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(Received for publication, December 16,
1994; and in revised form, January 6, 1995) From the
Melanosomes, the subcellular site of melanin synthesis and
deposition, may be related to the endolysosomal lineage of organelles.
To determine if melanosomes contain lysosomal hydrolases, we examined
the subcellular distribution of five of these enzymes in melanocytes
cultured from C57BL/6J mice. Analyses of Percoll gradient density
centrifugations demonstrated that Melanosomes are specialized, membrane-bound subcellular
organelles, unique to melanocytes, in which pigmented biopolymers
called melanins are synthesized and deposited. A relationship between
melanosomes and the endolysosomal lineage of organelles has long been
suspected. Cytochemical staining for the activity of acid phosphatase
demonstrated its presence within primary and compound
melanosomes(1, 2) . Melanosomes are accessible to
endocytosed receptor-bound melanocyte-stimulating hormone (3) as well as to phagosomes containing latex particles that
were previously introduced into the extracellular medium of cultured
melanocytes(4) . We have recently shown that a major
lysosomal integral membrane protein, lysosome-associated membrane
protein-1 (LAMP-1), ( We have now
combined cell biologic techniques with the use of genetic controls to
evaluate the presence of lysosomal hydrolases in melanosomes. Our
results demonstrate that melanosomes are the major repository of at
least five lysosomal hydrolases within melanocytes. Furthermore, we
have made the surprising observation that the activities of these
lysosomal hydrolases are elevated in melanocytes engaged in melanin
synthesis. Metabolic labeling studies suggest that these increased
levels are due in large part to an increased synthetic rate.
When the postnuclear supernatants of pigmented melanocytes
(melan-a cells) cultured from the skins of newborn, wild-type, C57BL/6J
mice were subjected to Percoll gradient density analysis, two peaks
(one dense, one light) of the lysosomal hydrolases
Figure 1:
Subcellular distribution of lysosomal
hydrolases in melanocytes. The subcellular distribution of five enzymes
with acidic pH optima was determined following fractionation of
disrupted murine melanocytes on Percoll density gradients (see
``Materials and Methods''). The bottom of each gradient
(densest fractions) is at the left. Upperpanels, distribution of enzymic activity in melan-a
cells, which actively synthesize melanin; lowerpanels, distribution of enzymic activity in melan-c
cells, which are genetically unable to synthesize melanin. Enzyme
activities: A,
Figure 2:
Subcellular distribution of lysosomal
hydrolases in melanocytes transfected with
v-ras
To explore the basis of the observed increase in
intracellular levels of these lysosomal hydrolases in pigmented
melanocytes, we subjected metabolically labeled cultured melanocytes to
pulse-chase analysis followed by immunoprecipitation with an antiserum
directed against the lysosomal hydrolase
Figure 3:
Biosynthesis of
The present study demonstrates that melanosomes are the major
repository of lysosomal hydrolases in melanocytes cultured from
C57BL/6J mice. Cells lacking melanized melanosomes due to mutations
that inhibit pigmentation such as the albino (c)
mutation or that are amelanotic because of repression of the expression
of the tyrosinase-related family of enzymes (due to transfection with
v-ras Our results suggest that melanosomes may
represent highly specialized members of the endolysosomal lineage of
organelles. It may be advantageous for the melanocyte to restrict
pigment deposition to a lysosome-like organelle. Nevertheless, the role
of lysosomal hydrolases within melanosomes remains obscure. Although
storage disorders due to the absence of individual hydrolases from
lysosomes are not generally associated with hypopigmentation,
diminished hair pigmentation may be a part of I-cell
disease(15) , in which mannose 6-phosphate residues are not
added to the glycosyl moieties of lysosomal hydrolases, resulting in
the failure of these enzymes to traffick to endosomes and lysosomes (16, 17) . It is also certainly possible that the
trafficking of lysosomal hydrolases to melanosomes proceeds mainly by a
pathway not dependent upon the mannose 6-phosphate receptor. Additional evidence linking melanosomes with lysosomes comes from
studies that demonstrate that the intramelanosomal pH is extremely
acidic(3, 18) . Acidification of intraorganellar pH is
believed to be a function of the presence of a vacuolar ATPase (and
related enzymes?), which imports H The relationship between
melanosomes and the endolysosomal lineage of organelles is further
supported by the existence of a group of coat color mutations in the
laboratory mouse, which also affect the secretion of kidney lysosomal
hydrolases into the urine(9) . A number of these mutations also
affect platelet aggregation and especially the presence of platelet
dense granules, suggesting that these organelles may also share
proteins in common with authentic lysosomes. We have previously
demonstrated the presence of the lysosomal membrane glycoprotein,
LAMP-1, in the delimiting membranes of
melanosomes(5, 6) . Subsequently, others have
corroborated this finding(20) . Furthermore, transfection of
fibroblasts with the gene encoding tyrosinase results not only in
measurable production of melanin within membrane-bound organelles but
also in the up-regulated expression of LAMP-1(20) . Whereas
melanosomes contain lysosomal membrane proteins and hydrolases, they
can be clearly distinguished from other organelles such as
peroxisomes(21) . Our results further establish a framework
by which those mutations that affect both melanosomes and lysosomes can
begin to be understood at a molecular level. In addition, they suggest
that melanocytes might respond to the intracellular synthesis of
melanin by up-regulating the synthesis of some (if not all) lysosomal
hydrolases. Future studies will undoubtedly uncover the signals that
direct proteins to melanosomes, and the cloning of the genes
responsible for melanolysosomal disorders will help elucidate the
pathogenesis of these fascinating diseases, which are characterized by
multiorganellar dysfunction.
Volume 270,
Number 9,
Issue of March 3, 1995 pp. 4213-4215
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-hexosaminidase,
-galactosidase, -glucuronidase, and cathepsins B and L all
co-sedimented with tyrosinase-rich densely sedimenting melanosomes. The
melanosomal distribution of these enzymes was confirmed in studies of
melanocytes cultured from albino mice and of melanocytes
rendered amelanotic by transfection with the
v-ras
oncogene (which lack
dense, melanized melanosomes). In these cells, only a less dense peak
of activity for each hydrolase was present. The level of each hydrolase
was elevated in black cells when compared with albino
cells. Metabolic labeling studies confirmed that the increase in
-glucuronidase in black versus albino
cells resulted mainly from increased synthesis of this enzyme. The data
suggest that melanosomes represent specialized lysosomes present within
melanocytes, that they contain a broad array of lysosomal hydrolases,
and that the levels of these hydrolases are elevated in cells actively
engaged in pigment production.
)is also present in the delimiting
membrane of melanosomes(5, 6) . Concurrently, studies
involving the transfection of cDNAs encoding tyrosinase and a related
protein, Trp-1, into fibroblasts (which lack authentic melanosomes)
demonstrated that the proteins encoded by the transfected genes
localized to intracellular vacuoles. These compartments stained
positively with antisera directed against LAMP-1 and
-glucuronidase, suggesting that the organelles to which these
proteins traffic in the absence of melanosomes represent either
lysosomes or late endosomes(7, 8) . Further support
for a relationship between melanosomes and lysosomes is provided by the
observation that mutations in at least 10 distinct genes in the
laboratory mouse result not only in a dilution of coat color but, also,
in an inhibition of the normally brisk secretion by kidney cells of
lysosomal hydrolases into the urine(9) . Similarly, a number of
autosomal recessive disorders in man, such as the Chediak-Higashi and
Hermansky-Pudlak syndromes, affect both melanosomes and lysosomes in a
variety of tissues (see (10) for review). If melanosomes and
lysosomes are indeed related, we reasoned that melanosomes might
contain an extensive array of lysosomal hydrolases.
Cell Culture
Pigmented melanocytes
cultured from C57BL/6J mice (melan-a cells(11) ), amelanotic
melanocytes cultured from mice homozygous for the spontaneously arising albino (c) mutation (melan-c cells)(11) , and
melan-a cells rendered amelanotic by transfection with the
v-ras
oncogene (melan-a-ras cells) (12) were obtained from Drs. D. Bennett (St. George's
Hospital, London, UK) and V. Hearing (NCI/NIH, Bethesda, MD) and
cultured as described(11, 12) .Subcellular Fractionation
Cultured
melanocytes were homogenized with a Dounce glass-glass homogenizer, and
postnuclear supernatants were prepared as described(5) . Equal
quantities of cellular protein were subjected to density fractionation
in self-forming 28% Percoll gradients(5) , and fractions were
collected from the bottom of each gradient.Enzymatic Assays
Following fractionation
on Percoll density gradients, fractions were either frozen directly for
assay of the tyrosine hydroxylase activity of tyrosinase (5) and immunologic determination of the presence of members of
the Trp family or were adjusted to pH 5.0 and assayed immediately for
the activities of lysosomal hydrolases. -Glucuronidase,
-hexosaminidase, and -galactosidase were assayed by
incubating 10-µl aliquots of each fraction with the respective
methylumbelliferone-conjugated substrate in 100 mM sodium
acetate buffer, 0.1% Triton X-100 at pH 4.5 for 15 min at 37
°C(13) . Cathepsins B and L were assayed with
Cbz-Arg-Arg-4MEC and Cbz-Phe-Arg-4MEC, respectively, in 100 mM sodium acetate buffer at pH 5.5 containing 1 mM EDTA and
2 mM dithiothreitol for 30 min at 37 °C(14) .
Assays were quantitated by fluorimetry with excitation at 350 nm and
emission at 460 nm using a Fluoroskan II (ICN) and results expressed as
fluorescent units generated under these incubation conditions.Immunoblotting
Fractions were tested for
the presence of tyrosinase protein by immunoblotting with a specific
antipeptide antiserum (obtained from Dr. V. Hearing, NCI/NIH) and
quantified by densitometry as described(5) .Immunoprecipitation
Following depletion
of cellular methionine/cysteine stores by culture for 1 h in medium
devoid of these amino acids, melanocytes were cultured for 1 h in the
presence of 200 µCi/ml methionine/cysteine
(Tran
S-label, Amersham, UK) (``pulse'') or were
radiolabeled and ``chased'' for an additional 4 h in
complete, nonradioactive medium. Cell pellets and conditioned media
were solubilized in 1% Triton X-100/phosphate-buffered saline with
protease inhibitors (1 µM pepstatin, 1 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride). The
pellets were normalized for trichloroacetic acid-precipitable counts of
incorporated [S]methionine from the pulse label
and subjected to immunoprecipitation with a rabbit polyclonal antiserum
to murine -glucuronidase obtained from Dr. Roger Ganschow
(Cincinnati, OH). Immune complexes were harvested with protein
A-Sepharose. Immunoprecipitates were solubilized with gel sample
buffer, separated on 10% SDS-polyacrylamide gel electrophoresis, and
equilibrated with ENHANCE (Amersham, UK) prior to exposure to a
PhosphorImager, as described (Molecular Devices, CA).
-glucuronidase,
-hexosaminidase, -galactosidase and cathepsins B and L were
observed (Fig. 1, A-E, upper panels). The dense
peak comigrated with the major peak of the key melanogenic enzyme
tyrosinase, a marker for melanized (Stage III-IV) melanosomes as we
have described previously (5) (Fig. 1F, upper panel). Analysis of melanocytes from albino
mice (melan-c cells) lacking dense Stage III-IV melanosomes due to a
point mutation inactivating the critical enzyme tyrosinase demonstrated
the presence of low levels of each hydrolase, limited to a lightly
sedimenting compartment (Fig. 1, lowerpanels). Results similar to those obtained with melan-c
cells were observed in homogenates of melan-a cells in which
melanogenesis was turned off due to transfection with the
v-ras oncogene (melan-a-ras) (Fig. 2, lowerpanels). As reported previously, these ras-transfected cells demonstrate decreased expression of
tyrosinase and the related protein Trp-1(12) . In both albino
and ras-transfected amelanotic cell types, the comigrating
dense peaks of -glucuronidase, -hexosaminidase, and
-galactosidase were absent ( Fig. 1and Fig. 2).
These results demonstrate the presence of lysosomal hydrolases within
the melanosomes of cultured murine melanocytes. A surprising
observation was the elevated level of these hydrolases in melan-a cells
when compared with lower levels in melan-a-ras cells and even lower
levels in melan-c cells. These results suggest that the levels of these
lysosomal hydrolases may be related to the ability of melanocytes to
synthesize melanin.
-hexosaminidase; B,
-galactosidase; C, -glucuronidase; D,
cathepsin B; E, cathepsin L. All activities are expressed as
fluorescent units of substrate hydrolyzed under conditions described
under ``Materials and Methods.'' In panelF, the distribution of tyrosinase is shown after
immunoblotting and quantitative densitometry (upperpanel, melan-a; lowerpanel,
melan-c).
. Melan-a cells and melan-a-ras
cells were subjected to subcellular fractionation as described in Fig. 1. The activities of -hexosaminidase and
-glucuronidase were assayed in each fraction from the Percoll
density gradients. Upperpanel, melan-a; lowerpanel, melan-a-ras. A, -hexosaminidase; B, -glucuronidase.
-glucuronidase.
Quantitation of biosynthesis of -glucuronidase was achieved by
scanning gel-resolved immunoprecipitates such as those shown in Fig. 3with a PhosphorImager. Two observations were made. First,
approximately three times as much radioactivity was found to be
incorporated into -glucuronidase by melan-a cells than by melan-c
cells during a 60-min pulse. Second, for melan-a cells, PhosphorImaging
revealed that 71% of the -glucuronidase labeled during the pulse
incubation could still be recovered from the cells after 4 h, while
approximately 8% of pulse-labeled enzyme was recovered from the culture
medium. By comparison, melan-c cells retained less newly synthesized
-glucuronidase (58% after 4 h) and secreted more of the enzyme
(24%). Taken together, these results show that after 4 h, melan-c cells
contain approximately 25% of the immunoprecipitable -glucuronidase
found in melan-a cells and that the elevated level of this enzyme in black melan-a cells compared with albino melan-c
cells is due mainly to increased synthesis of the enzyme.
-glucuronidase in
melanocytes. Melan-a and melan-c cells were biosynthetically labeled
for 1 h with [S]methionine as described under
``Materials and Methods.'' Samples of the labeled cells were
then chased for 4 h in complete medium, and -glucuronidase was
immunoprecipitated from the cell lysates and chase medium.
Immunoprecipitates were resolved by SDS-polyacrylamide gel
electrophoresis and visualized and quantitated by PhosphorImaging. Upperpanel: melan-a; lowerpanel:
melan-c. -Glucuronidase was immunoprecipitated from lysates of
cells pulsed (P) and chased (C) with
TranS-label and from chase medium (M). Each
immunoprecipitate with anti--glucuronidase antiserum (+) was
compared with a non-immune
control(-).
oncogene) lack the dense,
melanosomal peak of lysosomal hydrolases present in cells cultured from
wild-type C57BL/6J mice. More surprising is our observation that
melanized melanocytes express higher levels of certain lysosomal
hydrolases than do albino and ras-transfected
melanocytes not engaged in melanin synthesis. Whether the levels of
lysosomal hydrolases are truly coregulated with melanin synthesis
remains to be determined.
ions into
endolysosomal organelles(19) .
)
We thank Drs. D. Bennett, V. Hearing, and R. Ganschow
for providing cell lines and antisera, and J. Avins for preparation of
the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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