(Received for publication, August 25, 1994; and in revised form, October 27, 1994)
From the
The uptake of [H]cysteamine by
Percoll-purified human fibroblast lysosomes was investigated to
determine whether lysosomes contain a transport system recognizing
cysteamine. Lysosomal cysteamine uptake is a
Na
-independent process which rapidly attains a steady
state within 1 min at pH 7.0 and 37 °C. A biphasic Arrhenius plot
is observed for cysteamine uptake, giving a Q
of
2.2 from 17 to 26 °C and a Q
of 1.2 from 27
to 35 °C. The rate of lysosomal cysteamine uptake is maximal at pH
8.2, half-maximal at pH 6.8, and declines
50-fold from the maximum
to show very little transport at pH 5.0. Cysteamine uptake into
fibroblast lysosomes displays complete saturability with a K
of 0.88 mM and V
of 1410 pmol of
-N-acetylhexosaminidase/min at pH 7.0 and 37 °C.
Analog inhibition studies demonstrated that all analogs recognized thus
far by the cysteamine carrier are either aminothiols or aminosulfides
and contain an amino group and sulfur atom separated by a carbon chain,
2 carbon atoms in length. The K
constants
for these analogs as competitive inhibitors of lysosomal cysteamine
uptake are 2-(ethylthio)ethylamine (0.64 mM),
1-amino-2-methyl-2-propanethiol (0.74 mM),
2-dimethylaminoethanethiol (0.87 mM), thiocholine (1.6
mM), and bis(2-aminoethyl)sulfide (4.9 mM). L-Cysteine, D-penicillamine, and analogs lacking
either a sulfur atom or amino group are not recognized by the
cysteamine carrier including ethanolamine, choline, taurine,
-mercaptoethanol, ethylenediamine, cadaverine, spermine,
spermidine, histamine, dopamine, and 3-hydroxytyramine. In a
cystine-depletion assay, a 2-h exposure of cystinotic fibroblasts to 1
mM 1-amino-2-methyl-2-propanethiol lowers cell cystine levels
to the same low level obtained with cysteamine. Thus, all four
aminothiols, known to deplete cystinotic fibroblasts of their
accumulated cystine, are recognized as substrates by the lysosomal
cysteamine carrier, suggesting the importance of this transporter in
the delivery of aminothiols to the lysosomal compartment.
Lysosomes are a major intracellular site for the degradation of
a wide variety of macromolecules including proteins, nucleic acids,
complex carbohydrates, and lipids. The metabolites that form in
lysosomes from macromolecule degradation are recognized by specific
lysosomal transport systems which mediate metabolite exodus from the
lysosomal compartment. At the present time 20 different lysosomal
transport systems have been described including carriers for various
amino
acids(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) ,
monosaccharides(14, 15, 16, 17, 18, 19, 20) ,
nucleosides(21) , cobalamin(22, 23) ,
phosphate(24) , sulfate(25) , calcium(26) ,
dipeptides(27) , and methotrexate polyglutamates(28) .
However, not all lysosomal transport systems are believed to function
solely for facilitating metabolite egress from lysosomes. Previously,
we described a cysteine-specific lysosomal transport system serving as
a major route for the delivery of thiol into human fibroblast
lysosomes(13) . In addition, Rome and colleagues have
characterized a lysosomal membrane enzyme,
acetyl-CoA:
-glucosaminide N-acetyltransferase, which
catalyzes the transmembrane transfer of acetyl groups from cytosolic
acetyl-CoA to terminal
-linked glucosamine residues of heparan
sulfate located within the lysosomal compartment (29, 30).
The first detailed characterization of a lysosomal transport system was that of the lysosomal cystine carrier by Gahl et al.(1) and Jonas et al.(2) in 1982. These investigators demonstrated that the high levels of lysosomal cystine accumulation observed in the genetic disorder, nephropathic cystinosis, were due to a defect in lysosomal cystine transport. In the absence of treatment, nephropathic cystinosis leads to end stage renal failure by 10 years of age. In 1976, Thoene et al.(31) demonstrated that the addition of cysteamine to cultures of cystinotic fibroblasts produces rapid intralysosomal cystine depletion. Subsequent clinical studies have shown that cysteamine stabilizes renal function and improves linear growth in a large population of cystinotic patients(32) . Despite this knowledge of the therapeutic effects of cysteamine, however, the manner by which cysteamine enters lysosomes is not understood. In this report, we provide the first characterization of a lysosomal transport system which mediates the entry of cysteamine into human fibroblast lysosomes and raise questions regarding the role this transporter plays in lysosomal function.
We initially defined the effects of time, temperature,
substrate concentration, and pH on the rate of
[H]cysteamine uptake by Percoll-purified human
fibroblast lysosomes. Lysosomal cysteamine uptake at 37 °C and pH
7.0 occurs rapidly, attaining a steady state within 1 min following the
initiation of uptake (Fig. 1). Similar to all other lysosomal
transport systems, cysteamine transport was not affected by the
presence of 77 mM NaCl in the incubation mixtures (data not
shown), indicating that lysosomal cysteamine uptake is not a sodium
dependent process. In order for further kinetic analysis to reflect
initial rate kinetics, uptakes generally were performed for 30-45
s so as to occur during the linear portion of the uptake curve. The
effect of temperature on lysosomal cysteamine uptake revealed a
biphasic Arrhenius plot over the tested temperature range from 17 to 35
°C (Fig. 2). For the temperature range from 17 to 26 °C,
a rate-limiting step with a Q
of 2.26 is observed
which corresponds to an activation energy of 14 kcal/mol. At
temperatures above 26 °C, however, the activation energy for the
rate-limiting transport step decreases to 3.7 kcal/mol, corresponding
to a Q
of 1.2. These two different activation
energies may be a reflection of two completely different rate-limiting
steps involved in the process of lysosomal cysteamine translocation or
could correspond to essentially the same rate-limiting step occurring
more easily at temperatures above 26 °C due to substantial lowering
of a translocation barrier. Although we are not able to discriminate
among these possibilities, changes in membrane fluidity and structure
with changes in temperature could have a significant effect on the
energetics of transport. This is the first report of a biphasic
Arrhenius plot observed for a lysosomal transport system. All other
lysosomal carriers characterized until the present time have displayed
linear Arrhenius plots with Q
values ranging from
1.8 to 3.2, with a mean of 2.15 and a median of 2.0(37) .
Figure 1:
Time
course of 0.071 mM [H]cysteamine uptake
by human fibroblast lysosomes at pH 7.0. Time courses at 37 °C were
initiated by mixing a prewarmed 70-µl aliquot of 142 µM [
H]cysteamine in 20 mM Mops/Tris,
pH 7.0, buffer containing 0.25 M sucrose and 1 mM DTT
with a 70-µl aliquot of Percoll-purified
lysosomes suspended in 40 mM Mops/Tris, pH 7.0, buffer
containing 0.25 M sucrose. At the indicated times, 15-µl
aliquots were removed from the incubation mixture, added to 12 ml of
ice-cold phosphate-buffered saline, and filtered through a GF/A filter.
Filters were immediately washed twice with 12-ml portions of ice-cold
phosphate-buffered saline, mixed with 16 ml of Cytoscint scintillation
fluid, and counted for radioactivity. hex,
-N-acetylhexosaminidase.
Figure 2:
Arrhenius plot of 0.035 mM [H]cysteamine uptake by human fibroblast
lysosomes. Prewarmed lysosomes (8 µl) in 40 mM Mops/Tris,
pH 7.0, buffer containing 0.25 M sucrose were mixed with
24-µl aliquots of prewarmed 0.047 mM [
H]cysteamine in 33 mM Mops/Tris,
pH 7.0, buffer containing 0.25 M sucrose and 4.2 mM DTT. Uptakes were performed in triplicate for 30 s at a
temperature of either 17, 20, 23, 26, 29, 32, or 35 °C. At the
completion of each uptake, lysosomes were collected on glass fiber
filters and washed with ice-cold phosphate-buffered saline, and filters
were counted for radioactivity. hex,
-N-acetylhexosaminidase.
A
Michaelis-Menten plot of the initial rate of cysteamine uptake as a
function of the concentration of cysteamine demonstrates that lysosomal
uptake of cysteamine is a saturable process at pH 7.0 and 37 °C (Fig. 3). The Lineweaver-Burk plot is linear suggesting that
lysosomal cysteamine uptake is mediated by one transport route. The
kinetic parameters of K and V
were calculated using Cleland's HYPERL program yielding
values of K
= 0.88 mM ±
0.03 and V
= 1410 ± 30 pmol of
-N-acetylhexosaminidase/min. Although our data for these
short uptake time intervals conform well to a right rectangular
hyperbola characteristic of processes obeying Michaelis-Menten
kinetics, our analysis does not exclude the possibility that a small
nonsaturable component also may be associated with cysteamine uptake.
The observed V
for cysteamine uptake is one of
the largest rates of solute flux quantified thus far for a lysosomal
transport process.
Figure 3:
Kinetics of the initial rate of cysteamine
uptake into human fibroblast lysosomes as a function of the cysteamine
concentration. Fibroblast lysosomes were incubated for 30 s at 37
°C with [H]cysteamine of the indicated
concentration, in 35 mM Mops/Tris, pH 7.0, buffer containing
0.25 M sucrose and 30 mM DTT. At the completion of
the incubation period, lysosomes were collected on glass fiber filters
and washed with ice-cold phosphate-buffered saline, and filters were
counted for radioactivity. Determinations were performed in triplicate
at each concentration. The results are displayed in a Michaelis-Menten
plot of the initial rate of cysteamine uptake as a function of the
cysteamine concentration. The graph provided in the inset is a
double-reciprocal plot of the same data. hex,
-N-acetylhexosaminidase.
Lysosomal cysteamine uptake shows a dramatic
response to pH over the range from pH 5 to 8.5. An 50-fold greater
rate of cysteamine uptake is observed at the optimal pH of 8.2 compared
to the acidic pH of 5.0 (Fig. 4). If this pH dependence exists
on both sides of the lysosomal membrane, then once cysteamine is
transported into lysosomes from the cytosol, the intralysosomal pH of
5.3 (38) would not be favorable for cysteamine exodus from
lysosomes by this transport route. Thus the pH dependence of the
lysosomal cysteamine carrier appears to greatly favor net cysteamine
influx compared to efflux and may allow lysosomes to concentrate
cysteamine into this compartment when cystinotic cells are exposed to
therapeutic levels of cysteamine.
Figure 4:
pH dependence of 0.035 mM [H]cysteamine uptake by human fibroblast
lysosomes. Fibroblast lysosomes (8 µl) suspended in 0.25 M sucrose were incubated with 24 µl of 0.047 mM [
H]cysteamine in either 20 mM MES
or 20 mM MOPS buffers containing 0.25 M sucrose and
1.67 mM DTT and which had been titrated to the indicated pH
with Tris-free base. Incubations were performed at 37 °C for 0.75
min at which time 26.5-µl aliquots were removed from incubation
mixtures, lysosomes were collected and washed on GF/A filters, and the
filters were mixed with 16 ml of Cytoscint for measurement of
radioactivity. hex,
-N-acetylhexosaminidase.
To measure cysteamine efflux from
lysosomes, lysosomes were incubated with 157 µM [H]cysteamine for 10 min at 37 °C and
washed of unaccumulated radioactivity by centrifugation at 4 °C,
and the rate of lysosomal cysteamine egress was then measured at 37
°C. The results, shown in Fig. 5, indicate that
40% of
the radiolabeled cysteamine is rapidly released within the first 3 min
of incubation at 37 °C. Thereafter, only a slow rate of release is
observed for the remaining radioactivity accumulated within human
fibroblast lysosomes. Cysteamine is known to readily form mixed
disulfides by participating in sulfhydryl/disulfide exchange reactions (39, 40) . It is likely that much of the slowly
released radioactivity could represent mixed disulfides of cysteamine,
such as protein-bound cysteamine-cysteinyl-mixed disulfides, which
would be expected to form within lysosomes and require peptide bond
hydrolysis to be released as a free mixed disulfide, then transported
out of lysosomes by lysosomal system c(5, 6) .
Figure 5:
Release of radioactivity accumulated
within lysosomes following [H]cysteamine uptake.
Percoll-purified lysosomes were loaded with radioactivity by incubation
with 0.079 mM [
H]cysteamine in 29 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and
1.1 mM DTT, for 10 min at 37 °C. The lysosomal incubation
(0.1 ml) was then diluted to 1.5 ml with ice-cold 40 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose (wash
buffer), centrifuged at 15,600
g in an Eppendorf
microcentrifuge at 4 °C for 15 min. The supernatant was discarded,
and the lysosomes were washed once more by resuspension in 1.5 ml of
ice-cold wash buffer followed by centrifugation. The final lysosomal
pellet was resuspended to 200 µl with ice-cold wash buffer. To
assay for exodus of radioactivity from the
H-loaded
lysosomes, the lysosomal suspension was then incubated at 37 °C. At
the indicated time points, 15-µl aliquots were removed from the
incubation mixture, lysosomes were collected and washed on GF/A
filters, and filters were counted for radioactivity. hex,
-N-acetylhexosaminidase.
Several years ago, we used L-[S]cysteine uptake studies to
describe a cysteine-specific lysosomal transport system displaying an
extraordinary specificity for the amino acid, cysteine(13) .
During that investigation, we observed that cysteamine could strongly
inhibit the lysosomal uptake of L-[
S]cysteine, indicating that
cysteamine could be recognized by the cysteine-specific lysosomal
transport system. Whether cysteamine actually could be transported by
the cysteinespecific carrier, however, was not determined at that time
because of the unavailability of radiolabeled cysteamine. In the
present investigation, we now have applied analog inhibition analysis
to determine the specificity of the lysosomal transport system
mediating [
H]cysteamine uptake. For this
analysis, 30 different analogs, each at a concentration of 10
mM, were tested for their ability to inhibit 0.035 mM [
H]cysteamine uptake (Table 1).
Notably, L-cysteine, which is a model amino acid substrate for
the cysteine-specific carrier, has very little effect on lysosomal
cysteamine uptake, indicating that cysteamine is not transported into
fibroblast lysosomes by the cysteinespecific lysosomal transport
system. Six other analogs, however, were found to be well recognized by
the cysteamine carrier. These include cysteamine,
2-(ethylthio)ethylamine, 1amino-2-methyl-2-propanethiol,
2-dimethylaminoethanethiol, thiocholine, and bis(2-aminoethyl)sulfide.
All of these compounds share the general
structure,
where R = -H or -CH and X = -H,
-CH
-CH
, or
-CH
-CH
-NH
. The
amino group and sulfur atom are both required for substrate recognition
by the carrier. Replacing the SH group of cysteamine or thiocholine
with an OH group to give ethanolamine or choline results in poor
recognition by the cysteamine transporter. Likewise, replacing the SH
group of cysteamine with an SO
group to give taurine, or an
NH
group to give ethylenediamine, also yields structures
that are poorly recognized by the cysteamine transport system. A free
thiol is not required for recognition as indicated by the strong
inhibitory action of 2-(ethylthio)ethylamine and
bis(2-aminoethyl)sulfide, which contain a sulfur atom in the form of a
sulfide. The requirement for an amino group as part of the substrate
recognition unit is indicated by the lack of inhibitory effect of
-mercaptoethanol which contains the thiol of cysteamine but lacks
the amino group. Methylation of cysteamine's amino group is well
tolerated as indicated by the strong recognition of thiocholine and
2-dimethylaminoethanethiol. Acetylation of the amino group, however, is
not permitted as demonstrated by the complete inability of N-acetylcysteamine to inhibit
[
H]cysteamine uptake. As part of our analog
inhibition analysis, we determined if lysosomal cysteamine uptake could
be inhibited by various amines which are known substrates for amine
transporters in other biological membranes. These analogs included
tyramine, dopamine, spermine, spermidine, hydroxytryptamine,
acetylcholine, histamine, and cadaverine. Tyramine demonstrated a
modest inhibitory effect producing 50% inhibition. None of the other
amines, however, had any significant effect on lysosomal cysteamine
uptake including 3-hydroxytyramine which failed to show any inhibitory
effect on lysosomal cysteamine transport. Finally, the presence of an
-carboxyl group prevents recognition by the cysteamine transport
system as indicated by the failure of L-cysteine and D-penicillamine to inhibit cysteamine uptake, whereas their
decarboxylated analogs, cysteamine and 1-amino-2-methyl-2-propanethiol,
each strongly inhibit lysosomal transport of
[
H]cysteamine.
For the six analogs in Table 1that were found to be the strongest inhibitors of
lysosomal cysteamine uptake, further inhibition analysis revealed that
all six analogs act as competitive inhibitors of cysteamine transport.
In this analysis, five different concentrations of inhibitor were
chosen for their ability to inhibit either 0.025, 0.05, 0.1, 0.2, or
0.5 mM [H]cysteamine uptake. Results,
when displayed in a double-reciprocal plot, yielded inhibition lines
intersecting on the y axis. Fig. 6A demonstrates
such a plot for the inhibition of [
H]cysteamine
uptake by several different concentrations of thiocholine. When the
slopes of individual inhibition curves from the double-reciprocal
analyses were plotted as a function of the inhibitor concentration (Fig. 6B), a direct linear proportionality indicative
of pure competitive inhibition (41) was obtained for each
analog. The nearly identical K
values for
cysteamine and 2-(ethylthio)ethylamine, obtained from this kinetic
analysis (Table 2), indicate that the cysteamine transport system
does not discriminate whether the sulfur atom exists as a free thiol or
in the form of a sulfide. Moreover, the transport system easily
accommodates groups as long as an ethyl group bonded onto the non-amino
side of the sulfur atom without any loss in recognition. The addition
of an ethylamino group onto both sides of the sulfur atom, however,
results in an
8-fold decrease in recognition by the cysteamine
carrier as shown for bis-(2-aminoethyl)sulfide. Because of the
unavailability of suitable analogs, we have not been able to examine if
this large decrease in affinity of bis-(2-aminoethyl)sulfide compared
to 2-(ethylthio)ethylamine is due to the carrier having difficulty in
accommodating the overall length of bis-(2-aminoethyl)sulfide, is due
to the effect of the additional amino group, or is the result of both
structural changes significantly affecting substrate recognition.
Another possibility for the higher K
of
bis-(2-aminoethyl)sulfide is that, at pH 7.0, the carrier may only
recognize a minor ionic species of bis-(2-aminoethyl)sulfide such as
the species in which one amino group is positively charged and the
other amino group is uncharged. Branching at the
-carbon of
cysteamine has only a slight effect upon recognition by the cysteamine
transport system as demonstrated by the similar K
values for cysteamine and 1-amino-2-methyl-2-propanethiol.
However, di- and trimethylation of the substrate amino group modestly
diminishes recognition by the carrier as indicated by the larger K
values of 0.87 mM and 1.63 mM for 2-dimethylaminoethanethiol and thiocholine, respectively, as
compared to 0.58 mM for cysteamine.
Figure 6:
Inhibition of lysosomal cysteamine uptake
by thiocholine. Fibroblast lysosomes were incubated with either 0, 0.6,
1.2, 2.4, or 4.8 mM thiocholine (TC) for 30 s at 37
°C with [H]cysteamine of the indicated
concentration in 35 mM Mops/Tris, pH 7.0, buffer containing
0.25 M sucrose and 26 mM DTT. At the completion of
the incubation period, lysosomes were collected on GF/A filters, washed
with ice-cold phosphate-buffered saline, and the filters were washed to
determine the amount of radioactive cysteamine taken up. A,
double-reciprocal plot of the initial rate of
[
H]cysteamine uptake as a function of the
cysteamine concentration for the different concentrations of
thiocholine that were used. B, slope replot, the slope of each
curve from the above double-reciprocal plot is graphed as a function of
the thiocholine concentration. The negative of the x axis
intercept yields a K
of 1.6 mM for thiocholine. hex,
-N-acetylhexosaminidase.
Our analog inhibition
and K analyses in Table 1and Table 2indicate that the aminothiol,
1-amino-2-methyl-2-propanethiol (AMPT), also is recognized by the
lysosomal cysteamine transport system. Upon entering lysosomes, AMPT
would be expected to react with free cystine to form a cationic mixed
disulfide. Lysosomal system c, which has been shown to accept
a broad range of cationic substrates(6) , would likely
recognize this mixed disulfide and facilitate its exodus from the
lysosomal compartment. Thus, the aminothiol, AMPT, would appear to have
the necessary characteristics to deplete cystinotic cells of their
accumulated cystine. To test this possibility, cystinotic fibroblasts
were incubated in medium containing either 1 mM AMPT, 0.5
mM cysteamine or medium without any further additions. The
results shown in Fig. 7demonstrate that during a 2-h exposure
period, 1 mM AMPT is able to deplete cystinotic fibroblasts of
their accumulated cystine to the same low level as occurs when cells
are exposed to 0.5 mM cysteamine.
Figure 7: Comparison of the ability of cysteamine and 1-amino-2-methyl-2-propanethiol to deplete cystinotic cells of their cystine content. Confluent monolayers of cystinotic fibroblasts, grown in 60-mm dishes in Coon's modified Hams' F-12 medium containing 10% fetal calf serum, were washed three times with PBS and placed in cystine-free culture medium with 10% dialyzed fetal calf serum containing either 0.5 mM cysteamine (MEA), 1 mM AMPT, or no further addition. After a 2-h incubation, plates were washed three times with PBS, cells were scraped off plates with a rubber policeman, and cell protein and cystine content was quantified as described under ``Experimental Procedures.'' The experiment was performed in triplicate for each incubation condition. Results are expressed as a percent of the initial cystine content of the cystinotic cells prior to the 2-h incubation which was 9.97 ± 0.15 nmol of cystine/mg of protein.
In this report, we provide the first characterization of a
unique transport system that mediates cysteamine entry into human
fibroblast lysosomes. The substrate specificity of this lysosomal
cysteamine carrier is highly unusual among known transport systems in
that all analogs recognized thus far are either aminothiols or
aminosulfides, and contain a sulfur atom and amino group separated by a
carbon chain, 2 carbon atoms in length. The observation that
thiocholine, which contains a quaternary amine, is well recognized by
the cysteamine carrier provides strong evidence that the substrate
amino group is recognized in its positively charged form by the
carrier. Throughout the physiological pH range, the amino group of
cysteamine, with a pK of 10.5(42, 43) , also
would be expected to exist largely in its protonated form. Therefore,
the pH profile of lysosomal cysteamine uptake, showing 50-fold
greater cysteamine uptake at pH 8.2 than at pH 5.0, does not reflect
titration of the substrate amino group. Instead, this pH dependence
likely corresponds to either titration of an amino acid residue on the
carrier protein important for cysteamine transport, or reflects a
dependence of lysosomal cysteamine uptake on a transmembrane proton
gradient.
Cysteamine is therapeutically important in the treatment of nephropathic cystinosis by being able to enter lysosomes, react with free cystine to form the mixed disulfide of cysteamine and cysteine. These two products are then transported out of the lysosomal compartment by other functional lysosomal carriers(6, 13, 31, 32) . In addition to cysteamine, thiocholine and 2dimethylaminoethanethiol also have been shown to deplete cystinotic cells of their accumulated lysosomal cystine(6, 31) . How these aminothiols are able to enter lysosomes, however, has not been delineated. The results of the present investigation indicate that these three aminothiols and 1-amino-2-methyl-2-propanethiol are each recognized by the lysosomal cysteamine transport system and thus, carrier mediation by the cysteamine porter is an important step in the overall mechanism of cysteamine treatment of nephropathic cystinosis.
The biological importance of the lysosomal cysteamine transport system remains to be established. Cysteamine is the only naturally occurring aminothiol in man currently known to be recognized by the lysosomal cysteamine carrier. However, the levels of cysteamine generally detected in human fibroblasts are low(44) . It is noteworthy, however, that cysteamine is generated in animals as part of the coenzyme A degradative pathway(33, 44) . Some of the catabolic enzymes that comprise this pathway are lysosomal enzymes, and the cysteaminegenerating enzyme, pantethinase, appears to be located exclusively in the microsomal-lysosomal fraction in rat liver(33) . Although confusion remains as to the subcellular location of the complete coenzyme A degradative pathway, the lysosomal cysteamine transporter may serve for egress of cysteamine produced in lysosomes through the action of a lysosomal pantethinase.