(Received for publication, October 25, 1996, and in revised form, February 26, 1997)
From the Clinical Nutrition Research Unit, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Accumulation of products of proteolysis (e.g. dipeptides) in lysosomes may have pathological consequences. In the present experiment we have investigated the existence of a dipeptide transporter in a membrane preparation of liver lysosomes using Gly-3H-Gln as the probe. The results showed that (a) there was transport of Gly-Gln into an osmotically reactive space inside the lysosomal membrane vesicles; (b) transport was stimulated by acidification (pH 5.0) of the external medium; (c) there was a coupling between transport of protons and Gly-Gln with a stoichiometry of 1:1; (d) the presence of both acidic pH and membrane potential was necessary for uphill transport of Gly-Gln; (e) a single transporter with a Km of 4.67 mM mediated the uptake of Gly-Gln; and (f) Gly-Gln uptake was inhibited by dipeptides and tripeptides but not by amino acids. The results suggest the presence of a low affinity proton-coupled oligopeptide transporter in the liver lysosomal membrane which mediates transfer of dipeptides from a region of low dipeptidase activity (intralysosome) to a region of high dipeptidase activity (cytosol). In this manner, the transporter provides an active mechanism for completion of the final stage of protein degradation.
Lysosomes are major sites for protein degradation in the liver. Amino acids and dipeptides are the final products of proteolysis within this organelle (1, 2). These products must be exported into the cytosol if lysosomes are to retain their biological integrity. Failure to export may result in pathological consequences. This is evidenced by the genetic defect in the export of cystine, which results in accumulation of a high concentration of this amino acid in the lysosomes causing cystinosis. Cystinosis is a progressive systemic disease with many pathological expressions (3).
Although as yet no genetic disease of dipeptide accumulation in lysosomes has been described, there is evidence that such an accumulation would also have pathological effects. This is evidenced by the osmotic protection experiments which determine the rates of release of lysosomal enzymes. Lloyd (4) showed that incubation of lysosomes isolated from rat liver with 0.25 M solutions of dipeptides or their constituent amino acids increased the release of their enzyme contents. Furthermore, the release was greater with dipeptides than with their constituent amino acids. He suggested that amino acids and dipeptides enter the lysosomes, and the entry is more efficient for dipeptides than for amino acids. Recently, Bird and Lloyd (5) found that the rate of enzyme release was greater with dipeptides containing L-isomers than D-analogs, suggesting a stereospecificity in lysosomal permeation of dipeptides. However, as reviewed by Forster and Lloyd (6), the osmotic protection technique does not allow any conclusion regarding the mechanism which a solute uses for entry into the lysosomes. For example, the greater rate of enzyme release by dipeptides containing L- rather than D-amino acids could be explained by the hydrolysis of dipeptides within the lysosomes which is stereospecific (7). This is consistent with a more recent study of Bird and Lloyd (8) who found a positive correlation between ability of dipeptides to disrupt lysosomes and their susceptibility to hydrolysis by lysosomal enzymes.
To determine whether the liver lysosomal membrane indeed possesses a dipeptide transporter, in the present experiments we have used a membrane vesicle uptake technique which has been validated for the studies of solute transport by the liver lysosomes. Jonas and Jobe (9) showed that properties of rat liver lysosomal membrane, like the proton-pumping ATPase activity, are preserved in vesicle preparations. For the present experiment we used labeled glycylglutamine (Gly-Gln) as the probe because we have previously validated the use of this dipeptide for characterization of oligopeptide transport in membrane vesicles prepared from kidney and intestine (10, 11).
Adult male Harlan Sprague Dawley rats with body weights of 250-300 g were purchased from Zivic-Miller. Custom synthesized [glutamine-3,4-3H]glycylglutamine (49 Ci/mmol) was obtained from DuPont NEN. Amino acids, oligopeptides, carbonyl cyanide m-chlorophenylhydrazone (CCCP),1 and acridine orange dye were purchased from Sigma. Filters (type HAWP, 0.45 µm pore size) were purchased from Millipore Corp., Bedford, MA.
Preparation of Lysosomal Membrane VesiclesRats were killed
and their livers were removed for preparation of lysosomal membrane
vesicles (LMV). Vesicles were prepared as described by Symons and Jonas
(12). Briefly, livers were homogenized in buffer 1 (250 mM
sucrose, 20 mM Hepes, 1 mM EDTA, pH 7.0, with
Tris base) containing 1 µg/ml pepstatin and 2 µg/ml leupeptin,
using a Dounce homogenizer. The liver homogenate was suspended at an
average protein concentration of 35 mg/ml in buffer 1 and centrifuged
for 20 min at 750 × g. The nuclear pellet was discarded and the centrifugation was repeated. The resulting
supernatant was centrifuged for 10 min at 20,000 × g,
and the pellet was resuspended in buffer 2 (250 mM sucrose,
20 mM Hepes, pH 7.0, with Tris base) and recentrifuged for
10 min at 20,000 × g. The final pellet was suspended
in 9 ml of buffer 2 and mixed with 11 ml of isotonic Percoll (1 ml of
2.5 M sucrose, 200 mM Hepes/Tris, pH 7.0, mixed with 9 ml of Percoll). The resulting Percoll mixture was centrifuged for 90 min at 40,000 × g. The dense brownish lysosomal
band near the bottom of the gradient was removed and diluted with
buffer 2 and centrifuged for 10 min at 20,000 × g. The
resulting pellet containing lysosomes was incubated in freshly prepared
solution containing 5 mM methionine methyl ester, 2 mg/ml
bovine serum albumin, 2 mM magnesium chloride, 2 µg/ml
leupeptin, and 1 µg/ml pepstatin for 15-20 min at 37 °C.
Following this treatment, an equal volume of ice-cold isotonic Percoll
was added. The mixture was centrifuged for 30 min at 35,000 × g. A dark brown band located at the top of this dense
gradient containing purified LMV was removed and diluted in buffer 2 and centrifuged at 20,000 × g. The purified LMV pellet
was resuspended in transport buffer sufficient to provide 3-5 mg/ml
protein content. All of the above procedures were done at 4 °C.
Protein content of the preparation was assessed with the Bio-Rad
protein assay. The purity of the LMV preparation was investigated by
determining enrichment of organelle and membrane marker enzymes. The
activities of -hexosaminidase, 5
-nucleotidase, cytochrome
c oxidase, and
-glucosidase in liver homogenate and vesicle suspension were measured by the methods described previously (13-15).
Uptake studies by LMV were performed at 23 °C using a rapid filtration technique as described previously (10). Gly-Gln uptake was initiated by mixing 20 µl of membrane suspension (preloaded with 100 mM KCl, 100 mM sucrose, 20 mM Hepes/Tris at pH 7.0) preincubated with 50 µM valinomycin and incubated with 180 µl of transport buffer containing 0.1 mM Gly-3H-Gln. Composition of the transport buffer varied with different experiments and is described in the figure legends. Incubation of vesicles was terminated by injecting 20 µl of vesicle mixture into 2 ml of ice-cold stop solution (same composition of the transport buffer but without Gly-3H-Gln), followed by filtration. The filters were then washed with 5 ml of ice-cold stop solution. The radioactivity associated with filters was counted in a Beckman scintillation spectrometer. Nonspecific binding of the Gly-3H-Gln was determined by adding the transport solution and vesicles directly to the respective ice-cold stop solution, followed by filtering, washing, and counting.
Calculation and StatisticsKinetic constants of Gly-Gln transport were determined by applying a nonlinear regression method to the Michaelis-Menten kinetic equation using GRAFIT (Sigma): V = (Vmax·[S])/(Km + [S])), where V is Gly-Gln uptake in picomoles per milligram of protein per 45 s, [S] is the external Gly-Gln concentration in millimolar, Vmax is the maximal Gly-Gln uptake, and Km is the concentration of [S] that yielded one-half Vmax. Each rate of Gly-Gln uptake, which was corrected for nonspecific binding, is given as mean ± S.E. of three replicates. Significant differences between values were determined by Student's t test.
As shown in Table I, there was
no enrichment of either cytochrome c oxidase (mitochondria)
or 5-nucleotidase (plasma membrane). In contrast, there was a more
than 100-fold enrichment of a lysosomal membrane enzyme
(
-glucosidase). Furthermore, there was only a modest enrichment of
our preparation with a non-membrane-associated lysosomal enzyme
(
-hexosaminidase).
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Preliminary experiments
revealed that there was uptake of Gly-Gln by LMV. To determine whether
the observed Gly-Gln uptake by LMV was indeed transport into vesicles,
the following experiment was performed. The rates of Gly-Gly uptake
were measured as a function of medium osmolarity. Details of this
experiment are presented in the legend to Fig. 1. The
results showed that the rate of uptake decreased as a linear function
of the reciprocal of medium osmolarity. Extrapolation of the data to
infinite osmolarity showed no uptake, indicating the Gly-Gln was
entirely or mostly transport into an osmotically reactive space. The
data also indicate that the vesicles were properly resealed during
preparation for uptake studies.
Driving Force for Dipeptide Transport
To investigate the
driving force for dipeptide transport we determined lysosomal uptake of
Gly-Gln in the presence and absence of a sodium or a proton gradient.
Details of these experiments are presented in the legend to Fig.
2. As shown in this figure, the imposition of an
inwardly directed sodium gradient (Na+
concentrationout = 100, Na+
concentrationin = 0) had no effect, but the imposition of
an inwardly directed proton gradient (pHout
5.0/pHin 7.0) transiently stimulated Gly-Gln uptake by LMV.
During the initial 2 min of incubation all of the uptake values were
significantly (p < 0.01) greater when the outside pH
was 5.0 and the inside pH was 7.0 than when both pH values were
7.0.
To investigate whether the above proton gradient indeed served as the driving force for Gly-Gln transport, the following experiment was performed. Prior to imposition of the proton gradient, the vesicles were preincubated with a proton ionophore (CCCP). As shown in Fig. 2, CCCP did not reduce Gly-Gln uptake by LMV, suggesting that the proton gradient was not the driving force in this case.
To investigate whether membrane potential influences transport of
Gly-Gln by the liver LMV, the following experiment was performed. The
effect of K+-generated diffusion potential (inside
negative) in the presence and absence of an acidic pH (5.0) in the
transport medium was determined. The membrane potential was generated
by replacing the KCl in the transport medium with 100 mM
choline chloride while maintaining the inside concentration of KCl at
100 mM. Results showed that imposition of membrane
potential, when the pH of the transport medium was 7.0, transiently
stimulated Gly-Gln uptake by the liver LMV (Fig. 3). All
of the uptakes during the initial 2 min of incubation were
significantly (p < 0.01) greater in the presence than
in the absence of membrane potential. The stimulatory effect of
membrane potential was greatly enhanced by acidification of the
transport medium, resulting in a near 2-fold overshoot in transport of
Gly-Gln at 2 min. Therefore, the presence of a membrane potential and
an acidic pH was necessary for observing uphill transport of
Gly-Gln.
Characterization of Transport
To investigate the affinity and
the number of transporters involved in the uptake of Gly-Gln by
membrane vesicles we investigated the rate of uptake as a function of
dipeptide concentration. The rates of uptake by the liver LMV were
determined in the presence of both a membrane potential and an acidic
pH. Before this experiment, it was necessary to determine for how long
the rate of Gly-Gln transport was linear. The results showed that for
at least 50 s the rate was linear (data not shown); therefore,
45 s was chosen as the incubation time for the experiment detailed
in the legend to Fig. 4. Eadie-Hofstee plot of data
(Fig. 4) showed the presence of a single system for transport of
Gly-Gln. The calculation of kinetic constants by the Michaelis-Menten
equation showed a low affinity transporter with a Km
value of 4.67 ± 0.80 mM and a
Vmax value of 880.38 ± 3.81 pmol/mg
protein/45 s.
Relationship between Transport of Gly-Gln and H+
We used the fluorescent dye acridine orange
technique to determine whether there is a coupling between proton and
Gly-Gln transport. The results are shown in Fig. 5.
There was a less than 5% change in the fluorescence when the liver LMV
were added to the medium containing no dipeptide. The fluorescence
change became appreciably greater (more than 11%) when the medium
contained Gly-Gln.
The results of the above experiment suggested that Gly-Gln and
H+ are cotransported into the vesicles. To investigate the
kinetics of this interaction, we investigated the effect of a range of pH on Gly-Gln transport (Fig. 6). There was a steady
decline in the rate of Gly-Gln uptake by the liver LMV when the pH of
the transport medium was increased above 5.0 (Fig. 6, right
inset). There was a hyperbolic relationship between the rates of
Gly-Gln uptake and H+ concentration (Fig. 6). The linearity
of the Eadie-Hofstee plot of the data indicated a 1:1 stoichiometry
between Gly-Gln and H+ uptake (Fig. 6, left
inset). Calculation of the Hill coefficient also showed a coupling
ratio of 1 (1.036 ± 0.152). Calculation of kinetic constants by
the Michaelis-Menten equation showed a Km of
0.36 ± 0.02 µM
(pKa = 6.4) and
Vmax of 57.01 ± 1.43 pmol/mg protein/45
s.
Substrate Specificity of the Transporter
To investigate the substrate specificity of the transporter in the liver LMV, we determined the inhibitory effect of representative groups of amino acids, dipeptides, and tripeptides on Gly-Gln uptake. The results showed (Table II) inhibition by dipeptides and tripeptides but not by amino acids. Due to poor solubility of tetrapeptides at 30 mM concentration, the effect of these oligopeptides on Gly-Gln uptake was not studied.
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The present study is the first step in the functional characterization of a peptide transporter in the membrane of liver lysosomes. The information obtained reveals that there are functional similarities between this transporter and those located in the brush border membrane of kidney and intestine (16). For example, it is capable of uphill transport, it is stimulated by an acidic pH and membrane potential, its substrates include dipeptides and tripeptides but not amino acids, and it shows a 1:1 stoichiometry between cotransport of Gly-Gln and H+. The uphill transport may have been more pronounced if the membrane orientation of all the vesicles was inside-out. It has been shown (17) that with the current technique of preparation, half of the LMV are oriented inside-out and the other half are oriented outside-out.
The present results suggest that the driving force for uphill transport is membrane potential which normally exists in lysosomes (inside positive/outside negative). At the pH used in the present experiment (4.5-7.0) Gly-Gln is in zwitterionic form. Therefore, its uptake does not add any electrical charge to the transporter. On the other hand, binding of a proton adds a positive charge to the transporter, making it more available for the influence of membrane potential.
The present results may provide an explanation for the disparity among the results of previous studies looking for the presence of an oligopeptide transporter in the liver. Lombardo et al. (18), using functional analysis, did not find any evidence for the presence of a dipeptide transporter in plasma membrane of rat liver. On the other hand, Fei et al. (19), using Northern analysis, found expression of Pept-1 mRNA in the liver. In view of the present data, it is possible that the basis for a positive Northern analysis in the liver (19) is the presence of an oligopeptide transporter in lysosomes. This suggestion is based on reports of a strong homology between genes encoding different oligopeptide transporters (20).
We think that the physiological function of the transporter we have characterized in the lysosomal membrane is mainly to export rather than to import dipeptides because (a) previous studies have shown that the lysosomal carriers can be demonstrated by either uptake or efflux studies (21); (b) as already mentioned, the vesicles have both inside-out as well as outside-out orientations; and (c) it is generally believed that the conditions across lysosomal membranes are the determinant of solute flux. For example, the inside pH is highly acidic, while the outside pH is above neutral. As we have shown in the present study, these conditions greatly favor transport in the direction of cytosol.
The Km of lysosomal oligopeptide transporter (4.67 mM) is considerably greater than the Km of lysosomal amino acid transporters, which ranges between 0.01 and 0.5 mM (6). Usually a greater Km is associated with a greater transport capacity. This may explain the efficiency of dipeptides over amino acids in causing the release of enzymes when lysosomes were incubated with these substrates in vitro (4).
Finally, the present finding leads to a new concept in protein catabolism. Hydrolysis of proteins, whether in the gut lumen, renal tubules, or in liver lysosomes may result in production of considerable amounts of dipeptides (1, 2, 22, 23). However, the hydrolase activity against dipeptides as studied in the intestine (24, 25) is either totally or mostly located in the cell cytoplasm. Furthermore, rates of hydrolysis are much greater when dipeptides are incubated with liver homogenates than with liver lysosomes (26). Therefore, oligopeptide transporters, whether located in the brush border or lysosomal membranes, appear to serve an important physiological function. They provide an active mechanism for transporting dipeptides from a region of low to a region of high dipeptidase activity. In this manner, they participate in completion of the final stage of protein degradation.