant differences in Tyrp1 labeling were seen between the wild-type and OCA4 (or OCA1) melanocytes. The synthesis, processing and stability of Dct was also virtually identical in all three types of melanocytes (data not shown).
Western blot analysis revealed that tyrosinase was detectable predominantly in the mature 68 kDa form in the whole cell extract and only in the mature form in melanosomes of wild-type melanocytes, and none was detected in the secreted vesicle fraction (Fig. 3). By contrast, tyrosinase was detectable not only in the 68 kDa form in the cell extract and in melanosomes of uw-mutant melanocytes but also in the secreted vesicle fraction. Interestingly, Tyrp1, although processed correctly and found in melanosomes of uw-mutant melanocytes, was also present in abundance in the secreted vesicle fraction of those uw-mutant cells but was not found in the secreted vesicle fraction of wild-type melanocytes. Similar patterns of Dct distribution were seen in those vesicles (not shown).
|
Subcellular trafficking of tyrosinase in OCA4 melanocytes
To examine more closely the mechanism(s) by which tyrosinase is aberrantly
secreted from the uw-mutant melanocytes, we assessed the sensitivity of
tyrosinase to two different glycosidases. EndoH is an enzyme that removes high
mannose-type carbohydrates from N-linked glycoproteins, a conversion
that occurs in the medial Golgi region. When proteins are correctly processed
through the ER and Golgi, they become resistant to EndoH, and sensitivity to
EndoH indicates the presence of proteins that have not yet been processed
beyond the ER. By contrast, PNGaseF removes all carbohydrate residues and
reveals the native polypeptide size of the protein. When digested with EndoH
(Fig. 3), the mature 68 kDa
form of tyrosinase from the cell extract of uw-mutant melanocytes was almost
completely resistant to EndoH but was completely digested with PNGaseF,
showing a pattern virtually identical to that of wild-type melanocytes.
Western blot analysis of tyrosinase in melanosomes of wild-type and uw-mutant melanocytes revealed that the mature 68 kDa band was EndoH resistant, as expected, whereas digestion with PNGaseF generated a single strong 55 kDa band representing the fully deglycosylated form. Similarly, tyrosinase in the secreted vesicle fraction was resistant to EndoH but could be fully digested to the 55 kDa nascent polypeptide by PNGaseF. Thus, tyrosinase found in melanosomes and in vesicles secreted from uw-mutant melanocytes is EndoH resistant, showing that it has been processed correctly through the ER. Similar digestions of Tyrp1 (Fig. 3) and Dct (data not shown) showed comparable processing patterns.
Taken together, the sum of these results suggests that tyrosinase (and Tyrp1 and Dct) are processed correctly through the ER and Golgi in uw-mutant melanocytes but that a significant portion of those melanogenic enzymes are secreted from uw-mutant melanocytes. Thus, the subcellular distribution and enzyme functions of melanosomal proteins are distinctly affected by this mutation in the uw gene.
Subcellular distribution of melanogenic proteins in uw-mutant
melanocytes
To investigate the subcellular localization of melanogenic proteins in
uw-mutant OCA4 melanocytes, we used immunohistochemical staining to compare
their distribution with HMB-45, a marker for early (stage II) melanosomes
(Schaumburg-Lever et al.,
1991; Kushimoto et al.,
2001
) and KDEL, a marker for the ER
(Fig. 4). Tyrosinase, Tyrp1 and
Dct were detected by red fluorescence, and HMB-45 and KDEL were detected by
green fluorescence; in the merged images, yellow indicates colocalization of
the two signals.
|
In wild-type primary melanocytes, the majority of tyrosinase was found in
granules distributed throughout the cytoplasm (mainly in the dendrites), as
well as in the perinuclear area where the ER and stage I melanosomes are
found. Some tyrosinase colocalized with the ER marker KDEL but most of it was
in the particulate structures. Similar staining patterns were observed for
Tyrp1 and for Dct in those wild-type melanocytes. These data confirm that the
melanogenic proteins are processed in the ER and are then distributed
primarily in melanosomes of wild-type primary melanocytes according to their
expression level, as previously shown for immortalized melanocytes
(Toyofuku et al., 2001b;
Toyofuku et al., 2002
).
Confocal microscopy of uw-mutant melanocytes showed dramatic differences compared with wild-type melanocytes. As noted above, these cells were more dendritic and grew to much larger sizes than wild-type melanocytes, and this was particularly evident when they were cultured on glass. All three melanogenic proteins (Tyr, Tyrp1 and Dct) were distributed throughout the cytoplasm of uw-mutant melanocytes and accumulated in the perinuclear area, as in wild-type melanocytes. Colocalization of tyrosinase, Tyrp1 and Dct with the ER marker KDEL indicates that the melanogenic proteins reach the ER compartment in uw-mutant melanocytes. However, in contrast to wild-type melanocytes, there was little co-staining of tyrosinase with HMB-45, suggesting a trafficking defect later in the secretory pathway, before delivery of tyrosinase to the melanosomal compartment. Tyrp1 (and to a lesser extent Dct) was similarly disrupted from reaching the melanosomal compartment, and this was especially apparent at the periphery of the cells and in the dendrites, where they did not reach early stage II melanosomes (stained green with HMB-45).
Ultrastructural characterization of melanocytes and melanosomes
Electron microscopy revealed that melanocytes derived from wild-type mice
contain numerous highly melanized melanosomes (stages III and IV) with a very
well-organized structure (Fig.
5A). By contrast, uw-mutant melanocytes contain swollen, poorly
melanized melanosomes which show an abnormal arrangement of their internal
matrices, with the majority of them being at stages I or II
(Fig. 5B). DOPA reactivity of
uw-mutant melanocytes showed DOPA-positive reactions in some of the stage I or
II melanosomes, which is evidence that at least some active tyrosinase was
correctly delivered there (Fig.
5C). Ultrastructural analysis of the vesicles shed by uw-mutant
cells revealed the presence of stage I and II melanosomes exhibiting small
amounts of melanin and disorganized structures of their internal fibers
(Fig. 5D).
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Discussion |
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The biogenesis of melanosomes has gradually been elucidated, and the
earliest form of that organelle is the stage I melanosome, which is positive
for staining of gp100 with PEP13
(Kushimoto et al., 2001
). The
TRPs are not delivered to stage I melanosomes, but rather arrive there only
after the maturation of stage I to stage II melanosomes, which is linked to
the proteolytic cleavage of gp100 (which generates the internal fibrillar
matrix). Following that cleavage, gp100 undergoes a conformational change that
allows it to be recognized by the monoclonal antibody HMB45, which thus serves
as a specific probe for identifying stage II melanosomes
(Kushimoto et al., 2001
).
According to our results, uw-mutant cells express normal levels of
tyrosinase, which is, in part, delivered to and is active in melanosomes. At
least part of the active, correctly processed tyrosinase remains inside the
melanocytes and contributes to the low levels of melanin produced, thus
explaining the reduced but significant pigmentation seen in uw-mutant
melanocytes. However, uw-mutant melanocytes secrete into the medium dark
vesicles that contain tyrosinase, Tyrp1 and Dct. Although metabolic labeling
revealed that the amount of tyrosinase synthesized in wild-type and uw-mutant
melanocytes is approximately the same, tyrosinase activity in uw-mutant
melanocytes is reduced to about 20% of the level found in wild-type
melanocytes due to its misrouting along the secretory pathway. One can see
using confocal immunohistochemistry that while some of the tyrosinase in
uw-mutant cells reaches stage II (HMB45-positive) melanosomes, most of it does
not, thus leading to the conclusion that the uw-encoded protein (MATP)
functions in the sorting of tyrosinase (and Tyrp1) from the trans-Golgi
network to stage II melanosomes. Two relevant observations should be noted
about MATP in this regard. First, MATP obviously functions to sort only
melanocyte-specific proteins because if other common proteins were similarly
affected, the phenotype would be that of Hermansky-Pudlak syndrome rather than
of OCA (Sarangarajan et al.,
2001; Huizing et al.,
2002
). Second, a proteomic analysis of stage II melanosomes
recently published by our group (Basrur et
al., 2003
) identified all the early melanosome-specific proteins,
but MATP (and the P protein for that matter) was conspicuous by its absence,
which suggests that both of those transport proteins function in the sorting
pathway before melanosomes. Because only tyrosinase is secreted from OCA2
melanocytes, we hypothesize that the function of the P protein in the sorting
pathway precedes that of MATP, as depicted in
Fig. 6.
|
The uw-mutant melanocytes displayed a minimally pigmented melanosome
population at stages I and II; these had irregular shapes and the internal
fibers were not well organized (Fig.
5). Similar types of early immature melanosomes were seen in the
secreted vesicle fraction. DOPA staining of uw-mutant melanocytes showed that
active tyrosinase is localized in some of the early melanosomes of those
cells. This ultrastructural phenotype of uw-mutant melanocytes is in good
agreement with an earlier study (Lehman et
al., 2000), which reported similar features of melanosomes in
cultured melanocytes derived from mice carrying the uwd
(underwhite dense) mutation. The reduced melanin content and the irregular
shape of melanosomes in melanocytes derived from mice carrying different
mutations of the uw gene have been observed by others
(Sweet et al., 1998
;
Lehman et al., 2000
) and could
also be seen in the eyes of those mice. This phenotype is strikingly similar
to that previously reported for OCA2 melanocytes carrying mutations in the
p gene (Puri et al.,
2000
).
In conclusion, we show in this study that MATP plays a crucial role in modulating the processing and intracellular trafficking of tyrosinase and other melanosomal proteins. The uw mutation is associated with a disruption of this pathway, which results in the misrouting and abnormal release of tyrosinase and Tyrp1 into the medium. Because tyrosinase is the key rate-limiting enzyme of melanogenesis, this results in the hypopigmented phenotype of uw-mutant melanocytes. The similarity of functions of the P protein and MATP, both being predicted transport proteins, in the trafficking of melanosomal proteins, provides valuable clues as to the roles of such proteins in regulating mammalian pigmentation.
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Acknowledgments |
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