©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lysosomal Hydrolases Are Present in Melanosomes and Are Elevated in Melanizing Cells (*)

(Received for publication, December 16, 1994; and in revised form, January 6, 1995)

Stephanie Diment (1) Michael Eidelman (2) G. Marcela Rodriguez (1) Seth J. Orlow (2) (3)(§)

From the  (1)Department of Pathology, (2)Ronald O. Perelman Department of Dermatology, and (3)Department of Cell Biology, New York University School of Medicine, New York, New York 10016

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 beta-hexosaminidase, beta-galactosidase, beta-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 beta-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.


INTRODUCTION

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), (^1)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 beta-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.

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.


MATERIALS AND METHODS

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. beta-Glucuronidase, beta-hexosaminidase, and beta-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 (TranS-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 beta-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).


RESULTS

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 beta-glucuronidase, beta-hexosaminidase, beta-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 beta-glucuronidase, beta-hexosaminidase, and beta-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.


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, beta-hexosaminidase; B, beta-galactosidase; C, beta-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).




Figure 2: Subcellular distribution of lysosomal hydrolases in melanocytes transfected with v-ras. Melan-a cells and melan-a-ras cells were subjected to subcellular fractionation as described in Fig. 1. The activities of beta-hexosaminidase and beta-glucuronidase were assayed in each fraction from the Percoll density gradients. Upperpanel, melan-a; lowerpanel, melan-a-ras. A, beta-hexosaminidase; B, beta-glucuronidase.



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 beta-glucuronidase. Quantitation of biosynthesis of beta-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 beta-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 beta-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 beta-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 beta-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.


Figure 3: Biosynthesis of beta-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 beta-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. beta-Glucuronidase was immunoprecipitated from lysates of cells pulsed (P) and chased (C) with TranS-label and from chase medium (M). Each immunoprecipitate with anti-beta-glucuronidase antiserum (+) was compared with a non-immune control(-).




DISCUSSION

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 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.

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 ions into endolysosomal organelles(19) .

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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant AR41880 (to S. J. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Dermatology, Rm. H-100, NYU Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-5070; Fax: 212-263-8752.

(^1)
The abbreviations used are: LAMP-1, lysosome-associated membrane protein-1; Trp-1, tyrosinase-related protein; Cbz, benzyloxycarbonyl; 4MEC, 4-methylcoumarin.


ACKNOWLEDGEMENTS

We thank Drs. D. Bennett, V. Hearing, and R. Ganschow for providing cell lines and antisera, and J. Avins for preparation of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.