(Received for publication, August 18, 1995; and in revised form, October 17, 1995)
From the
In this study, we identify a transport system for tyrosine, the
initial precursor of melanin synthesis, in the melanosomes of murine
melanocytes. Melanosomes preloaded with tyrosine demonstrated
countertransport of 10 µM [H]tyrosine, indicating carrier-mediated
transport. Melanosomal tyrosine transport was saturable, with an
apparent K
for tyrosine transport of 54
µM and a maximal velocity of 15 pmol of tyrosine/unit of
hexosaminidase/min. Transport was temperature-dependent (E
= 7.5 kcal/mol) and showed
stereospecificity for the L-isomer of tyrosine. Aromatic,
neutral hydrophobic compounds (such as tryptophan and phenylalanine),
as well as the small, bulky neutral amino acids (such as leucine,
isoleucine, and methionine) competed for tyrosine transport. Tyrosine
transport was inhibited by the classical system L analogue,
2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and by
monoiodotyrosine, but not by cystine, lysine, glutamic acid, or
2-(methylamino)-isobutyric acid. Tyrosine transport showed no
dependence on Na
or K
, and did not
require an acidic environment or the availability of free thiols. These
results demonstrate the existence of a neutral amino acid carrier in
murine melanocyte melanosomes which resembles the rat thyroid FRTL-5
lysosomal system h. This transport system is critical to the
function of the melanosome since tyrosine is the essential substrate
required for the synthesis of the pigment melanin.
Pigmentation in mammals occurs within melanocytes and results
from the deposition of melanin pigment in cells of the skin, hair, and
eye (1, 2) . Some important biological functions of
melanin include protective coloration(3) , shielding from
ionizing radiation(4) , trapping of toxic
metabolites(4) , sexual attraction within species (3) ,
and proper neurological (ocular) development(5) . Melanin
synthesis involves a complex series of biochemical reactions that are
restricted to melanocyte-specific, membrane-bound organelles termed
melanosomes(1, 2, 3, 4) . The
initial, rate-limiting step in melanogenesis is the hydroxylation of
tyrosine to 3,4-dihydroxyphenylalanine (DOPA) ()by the
enzyme tyrosinase (monophenyl monooxidase, EC 1.14.18.1). Subsequent
metabolism of DOPA and its derivatives by various melanocyte-specific
enzymes, including tyrosinase, tyrosinase-related protein-1 (TRP-1),
and tyrosinase-related protein-2 (TRP-2) results in the synthesis of
eumelanin, a brown-black pigment(3) . The synthesis of
phaeomelanins (yellow to red pigments) is poorly understood, but
involves the production of cysteinyldopa conjugates(6) , also
following the production of DOPA from tyrosine. With complete
pigmentation, mature melanosomes are extruded from dendritic processes
of the melanocyte and taken up by neighboring
keratinocytes(4) .
Since melanin production requires tyrosine, some mechanism, such as transmembrane import, must supply this amino acid to the melanosome and may be a critical element limiting pigment synthesis. We have previously characterized the transport of various ligands across the membranes of lysosomes, which are intracellular vesicles closely resembling early stages of melanosomes. Carrier-mediated lysosomal membrane transport systems are known to exist for cystine (7, 8) and sialic acid(9) , and a tyrosine/monoiodotyrosine transporter effecting iodine salvage has been characterized in the lysosomes of rat FRTL-5 thyroid cells(10) . In view of these findings, it has been proposed that a tyrosine transport system must exist within the melanosomal membrane (11) and, furthermore, it has been hypothesized that this carrier may be defective in the specific disorder known as oculocutaneous albinism type II(5) . However, recent evidence from our laboratories, using cultured melanocytes from the murine model of oculocutaneous albinism type II, the pink-eyed dilution mouse(12) , has revealed no significant decrease in plasma or melanosomal membrane tyrosine transport attendant to this mutation(13) .
In this study, we have identified a neutral amino acid transporter in murine melanocytes that is responsible for melanosomal tyrosine transport. We have characterized the carrier's ligand specificity, energy of activation, trans stimulation, pH optimum, ion effects, among other parameters. In view of the requirement for tyrosine within the melanosome to facilitate pigment production, this transporter may serve a critical function in regulating melanogenesis.
To
differentiate between true uptake and nonspecific binding, granular
fractions were sonicated and incubated with 10 µM [H]tyrosine in 0.25 M sucrose, 10
mM HEPES for 1 min at 37 °C. Negligible (i.e. less than 8% of control) [
H]tyrosine uptake
was detectable under these conditions. In addition, incubation of
granular fractions on ice yielded 0-10% of the activity measured
at 37 °C.
Competition for 10 µM [H]tyrosine uptake by various ligands was
determined by adding a competing ligand at the appropriate
concentration to the incubation mixture prior to adding the
melanosomes. With each group of competition experiments, controls with
no competing ligand were simultaneously performed. Tyrosine transport
in the presence of each competing ligand was then expressed as a
percent of control tyrosine uptake.
For experiments involving ionophores and various cations, carbonyl cyanide m-chlorophenylhydrazone (CCCP), nigericin, or monensin were dissolved in acetone, placed into empty incubation tubes, and the acetone was evaporated before the incubation mixture was added; an equal volume of acetone was evaporated from the control tube.
Centrifugation of a postnuclear supernatant derived from normal murine melanocytes yielded a pigmented granular fraction highly enriched in melanosomes, as indicated by their pigmentation and elliptical shape (Fig. 1, A and B), as well as by the presence of tyrosinase activity (data not shown). The lysosomal enzyme hexosaminidase (8, 16) was also present in this fraction, an activity that has been previously shown to co-purify with melanosomes(17, 18) . Fragmented mitochondrial membranes were the major contaminant, but no lysosomal structures were seen in this granular fraction, which was subsequently examined for tyrosine transport activity.
Figure 1:
Ultrastructure of melanosomes purified
from melan-a melanocytes. A and B, granular fraction
preparations 9,324 (A) and
18,500 (B). C and D, sucrose gradient
1.8 M fraction
preparations
11,840 (C) and
18,500 (D).
Melanosomes are elliptical in shape, and appear as highly
electron-dense organelles in different stages of maturation
(pigmentation), indicative of melanin deposition. Bars represent 1 µm.
At pH 7.0 and 37 °C,
[H]tyrosine uptake was linear for at least 1 min
of incubation (data not shown), allowing determination of an initial
velocity of tyrosine uptake. This rate increased linearly with tyrosine
concentration and then leveled off, indicating saturation kinetics (Fig. 2). The apparent K
for melanosomal
tyrosine uptake at pH 7.0 and 37 °C was 54 µM, with a V
of 15 pmol of tyrosine/unit of
hexosaminidase/min. An Arrhenius plot at pH 7.0 yielded an energy of
activation of 7.5 kcal/mol and a Q
of 1.5 (Fig. 3), which is consistent with carrier-mediated
translocation of tyrosine. The initial velocity of tyrosine uptake into
melanosomes also varied with extramelanosomal pH (Fig. 4).
Uptake was maximal at pH 6.5, 20% higher than at pH 7.0, with the
extremes of the pH range (6.0-7.5) showing decreased transport.
Figure 2:
Initial velocity of tyrosine uptake into
melanosomes as a function of tyrosine concentration. Melanosomes
(granular fractions) were added to mixtures of 0.25 M sucrose,
10 mM HEPES and different concentrations of
[H]tyrosine at 37 °C. Aliquots were removed
(zero time), diluted into ice-cold 0.25 M sucrose, 10 mM HEPES and collected onto glass fiber filters. After 1 min at 37
°C, further aliquots were removed and treated similarly. Results
plotted represent the differences between 1 min and 0 time points,
converted to picomole of tyrosine (based upon the specific
radioactivity of tyrosine) and are the means of triplicate
determinations at each concentration performed on different
days.
Figure 3:
Arrhenius plot for
[H]tyrosine uptake by melanosomes. Melanosomes
(granular fractions) were added to 0.25 M sucrose, 10 mM HEPES medium containing 10 µM [
H]tyrosine at 37, 30, or 25 °C for 1
min. The initial velocity of [
H]tyrosine uptake
per unit of hexosaminidase was plotted as a logarithmic function of
reciprocal temperature in degrees Kelvin.
Figure 4:
pH curve for tyrosine uptake by
melanosomes. Buffers were either 50 mM MES (pH 6.0-6.8)
or 50 mM HEPES (pH 7.0-7.5). Uptake rate at each pH was
determined relative to the rate at pH 7.0 (100%). Points represent
means of 3-5 determinations. The mean rate of 10 µM [H]tyrosine uptake at pH 7.0 was 19.5
± 5 pmol/min/mg protein or 1.0 ± 0.2
pmol/min/hexosaminidase unit (n =
5).
The structural requirements for ligand recognition by the tyrosine
transporter were assessed by measuring competition of various amino
acids and analogues with 10 µM [H]tyrosine for melanosomal uptake. The
murine tyrosine transporter resembled the lysosomal system h, first described in rat FRTL-5 thyroid cell lysosomes(10) ,
in its pattern of ligand specificity (Table 1). It showed
stereospecificity for the L-stereoisomer of tyrosine, with
minimal competition by D-tyrosine. D-Tryptophan also
competed poorly compared to L-tryptophan. Both the murine
melanosomal and rat lysosomal carriers also showed the greatest
affinity for large, hydrophobic aromatic compounds (i.e. tyrosine, tryptophan, and phenylalanine), as well as bulky neutral
amino acids such as leucine and isoleucine. In addition, competition
for tyrosine uptake was apparent using the classic system L analogue,
BCH(20) . The melanosomal carrier also had a high affinity for
monoiodotyrosine, which is transported by the thyroid cell lysosomal
transporter(19) . 3-(p-Hydroxyphenyl)-propionic acid,
which lacks the amino group of tyrosine, did not compete with
[
H]tyrosine for uptake, indicating that the
-amino acid configuration is necessary for ligand recognition.
Removal of the
-carboxyl, as in tyramine, also resulted in lack of
competition. Addition of a methylene group to the aliphatic chain
(homophenylalanine) had an inhibitory effect on tyrosine uptake similar
to that of phenylalanine, suggesting that the length of the aliphatic
chain is not a strict requirement for recognition. Adding substituents
on the aromatic ring which confer less hydrophobicity also reduced
competition compared to phenylalanine, as demonstrated by DOPA. DOPA, a
melanin precursor and also a substrate for tyrosinase, inhibited
tyrosine uptake by approximately 80%. However, other potential
competitors examined, including L-cystine, L-alanine, L-glutamate, and L-lysine, had little or no
competitive nature toward this transporter.
Melanosomal tyrosine transport was not affected by the presence of 20 mM ammonium chloride, 5 mMN-ethylmaleimide, or by high concentrations of sodium or potassium (Table 2). CCCP and monensin (in the presence or absence of KCl or NaCl, respectively) failed to affect transport, while treatment of melanosomes with the ionophore nigericin in the presence or absence of KCl resulted in a modest decrease in tyrosine transport.
In order to address the
possible contribution of contaminating lysosomes to the measured
tyrosine transport by melanosomes in the granular fraction, further
purification on a discontinuous sucrose gradient was performed,
yielding highly purified melanosomes at density 1.8 M (Fig. 1, C and D). Over 90% of the
organelles in these preparations were estimated visually to be
melanosomes and no lysosomal structures were seen in these fractions (i.e. with density
1.8 M). When comparable
subcellular fractions derived from non-pigmenting melanocytes (e.g. pink-eyed dilution melanocytes(13) , or albino melan-c (21) melanocytes) were similarly purified by sucrose density
gradients, no hexosaminidase activity could be found in the
1.8 M fractions (data not shown). Since these non-pigmented
melanocytes contained a normal lysosome content, the lack of
hexosaminidase activity reflects a lack of authentic lysosomes
sedimenting at sucrose densities
1.8 M.
Melanosomes were found at that density, however, and possessed an esterase activity necessary to convert amino acid methyl esters to the free amino acids(22) ; this allowed melanosomes to be loaded for more definitive countertransport experiments. In fact, free tyrosine concentrations of 12.2 nmol/mg protein and 2.5 nmol/mg protein could be achieved for granular fraction melanosomes and sucrose gradient purified melanosomes, respectively, after incubation in 1 mM tyrosine methyl ester for 25 min at 37 °C. These levels represented 15-30-fold increases in tyrosine content compared to controls not exposed to tyrosine methyl ester, and allowed for a demonstration of trans stimulation, or countertransport, of tyrosine across the melanosomal membrane.
In countertransport, which
serves as classical evidence for carrier-mediated rather than
diffusional transport(23) , tracer amounts of a radiolabeled
substance will cross a membrane at an increased rate if there is a
substantial concentration of the nonradioactive substance on the
opposite side of the membrane. Once the radioactive tracer has moved
across the membrane it is effectively trapped due to competition with
saturating amounts of the nonradioactive compound within; it therefore
cannot exit the vesicle(16) . In this study, the radioactive
tracer used outside of the melanosomes was 10 µM [H]tyrosine, and the nonradioactive
competitor was tyrosine placed inside the melanosomes by pre-exposure
to 1 mM tyrosine methyl ester. The tyrosine-loaded melanosomes
took up much more [
H]tyrosine than did non-loaded
melanosomes in both the granular fraction (Fig. 5A) and
in the sucrose gradient purified fraction (Fig. 5B).
The countertransport activity of the highly purified melanosomes
derived from the
1.8 M sucrose gradient fraction amounted
to 10% of the total tyrosine transport activity of the postnuclear
supernatant. When tyrosine transport activity was expressed as units, i.e. picomoles of tyrosine/min/unit of hexosaminidase, the
values of the granular and sucrose fractions were essentially the same
(1.4 and 0.8, respectively), verifying carrier-mediated tyrosine
transport in a melanosomal fraction.
Figure 5:
Countertransport of
[H]tyrosine into melanosomes of the granular
fraction and of the sucrose density gradient purified fraction. A, melanosomes (granular fractions) preincubated in the
presence (
) or absence (
) of 1 mM tyrosine methyl
ester for 25 min at 37 °C were added to sucrose/HEPES medium
containing 10 µM [
H]tyrosine at 37
°C. Aliquots were removed after 0.5, 1, 2, and 3 min, centrifuged,
diluted into ice-cold sucrose/HEPES, and assayed for tyrosine uptake.
Tyrosine loading of the melanosomes was 12.2 nmol of tyrosine/mg of
protein. Radioactivity at time 0 was subtracted from all time points. B, experimental design as in A, but using sucrose
density purified melanosomes; tyrosine loading of these melanosomes was
2.5 nmol of tyrosine/mg of protein.
The primary function of melanosomes within melanocytes is to
produce melanin, whose synthesis is initiated by the
tyrosinase-catalyzed conversion of tyrosine to
DOPA(1, 2, 3, 4) . This process
creates a tyrosine ``sink'' by consuming the amino acid, and
emphasizes the critical nature of tyrosine entry into melanosomes in
order for pigmentation to occur. We now show that tyrosine entry into
melanosomes is carrier-mediated, as demonstrated by its saturability (Fig. 2), its ligand competition (Table 1), and its trans stimulation, or countertransport, both in
melanosome-rich granular fractions and in sucrose-gradient purified
melanosomes (Fig. 5, A and B). Furthermore,
melanosomal tyrosine transport displayed a temperature dependence (Q = 1.5, Fig. 3) consistent with
carrier-mediated uptake rather than other mechanisms such as diffusion
or movement via a membrane channel. Recently, Pisoni et al. described a cysteamine transporter in human fibroblast lysosomes
with a Q
value similar to that of this
melanosomal tyrosine transporter(24) .
It is not surprising
that a carrier for amino acid transport resides within the melanosomal
membrane, in view of the many lysosomal transporters known to exist (20, 22) and the similarities in organellogenesis
between melanosomes and lysosomes(17, 18) .
Melanosomes contain the lysosomal integral membrane protein LAMP-1 (25) , as well as acid phosphatase (26) and various
other ``lysosomal'' hydrolases such as
-N-hexosaminidase and cathepsins B and L(17) . In
this study, we have demonstrated the presence of an esterase activity
within murine melanosomes which cleaves tyrosine methyl ester to yield
free tyrosine. How the various lysosomal enzymes are directed to the
melanosome remains to be determined; they may enter by the same
mechanism used by the melanocyte-specific enzymes tyrosinase, TRP-1,
and TRP-2. These proteins are targeted to ``premelanosomes''
via coated vesicles, which originate from the trans-Golgi
network(27) , and apparently lack mannose 6-phosphate receptor
binding sites(28) . Alternatively, the tyrosine carrier itself
may be directed to the melanosomal membrane via mechanisms independent
of small vesicle delivery. For example, the melanocyte-specific
protein, Pmel 17/silver (29) , has been shown to co-purify with
immature and mature melanosomes but is not detected in small vesicle
fractions and presumably is targeted to melanosomes via another
processing route(30) .
Using tyrosinase as a marker for melanosomes, only a 2-fold purification of melanosomes is achieved in the granular fraction and sucrose gradient fraction compared with the whole cell homogenate (data not shown). This may be due to the fact that tyrosinase, and even typical lysosomal enzymes such as hexosaminidase, sediment also in a light, coated vesicle fraction which does not sediment with either the granular fraction or the denser sucrose gradient fraction(27) . Additionally, as melanosomes become highly pigmented and travel to the periphery of the melanocyte (i.e. reach maturation), they may lose their tyrosinase activity due to dilution or inhibition of enzymatic activity. They may also lose their esterase activity, and this may be the reason for the 5-fold higher level of tyrosine loading in the less pure (and less mature) granular fraction compared with the sucrose gradient purified melanosomes. The greater manipulation involved in preparing the sucrose gradient purified melanosomes may also have influenced its tyrosinase and esterase activities.
The biochemical similarities between
melanosomes and lysosomes (31) make it difficult to eliminate a
small lysosomal contribution to tyrosine transport in our melanocyte
granular fraction, since both melanosomes and lysosomes contain
hexosaminidase activity(8, 16, 17, 18) and other shared hydrolases(16, 17) .
Therefore, we set out to demonstrate that highly purified melanosomes (i.e. purified by high density sucrose gradient
ultracentrifugation) exhibited tyrosine countertransport. In fact, this
fraction contained a specific activity of tyrosine countertransport,
expressed as picomole/min/unit of hexosaminidase, similar to that of
the cruder granular fraction. Yet these highly purified melanosomes
lacked discernible lysosomes whether morphological (Fig. 1, C and D) or genetic/biochemical means were employed
to identify them. Specifically, non-pigmented murine melanocytes
(pink-eyed dilution and melan-c, both of which have mutations that
affect pigmentation but not their lysosome content) exhibited no
hexosaminidase activity (i.e. they had no melanosomes or lysosomes) in the sucrose fractions where pigmented melan-a melanosomes sediment at 1.8 M (data not
shown). Therefore, to the best of our ability to detect them, lysosomes
are not present in the pigmented melanosomal fraction exhibiting
tyrosine countertransport. Based on the morphological and biochemical
evidence presented above, we conclude that the tyrosine transport we
measured in these studies is largely melanosomal. It is critically
important to demonstrate that melanosomes (as opposed to possibly
contaminating lysosomes) effect tyrosine transport because
pigmentation, which utilizes enzymes not associated with lysosomes,
occurs only in melanosomes. This organelle has a unique
requirement for the import of tyrosine in order for the process of
melanogenesis, or pigment production, to proceed.
Melanosomal tyrosine transport resembled lysosomal tyrosine transport in many ways. It was not affected by alkalinization of the melanosome with ammonium chloride(32) , by blocking of free thiols with N-ethylmaleimide(7) , or by high concentrations of sodium or potassium (Table 2). Therefore, there was no evidence that the carrier operated through a structural change involving a protonated intermediate or that it required free thiols for binding, translocation, or release of tyrosine; furthermore, the system did not appear cation-dependent. The ionophores CCCP and monensin, with or without potassium or sodium, respectively, had no effect on tyrosine transport. Nigericin alone, or in the presense of KCl, effected a modest decrease in tyrosine uptake, perhaps indicating a small dependence on membrane potential. Since high concentrations of potassium did not inhibit tyrosine transport, nigericin was presumably responsible for this decrease.
The melanosomal tyrosine carrier showed a preference for the large, aromatic compounds tyrosine, phenylalanine, and tryptophan, as well as smaller neutral amino acids such as leucine and isoleucine and to some extent, methionine and histidine. It also displayed a lack of dependence upon extramelanosomal sodium, and was inhibited by the classical system L analogue, BCH, suggesting a similarity to rat thyroid lysosomal system h and human fibroblast lysosomal system l. The melanosomal tyrosine carrier did not recognize the classic plasma membrane system A analogue, MeAIB, or other amino acids which primarily utilize carriers other than lysosomal systems l and h (see Refs. 11, 20, and 22 for reviews), including the cystine carrier, system c for lysine, systems e and f for alanine, or system d for glutamate (Table 1). Collarini et al.(33) described the existence of a membrane transporter in human fibroblast lysosomes, termed system t, that is more selective for aromatic amino acids than systems l and h, having a higher affinity for tyrosine, tryptophan, and phenylalanine. Other characteristics of system t include affinity for D-isomers of aromatic amino acids, and sensitivity to the sulfhydryl-reactive agent, N-ethylmaleimide. In contrast, the melanosomal tyrosine carrier characterized in this study displayed a strong preference for the L-stereoisomer of tyrosine, and was not inhibited by D-tyrosine and only modestly by D-tryptophan. Furthermore, melanosomal tyrosine transport was not sensitive to N-ethylmaleimide, suggesting that in melanocytes, the primary route for tyrosine entry into melanosomes is via a single carrier that resembles systems h and l. Genetic similarities between the rat and mouse may explain the lack of system t in these species, while human fibroblasts may have evolved more differentiated transport systems.
While most lysosomal carriers transport the small molecule products of lysosomal hydrolysis out of vesicles(20, 22) , melanosomes appear to effect a net inward flux of tyrosine for melanogenesis. The melanosomal tyrosine carrier does resemble one lysosomal amino acid transport system in this respect; the cysteine transporter appears to favor a net inward flux, which Pisoni et al.(34) have proposed promotes proteolytic digestion within lysosomes. In melanocytes, the inward flux of cysteine could have a physiological basis since cysteine is required for the synthesis of the cysteinyldopa conjugates which eventually form the yellow to red pigment, phaeomelanin(6) . It remains to be determined if the melanosomal membrane possesses a carrier capable of transporting cysteine.
The melanosomal tyrosine
carrier strongly resembles the lysosomal system h first
described in rat FRTL-5 thyroid cell lysosomes(10) . The two
systems share ligand specificities, even for monoiodotyrosine (16) (Table 1), and have similar apparent K values for tyrosine and similar energies of
activation, 7.5 kcal/mol (Fig. 3) and 9.7 kcal/mol(10) .
But most intriguing is the role that each vesicular transport system
plays in achieving the differentiated function of its specific cell
type. While membrane transport is bidirectional and its flux is
determined by ligand concentrations on the two sides of the
membrane(20, 22) , tyrosine and monoiodotyrosine egress from lysosomes is required for salvage of iodine and
tyrosine for subsequent re-incorporation into thyroglobulin in thyroid
cells(16) ; tyrosine entry into melanosomes is
necessary for the synthesis of melanin in melanocytes. Both of these
processes are fostered by a sink on one side of the membrane, and both
processes highlight how physiologic conditions and the functional
imperative of the cell dictate the net direction of movement across
membranes. In addition, both processes may be subject to hormonal
regulation. In rat FRTL-5 cells, thyroid-stimulating hormone (or
thyrotropin) markedly up-regulates the lysosomal
tyrosine/monoiodotyrosine carrier at the transcriptional
level(35) . In the mouse, the expression of various
melanocyte-specific enzymes localized to the melanosome and involved in
melanogenesis is regulated in response to
-melanocyte-stimulating
hormone(2) . Whether melanosomal tyrosine transport is also
regulated by these or other factors remains an interesting possibility
to be determined.
Finally, since both melanosomes and lysosomes contain amino acid carriers and known lysosomal transport defects result in certain diseases (i.e. cystinosis and Salla disease), some tyrosinase-positive forms of human albinism may be due to impaired tyrosine uptake across the melanosomal membrane.