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Proton-coupled solute transport in the animal cell plasma membrane
Proton-coupled
solute transport in the animal membrane
Vadivel
Canapathy
Medical
College
and Frederick
of Georgia,
Augusta,
cell plasma
H. Leibach
Georgia,
USA
This review focuses primarily on the progress made in the last couple of years in the understanding of the intestinal peptide transporter, a prototype for H+-coupled solute transport systems in the animal cell plasma membrane. The impressive number of transport systems currently known to be energized by the components of the proton-motive force indicates that the role of H+ as the coupling ion for active transport has not been lost during evolution. Current
Opinion
in Cell Biology
Introduction
In biological systems, active transport of organic solutes is coupled primarily to simu1taneous transport of inorganic ions. The free energy released during the movement of the inorganic ions down a concentration gradient is used as the driving force to move the organic solutes against a concentration gradient. In bacterial systems, the principal inorganic ion used for this purpose is H+. The electrochemical H+ gradient (proton-motive force, ASH), the energy source for the H+ -coupled transport systems, is located across the bacterial cell membrane and consists of an electrical component (AY) and a chemical component (ApH). This gradient is generated either by respiration or ATP hydrolysis. The Escbticbia coli lactose-pennease system can be considered as the prototype of H + coupled solute transport systems in bacteria [ 11. The principal coupling ion in animal cells appears to be Na+ rather than H+ [2]. The electrochemical Na+ gradient (sodium-motive force, AuNa), the energy source for the Na +-coupled transport systems, exists across the animal cell plasma membrane and is generated by the Na+-K+ pump. In recent years, evidence has accumulated which indicates that the difference in the preferred inorganic ion between bacterial (H+> and animal (Na+> systems is not absolute. A number of Na+-coupled solute transport systems have recently been discovered in bacteria [3]. Likewise, a number of organic-solute transport systems in animal cells have been shown to be coupled to H+ rather than Na+. The animal cell plasma membrane possesses mechanisms to generate the chemical as well as the electrical components of the proton-motive force (Fig. 1). The Na+-K+ pump converts the free energy released from ATP hydrolysis into the chemical and the electrical gradients of Na+ (ApNa and AY). The Na+-H+ exchanger, known to be present in most cell types, converts ApNa to ApH. Thus, the concerted actions of the Na+-K+ pump and the Na+-H+ exchanger result in the produc@ Current
Biology
1991, 3:695-701
tion of the proton-motive force. The H+ pump, which has been shown to be present in at least some cell types, directly generates ASH and AY from ATP hydrolysis. The proton-motive force is the energy source for many solute transport systems in animal cells. Among these, the peptide transporter can be considered as the prototype of H+ coupled solute transport systems of the animal cell plasma membrane. This transporter is of major physiological importance because of its participation in the absorption of an important group of nutrients in the small intestine and the kidney. It uses both the chemical and the electrical components of the proton-motive force as its energy source, and the progress made in understanding the molecular characteristics of this transporter has been more impressive than that made with other H+ coupled solute transport systems [4-S]. The last 2 years have seen dramatic breakthroughs in different aspects of this transporter. Significant progress has also been made in understanding other H+ coupled transport systems. The purpose of the current review is to highlight these recent advances in the area of H+ coupled solute transport systems in animal cells.
The peptide
transporter
Direct evidence for the coupling of the proton-motive force to active transport of peptides in the small intestine
Although a number of studies have unequivocally demonstrated proton-motive-force-dependent, active transport of small peptides in brush-border membrane vesicles derived from the kidney, the results with intestinal brushborder membrane vesicles have been conflicting. The stimulation of peptide transport in these vesicles by an inwardly directed H+ gradient (APH) has been shown in many studies. Similarly, the electrogenic nature of the H+ -dependent peptide transport has also been demonstrated in different laboratories. However, the overshoot Ltd ISSN 0955-0674
695
6%
mmbrane
permeability
Na+
Scheme’
2K+
Na+-K+
Energy
transduction
3Na’
pump
ATP + ApNa + A’?
ADP
H’
4 N
Nat
Hi
ApNa
H’
exchanger
tf
ApH
phenomenon, which is indicative of active transport against a concentration gradient in membrane vesicles, has been observed in some studies but not in others. (Readers are referred to [9] for a detailed analysis of available information on this particular aspect of peptide transport.) The absence of uphill transport in such studies might have resulted from an inability to maintain the experimentally imposed proton-motive force in isolated membrane vesicles. Two recent reports, however, by Dantzig and Bergin [lo**] and Calonge et al. [ll**], have provided unequivocal evidence for the energization of the intestinal peptide transporter by the proton-motive force. Dantzlg and Bergin used the Caco-2 ceU line as a model for studying the characteristics of the intestinal peptide transporter. In these cells, uptake of cephalexin, a @lactarn antibiotic known to be transported via the peptide transporter, was Na + -independent and exhibited an acidic extracellular pH optimum. A cellmedium ratio greater than one was observed in a Na+ -free buffer with an extracellular pH of 6.0. The uptake was shown to be dependent on metabolic energy because poisons such as oligomycin, azide and dinitrophenol signiUcantly inhibited it. Protonophores, which collapse a transmembrane H+ gradient, also caused marked inhibition of the uptake. These data provide direct evidence for the energization of the peptide transporter by an inwardly directed ttansmembrane H+ gradient in Caco-2 cells. Although the role of a tr-ansmembrane potential difference in the catalytic function of the transporter was not investigated in this study, as the experiments were conducted on intact cells it is most likely that the electrical component of the proton-motive force also contributed to the energization of the peptide transporter. Calonge et al. [ ll**] demonstrated uphill transport of a dipeptide in the small intestine with the proton-motive force as the energy source using ATP-depleted chicken intestinal cells, which behave like large membrane vesicles. This experimental system has numerous advan-
H+-pump
ATP
+
ApH
+ A’+’
Fig. 1. Transport systems which generate ApH+. AY is the electrical component of the electrochemical gradient. ApH and ApNa represent the chemical component.
tages over isolated intestinal brush-border membrane vesicles. The chicken intestinal cells have a large intracellular volume and are not subject to the possible alterations that normally occur during the isolation of brushborder membrane vesicles, such as cell lysis, divalent cation aggregation and resealing. In these ATP-depleted cells, uptake of Gly-Sar was Na+-independent, but stimulated by an inwardly directed H+ gradient. Generation of an inside-negative membrane potential also increased Gly-Sar uptake. Although concentrative uptake was not evident in the presence of either the chemical component (A~H) or the electrical component (AW of the protonmotive force, when both components were simultaneously present there was clear evidence for a transient uphill accumulation of Gly-Sar. This study provided direct evidence for the energization of the intestinal peptide transporter by the proton-motive force. The same investigators had previously demonstrated in intact chicken enterocytes that Gly-Sar uptake was active, partially dependent on Na+ , showed an acidic extracellular pH optimum and was sensitive to inhibition by amiloride, an inhibitor of the Na+-H+ exchanger [ 121. This can be taken as strong evidence for the role of the Na+-H+ exchanger in the generation of the transmembrane H+ gradient across the intestinal brush-border membrane in metabolically active, intact enterocytes. At the present time, it appears that the idea of the protonmotive force being the energy source for intestinal peptide transport has not been widely accepted among investigators in the area of intestinal absorption. This apparent skepticism is evident when one peruses the currently available textbooks on gastrointestinal physiology. Many of these textbooks still either state that intestinal peptide transport is energized by an electrochemical Na+ gradient or seem to have taken a somewhat ‘middle of the road position’ stating that the mechanism of peptide transport is controversial. It is our hope that the two reports men-
Proton-coupled
solute transport
tioned above will enhance the acceptance of the protonmotive force as the driving force for the intestinal absorption of small peptides and remove the skepticism that currently exists in the minds of the textbook writers.
Tripeptides
as substrates
for the peptide
transporter
In the past, most studies on peptide transport used only dipeptides as substrates and any conclusion drawn about whether the peptide transporter recognizes longer peptides as substrates was based merely upon competition experiments. However, because these competition experiments were all done under conditions in which peptidases associated with the brush-border membrane can potentially hydrolyze the test peptides to generate dipeptides, the possibility exists that the observed competition in these experiments was not real. Non-availability of radioactive tripeptides and hydrolysis of most tripeptides by brush-border peptidases are the primary reasons for the lack of direct studies on tripeptide transport. Recently, however, four papers have appeared in the literature that directly demonstrate intact transport of tripeptides and its energization by a proton-motive force. In 1989, Wilson et al. [ 131 reported on the characteristics of the transport of an intact tripeptide, GlyGly-Pro, in isolated intestinal brush-border membrane vesicles. In this report, hydrolysis of the test peptide was prevented by differential removal of the membrane-bound peptidases by papain digestion. Uptake of intact Gly-Gly-Pro could be demonstrated in these peptidase-depleted membrane vesicles. One year later, our laboratory reported similar results in renal brush-border membrane vesicles with Phe-Pro-Ala as the tripeptide substrate, though in this study hydrolysis of the peptide was prevented by irreversibly inhibiting dipeptidyl peptidase N, the enzyme responsible for breakdown of X-ProY type peptides [ 141. More recently, we took another approach which enabled us to investigate directly the transport of peptides containing three or more amino acids without interference from peptide hydrolysis [ 151. In this approach, a novel rat strain (Japanese Fisher 344) [ 161, which exhibits a genetic deficiency of dipeptidyl peptidase N was used. Because of the absence of the enzyme, purified renal brush-border membrane vesicles were unable to hydrolyze Phe-Pro-Ala. With these membrane vesicles, we could demonstrate that the tripeptide was transported into the vesicles in intact form [ 17**]. Uptake of the tripeptide was found to be Na+-independent, but was stimulated by an inwardly directed H+ gradient. Transient accumulation of the peptide against a concentration gradient was evident in the presence of the H+ gradient. The H+ -gradient-dependent uptake was electrogenie. This was the first demonstration of H+ -gradientdriven tripeptide uptake. Availability of this unique system for studying tripeptide transport made it possible for further investigations to delineate the competitive interaction between dipeptides and tripeptides with the peptide transporter [ 180~1. These studies have unambiguously demonstrated that a common transporter is responsible for the uptake of di- and tripeptides containing neutral amino acids.
E
in the animal
cell plasma
membrane
Ganapathy and Leibach
Because the Japanese Fisher 344 rats lack dipeptidyi peptidase N not only in the kidney but also in the small intestine, this particular rat strain will become very useful in future studies for investigating the characteristics of tripeptide transport in the small intestine. Moreover, uptake of longer peptides containing four or five ammo acids can also be studied in intestinal and renal brushborder membrane vesicles isolated from this rat strain without interference from hydrolysis by carefully selecting peptides of particular amino acid composition and sequences. Studies along these lines are currently underway in our laboratory.
The chemical
nature
of the peptide
transporter
One of the primary goals of studies on a transport system is to analyze the chemical nature of the proteins responsible for the transport function in order to understand the chemical basis of the transport mechanism. This can be achieved by purifying the membrane proteins responsible for the transport function to homogeneity and/or cloning the genes coding for these proteins. Significant progress has been made in the last year in this area with respect to the intestinal peptide transporter. Kramer et al. [ 19.01 have apparently purified the intestinal peptide transporter. The molecular weight of the transport protein is 127 kD. The evidence for its identity with the peptide transporter is as follows: the protein is preferentially labelled by photoreactive derivatives of dipeptides and j3-lactam antibiotics; the photolabelling is specifically inhibited by substrates of the transporter; and the polyclonal antibodies raised against this protein are able to inhibit the catalytic function of the peptide transporter in isolated intestinal brush-border membrane vesicles and also block the photoaffinity labelling of the protein. However, the final and unequivocal proof for the identity of this protein with the peptide transporter will be to demonstrate that this protein, when incorporated into art&ial membrane vesicles, does indeed carry out a peptide transport function. But this evidence is yet to come. Kramer et al [20*] have also demonstrated that the 127kD protein is not part of a brush-border peptic&e. The protein could be physically separated from the major peptidases of the brush-border membrane, namely aminopeptidase N and dipeptidyl peptidase N. These Sndings are interesting because it has often been hypothesized in the past that the peptic&es of the intestinal and renal brush-border membranes may indeed catalyze the transmembrane movement of amino acids and/or peptides. The study by Kramer et al [20*] disproves this hypothesis. Of relevance to this particular point are our own observations that the renal brush-border membrane vesicles isolated from the Japanese Fisher 344 rats do not possess the dipeptidyl peptidase N protein, but exhibit normal peptide transport activity [15]. We have recently succeeded in expressing the intestinal peptide transporter in Xen0pu.r laev& oocytes in a functional form [21**]. Following microinjection of rabbit intestinal poly(A) + mRNA, expression of an exogenous
697
698
Membrane
permeability
peptide transporter in the oocytes can be demonstrated by measuring Gly-Sar uptake. The expressed transporter exhibits characteristics similar to those of the transporter studied in rabbit intestinal brush-border membrane vesicles. The transporter is speciiic for peptides and its activity is stimulated by an inwardly directed H+ gradient. This oocyte system can become a useful assay technique for the expression cloning of the intestinal peptide transporter. Although the brush border membranes isolated from the small intestine and the kidney possess peptide transport activities with similar properties, there is accumulating evidence that the proteins responsible for the transport function in these membranes may not be identical. The labelling pattern with the photoreactive derivatives of transporter substrates in intestinal and renal brush-border membranes are markedly different [ 22,231. The two transport systems also differ in their inhibition by various amino-acid-group-specilic reagents [ 23-261.
Other
interesting
aspects of peptide
transport
It is widely accepted that while a majority of proteindigestion end products enter the enterocytes across the brush-border membrane in the form of small peptides, they exit across the basolateral membrane predominantly as free amino acids. This happens because of the efficient intracellular hydrolysis of peptides by cytosolic peptidases. However, there is substantial evidence in intact tissue preparations that a small, but significant, amount of intact peptides do cross the basolateral membrane. This suggests that the basolateral membrane may possess mechanisms for peptide transport. But, until recently, there was no direct study on the characteristics of peptide transport across the basolateral membrane. The report by Dyer et al [27*] is the first to investigate basolateral membrane peptide transport. The basolateral membrane vesicles used in this study were highly enriched in ouabain-sensitive, K+ -activated phosphatase with no detectable contamination from brush-border membranes as assessed by cross-reaction with antibodies against the sodium-glucose symporter. Uptake of Gly-Pro in these vesicles was carrier-mediated and was stimulated by an inwardly directed H+ gradient. This stimulation was inhibited by protonophores.The results of the study thus indicate that there is a peptide transporter in the enterocyte basolateral membrane. It is very likely that this transporter plays a role in the exit of intact peptides from the cells across the basolateral membrane. Another recent paper on peptide transport, by Bird and Lloyd [28**], deserves attention as it provides evidence for the presence of a peptide transporter in the lysosomal membrane. Lysosomes participate in the breakdown of cellular proteins which enter this organelle by processes involving endocytosis and pinocytosis. There is no definite information available on the nature and fate of end products of lysosomal protein digestion. We speculated almost a decade ago that, similar to protein digestion and absorption in the small intestine, breakdown of proteins within the lysosomes may not be complete and
that the end products may in fact consist of free amino acids as well as small peptides which exit the lysosomes via specific transporters located in the lysosomal membrane [29]. This idea was very diIferent from the generally held view at that time that free amino acids were the primary end products of lysosomal protein digestion and that these end products diRused out of lysosomes to be used by the cell, Between that time and now, numerous papers have appeared in the literature demonstrating carriers for a variety of amino acids in the lysosomal membrane. In fact, the different amino acid transport systems now known to be present in the lysosomal membrane are as many and diverse as those of the plasma membrane. Although handling of dipeptides by lysosomes has been investigated in the past, the existence of a specific transporter for these compounds was never invoked. The recent discovery of transport systems not only for amino acids but also for compounds such as glucose and sialic acid has led Bird and Lloyd [28**] to reinvestigate the lysosomal handling of dipeptides. Using an ‘osmotic protection’ method, these investigators analyzed the passage of dipeptides across the lysosomal membrane. It was concluded from the study that lysosomes possess a transport mechanism which accepts L-dipeptides as substrates but excludes D-dipeptides. It is very likely that this transporter catalyzes extrusion of dipeptides arising from intra-lysosomal protein breakdown. The involvement of H+ in the catalytic activity of the lysosomal dipeptide transporter has not been investigated in this study. However, it is tempting to speculate that, because the intenor of the lysosome has an acidic environment with a H+gradient across the lysosomal membrane in the direction of lysosome-to-cytoplasm, the H+ gradient is the most logical driving force for active extrusion of dipeptides from the lysosome.
Other
H+-coupled
solute transport
systems
Evidence has been presented in recent years that the system ~-amino acid transporter, which is Na+ -independent, is indeed energized by the proton-motive force in certain animal cells [30]. This system operates via the amino acid-H+ symport mechanism and uses both the chemical and the electrical components of the proton-motive force as the energy source. This transport system has recently been reconstituted in proteoliposomes and the reconstituted transporter exhibits H+ dependence [31]. The lactate transporter is another system which uses a transmembrane H+ gradient as the driving force. The lactateH+ symport (or phenomenologically indistinguishable lactate-hydroxyl exchange) is electroneutral and hence is not inlluenced by transmembrane electrical potentials. Those cells which actively produce lactic acid possess this transport system. The transporter most probably plays a role in the protection of the cell from potential acid-loading as a result of accumulation of lactic acid. The presence of this transporter has been documented in erythrocytes, cardiac muscle, skeletal muscle, small intestine and placenta [32-341. Intestinal absorption of folate is also
Proton-coupled
X
solute
transport
H’
X-
in the animal
cell plasma
H+
membrane
Canapathy and Leibach
X-
OH
X
H’
X-
OH-
Xc
H+
Scheme
Mechanism
Example
Substrate-H’
symport
Substrate-H’
Peptide transporter System r-amino acid transporter
symport
Lactate
ApH + A't'
Driving force
2. Transport
systems
which
use
transporter,
Substrate-OH-
folate
antiport
Substrate-H+
transporter
Organic
APH
1 Fig.
or
antiport
cation
transporter
APH -L
ApH+.
H+ -gradient dependent, and the mechanism of transport is either folate-hydroxyl exchange or its phenomenological equivalent, folate-H+ symport [35]. Another interesting system which is coupled to a transmembrane H+ gradient is the organic cation-H + antiporter [ 361. This system is electrically silent and has been described in the kidney, intestine and placenta. The mechanistic aspects of the transport systems discussed above are illustrated in Fig. 2. Ln addition to the above-mentioned transport systems which exclusively use the proton-motive force components as the driving force, there are other systems in arimal cells which accept either Na+ or H+ as the co-substrate. This includes the glucose transporter in the intestinal brush-border membrane [37], and various amino acid transport systems in the renal brush-border membrane [38,39].
Conclusions The plasma membrane of animal cells possesses necessary energy-transducing mechanisms to convert metabolic energy into the proton-motive force. It has been established beyond doubt in recent years that many solute transport systems in this membrane are energized by either the chemical component alone (ApHI or by a combination of the chemical and electrical components (ASH + AY) of the proton-motive force (Fig. 2). The direction of solute movement via these H+ -coupled transport systems is dictated by the direction of the electrochemical H+ gradient as well as the solute gradient
across the plasma membrane under normal physiological conditions. The net charge on the solute molecule appears to play a crucial role in determining the operational mechanism of the transport system. The substrates of the peptide transporter and the system ~-amino acid transporter normally exist as zwitterions at physiological pH. These transport systems operate via the solute-H + symport mechanism. They serve to transport the solutes into the cell against a concentration gradient and derive energy from the H+ gradient as well as the membrane potential. The substrates of the lactate transporter and the folate transporter are anions at physiological pH. Both transport systems are energized by a transmembrane H+ gradient, but are not influenced by the membrane potential. The electrically silent nature of these systems is the result of either solute-H+ symport or solute-OH-antiport. These two operational mechanisms, however, are phenomenologically indistinguishable. The lactate transporter can operate to move lactate either into or out of the cell, depending upon the intracellular and extracellular concentrations of lactate. The tissues such as erythrocyte, muscle and placenta, which generate lactate from cellular metabolism, transport lactate out of the cell via this transport system as a means of eliminating the potentialfy harmful acid load. On the other hand, under the conditions in which the cellular production of lactate is low, circulating lactate can be transported into these tissues via the same transporter for use. The folate transporter primarily functions to supply the cells with folate. The intestinal folate transporter is responsible for the absorption of dietary folate. The substrates of the organic cation transporter are positively charged at physiological pH. The transporter in most
699
cases functions to actively eliminate organ? cations from the cell via substrate-H+ antiport. Many compounds, exogenous (mostly xenobiotics) as well as endogenous, act as substrates for the system. The transporter derives its energy from a transmembrane H+ gradient. Because the system is electroneutral, its activity is not inlluenced by a membrane potential. In summary, the old dogma that the sodium-motive force has completely replaced the proton-motive force as the energy source for active transport of organic solutes in animal cells is no longer tenable. Prior to the discovery of the existence of energy-transducing mechanisms in the animal cell plasma membrane to generate the protonmotive force, there appeared to be no reason to question the idea of Na+ being the sole coupling ion in solute transport in animal cells. With the identification of the Na+-H+ exchanger and the H+ pump and with the im pressive number of H+-coupled solute transport systems which are now known to be present in the animal cell plasma membrane, there is enough reason to believe that H+ is also an important coupling ion for solute transport in animal cells.
man Intestinal Ceil Line, Caco-2. &o&m 1027:211-217. This report demonstrates of cephalexin, a substrate Caco-2 cell line. 11.
..
and recommended
12.
13.
14.
reading
Papers of special interest, published within the annual period of review, have been highlighted as:
. ..
of interest of outstanding
1.
KABACK HR: Molecular Biology of Membrane Transport From Membrane to Molecule to Mechanism. Hurug~ Lect 1989, 83:77-105.
2.
16.
interest
CRANE RK: The Gradient Hypothesis and Other Models of Carrier-mediated Active Transport. Rev Pbysiol Birxbem Phmacol
1977,
78:9’+159.
3.
Stculrlc~n, VP: Membrane-Linked Energy Transductions. Bioenergetic Functions of Sodium: H+ is not Unique as a Coupling Ion. Eurj Biccbem 1985, 151:199-208.
4.
GANAPATHYV, IXIFiACH FH: Is Intestinal Peptide Trans. port Energized by a Proton Gradient? Am J Pbysiof 1985, 249:G153G160.
5.
Jpn
J Plysiol
1985,
35:179191.
6.
GAN~PATHYV, LEtBACHFH: Carrier Mediated Reabsorption of SmaII Peptides in Renai Proximal Tubule. Am J PLysio/ 1986, 251:F94>F953.
7.
GANMATHY
V, MNAMOTO Y, LE~BACHFH: Driving Force for Peptide Transport in Mammaiian Kkirtey and Intestine. ConOib InJkdn
0.
9.
10.
..
l%er
Clin Nutr
1987,
17:54-&L
GANAPATHYV, Mxv~o’ro Y, IEIBACHFH: Properties and Phys iologicai Functions of the Mammalian RenaI Peptide Transport System. Mu Bbsci 1987, 65:91-98. Mmmaws DM: Protein Absorption. Development and Present State of the Subject. 1991, New York, NY: Wiley Iiss. D,wtzt~ AH, BERGH L Uptake of the Cephalosporin. Ccpbalexitt, by a Mpeptide Transporter Carrier In the Hu-
1990,
uphill transport transporter, in the
CALONGEML ILUNDAINA, BOLLJFER J: Giycyl-tSarcosIne Transport by ATPdepleted Isolated Enterocytes from Chicks. Am J Pbyciol 1990, 259zG775G780.
ONCE ML ILLINDAINA, BOLLIFERJ: Ionic Dependence of Glycylsarcosine Uptake by Isolated Chicken Enterocytes. / Cell P@siol 1989, 138:579-585. WIIX)N D, Bm J& RAMAs\vAhtvK: Characteristics of Tripeptide Transport in Human JejunaI Brush-border Membrane Vesicles. Biocbim Biophvs Acta 1989, 986123-129. TIRLIPPATHIC, KLI~ L P, GANAPATHYV, LEIBACHRI: Evidence for Tripeptide-H+ Cotransport in Rabbit Renai Brush Border Membrane Vesicles. Eiocbem J 1990, 268:27-33. T~RLIPPATHIC, MNAMOTO Y, GANAPATHY V, ROE~EL RA, Wurrroao GM, ~XIBACH FH: Hydroiysis and Transport of Proline-containing Peptides in Renal Brush-border Membrane Vesicles born Dipeptidyl Peptidase IV-positive and Dipeptidyl Peptidase IV-negative Rat Strains. J Biol &em 1990, 265:1476-1483. WATANABEY, KOJ~MAT, FUJ~MOTO Y: Deficiency of Membranebound Dipeptidyl Aminopeptidase IV in a Certain Rat Strain.
17. ..
Experientiu
1987. 43:4C+401.
T~RUPPATHIC, GANAPATHYV, &BACH FH: Evidence for Tripeptide-Proton Symport in Renal Brush Border Membrane Vesicles. Studies in a Novel Rat Strain with a Genetic Absence of Dipeptidyl Peptidase IV. J Biol Cbem 1990,
265:2048-2053. This report provides the first direct Aidence for the coupling of protonmotive force and tripeptide transport in the kidney. This study ‘was conducted in a unique rat strain (Japanese Fisher 344) which does not possess dipeptidyl peptidase IV activity. Because of the absence of the enzyme, tripeptides of the X-Pro-Y type remain unhydrotyzed during incubation with renal brush border membrane vesicles making it possible to investigate the properties of intact tripeptide transport. 18.
HOSHI T: Proton-Coupled Transport of Organic Solutes in AnitnaI CeU Membranes and Its Relation to Na+ Transport.
Acta
These investigators used a unique experimental system to demonstrate the energization of the intestinal peptide transporter by the protonmotive force. When chicken enterocytes were depleted of their ATP content, they behaved like large membrane vesicles and were suitable for uptake measurements. In these vesicles, the uptake of a dipeptide, Gly-Sar, was found to be Na+-independent. H+.dependent and electrogenic. The uptake was concentrative when the chemical (inwardly directed H+ gradient) and the electrical (inside-negative membrane potential) components of the proton-motive force were simultaneously present.
15.
References
the H+-gradient-dependent for the intestinal peptide
Biqdys
..
TIRUPPATH~C, GANAPAIHY V, IXIBACH FH: Kinetic Evidence for a Common Transporter for GlycyIsarcosine and Phenylalanylprolykakanine in Renal Brush Border Membrane Vesicles. J Biol &em 1990, 265:1487&14874.
In this investigation, interaction between a dipeptide, Gly-Sar, and a tripepride, Phe-Pro-Ala, during their transport in renal brush border membrane vesicles isolated from Japanese Fisher 344 rats wds studied. These rats do not possess dipeptidyl peptic&e N and, therefore, uptake of intact Phe-Pro-Ala could be studied. The classic A-B-C test was employed to determine whether a common transporter is involved in the transport of ci- and tripeptides. The results of the study suggest that transport of neutral di- and tripeptides across the renal brush-border membrane occurs via a common transporter. 19.
..
KRAMERW, Gtrrwnt U, GIRBIG F, IEIPE I: Intestinal Absorption of Dipeptides and j%Lactam Antibiotics. II. Puriftcation of the BindIng Protein for Dipeptides and B-Lactam Antibiotics from Rabbit SmaR Intestinal Brush Border Membranes. Bicdim BiqLys Acka 1990, 1030~50-59.
This is the first report to claim puritication of the intestinal peptide transporter. The purified protein has a molecular weight of 127 kD and is preferentially labelled with phototeacdve derivatives of the substrates of the peptide transporter. Po!yclonal antibodies raised against this pro-
Proton-coupled
solute
transport
tein are able to block the catalytic function of the peptide transporter in intestinal brush border membrane vesicles. 20. .
KRAMERW, DECHENTC, GIRFJIGF, GUTJAHRU, NEUBAUERH: intestinal Uptake of Dipeptldes and p-Lactam Antibiotics. 1. The Intestinal Uptake *tern for Dipeptides and p-Lactam Antibiotics is not Part of a Brush Border Membrane Peptidase. Biocbim BiqLys Acka 1990 1030:41-@. This study documents the physical separation of the putative dipeptide transport protein of the intestinal brush-border membrane from two of the major brush-border peptidases, aminopeptidase N and dipeptidyi pepridase Iv. 21. ..
MNMOTO Y, THOMP.%~NYG, HOWARD EF, GANAPATHYV, hIBACH FH: Functional Expression of the Intestinal PeptideProton Cotransponer in Xenopus laevis Oocytes. J Biol Cbem 1990, 265~4742-4745. This paper describes functional expression of the intestinal peptide transporter in Xenq~us oocytes following injection of poly(A)+ mRh!A isolated from rabbit intestinal mucosal cells. The expressed transporter exhibits the substrate specilicity and the H+.dependence chatacteristics similar to the native rabbit intestinal peptide transporter. 22.
KRAMERW, II~~E I, PE’IZOLDTE, G~RBIGF: Characterization of the Transport System for /3-Lactam Antibiotics and Dipep tides in Rat Renal Brush Border Membrane Vesicles by PhotoalTinity Labeling. Biocbim Biq2ys Acfu 1988, 939:167-172.
23.
KRAMERW, DURCKHE~MER W, G~IG F, GUTJAHRU, LEIPE I, OEKONOMOPU~~~RI InlIuence of Amino Acid Side-chain Modification on the Uptake System for p-Lactam Antibiotics and Dipeptides t%om Rabbit Small Intestine. Biochim Sic&s Acka 1990, 1028:174-182. MNAMO’T’OY, GANAPA-IHYV, LEIBACHFH: Identification of Histidy1 and Thiol Groups at the Active site of Rabbit Renal Dipeptide Transporter. J Biol &em 1986, 261:16133-16140.
24.
25.
IQ70 M, MAEGAWAH, OKANO T, INUI KI, Horn R: Effect of Various Chemical Modifiers on H+ Coupled Transport of Cepharadine via Dipeptide Carriers in Rabbit lntestinal Brush Border Membranes: Role of Hlstidine Residues. J Phmacol .!3$1 k 1989, 251:74+749.
26.
MNA~%OTOY, TIRUPPA’IHIC, GANAPATHYV, LEBACH FH: Involvement of Thiol Groups in the Function of the Dipep tide/Proton Cotranspott System in Rabbit Renal BrushBorder Membrane Vesicles. Biocbim Biqpbys Acta 1989, 978:2>31.
27. .
DYER J, BEECHJZYRB, GORVEL JP, Shlrm RT, WINSTON R, SHIRAZI-BEECH!ZY SP: Giycyl-L-Proline Transport in Rabbit Enterocyte Basolateral Membrane Vesicles. Biocbem J 1990, 269:56>571. Until now, there has been no direct study on peptide transport in isolated intestinal basolateral membrane vesicles. This paper is the first to demonstrate the presence of a peptide transporter in this membrane. Interestingly, this carrier also exhibits the H+-dependence characteristic, as does the brush border membrane peptide transporter. 28 ..
Bou, SJ, ~YD JB: Evidence for a Dipe.ptide Porter in the Lysosomal Membrane. Ebchim Bic@ys Acfu 1990, 1024267-270.
in the animal
cell plasma
membrane
Ganapathy
and Leibach
This study is, in a way, a reinvestigation of the earlier observations on the msomal handling of dipeptides. The presence of a transport mechanism for dipeptides was never invoked in earlier SNdieS. In contrast, experimental data from the present Study provide a strong argument in favor of the existence of a specific transport mechanism for dipeptides in the lysosomal membrane. GANAPATHY V, PA~HUZYDH, FONTELESMC, LEIBACHFH: Pepti29. dases and Peptide Transport in Mammalian Kidney. Cunti Nephrol 1984, 42:10-18. M~LIMOTO Y, SATO K, OH-YASHKIT, MOHRIT: Leucine-Proton 30. Cotransport System in Chang Liver Cell. J Bid C&m 1986, 261:4549-4554. 31. M~UMOTO Y, MOHRI T: Reconstitution of the L-LeucineH+ Cotransponer of the Plasma Membrane from Chang Liver Cells into Proteoliposomes. Biocbim Biqdys Acta 1991, 1061:171-174. TIRUPPATHIC, B~LKO~E’IZ DF, GANAPATHYV, MNAMOTO Y, 32. IIIEIACH FH: A Proton Gradient, not a Sodium Gradient, is the Driving Force for Active Transport of Lactate in Rabbit Intestinal Brush-Border Membrane Vesicles. Bkxbem J 1988, 256:21‘+233. B~ow-rz DF, LEIEZACH FH, MN~E~HVB, GANA~ATHYV: A Pro33. ton Gradient is the Driving Force for Uphill Transport of Lactate in Human Placental Brush-Border Membrane Vesicles. J Biol Gem 1988, 263:1382%13830. HALESTRAP AP, POOLERC, CRANMERSL: Mechanisms and Reg 34. ulation of Lactate, Pyruvate and Ketone Bcdy Transport Across the Plasma Membrane of Mammalian Cells and their Metabolic Consequences. Biocbem Sot Truns 1990, 18:1132-1135. 35. SCHRONCM, WASHINGTONC JR, BLAZER BL: The Transmembrane pH Gradient Drives Uphill Folate Transport in Rabbit Jejunum. Direct Evidence for Folate/Hydroxyl Exchange in Brush Border Membrane Vesicles. J Clin Invest 1985, 76:203%2033. Ross CR, HOWHAN PD: Transport of Organic Anions and 36. Cations in Isolated Renal Plasma Membranes. Annu Reu Pbannacol Toxicol 1983, 23:65-85. HOSHIT, TAKLKVAN, ABE M, TAJIMAk Hydrogen Ion-coupled 37. Transport of ~-Glucose by Pblorizin-sensitive Sugar Carrier in Intestinal Brush-border Membranes. Biocbim BiqXys Acta 1986, 861:483-Q%. ROIGAARD-PETERSEN H, JACOBSENC, SHEIKH Ml: H+-L-Proline 38. Cotransport by Vesicles from Pars Convoluta of Rabbit Proximal Tubule. Am J P&siof 1987, 253:F15-F20. RAJENDRAN V-M. BARRYJA, KLEINMANJG, RAMASWAMYK: Pro39. ton Gradient-dependent Transport of Glycine in Rabbit Renal Brush-border Membrane Vesicles. J Biol &em 1987, 262314974-14977.
V Ganapathy and FH Leibach, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912, USA
701
solute transport in the animal membrane
Vadivel
Canapathy
Medical
College
and Frederick
of Georgia,
Augusta,
cell plasma
H. Leibach
Georgia,
USA
This review focuses primarily on the progress made in the last couple of years in the understanding of the intestinal peptide transporter, a prototype for H+-coupled solute transport systems in the animal cell plasma membrane. The impressive number of transport systems currently known to be energized by the components of the proton-motive force indicates that the role of H+ as the coupling ion for active transport has not been lost during evolution. Current
Opinion
in Cell Biology
Introduction
In biological systems, active transport of organic solutes is coupled primarily to simu1taneous transport of inorganic ions. The free energy released during the movement of the inorganic ions down a concentration gradient is used as the driving force to move the organic solutes against a concentration gradient. In bacterial systems, the principal inorganic ion used for this purpose is H+. The electrochemical H+ gradient (proton-motive force, ASH), the energy source for the H+ -coupled transport systems, is located across the bacterial cell membrane and consists of an electrical component (AY) and a chemical component (ApH). This gradient is generated either by respiration or ATP hydrolysis. The Escbticbia coli lactose-pennease system can be considered as the prototype of H + coupled solute transport systems in bacteria [ 11. The principal coupling ion in animal cells appears to be Na+ rather than H+ [2]. The electrochemical Na+ gradient (sodium-motive force, AuNa), the energy source for the Na +-coupled transport systems, exists across the animal cell plasma membrane and is generated by the Na+-K+ pump. In recent years, evidence has accumulated which indicates that the difference in the preferred inorganic ion between bacterial (H+> and animal (Na+> systems is not absolute. A number of Na+-coupled solute transport systems have recently been discovered in bacteria [3]. Likewise, a number of organic-solute transport systems in animal cells have been shown to be coupled to H+ rather than Na+. The animal cell plasma membrane possesses mechanisms to generate the chemical as well as the electrical components of the proton-motive force (Fig. 1). The Na+-K+ pump converts the free energy released from ATP hydrolysis into the chemical and the electrical gradients of Na+ (ApNa and AY). The Na+-H+ exchanger, known to be present in most cell types, converts ApNa to ApH. Thus, the concerted actions of the Na+-K+ pump and the Na+-H+ exchanger result in the produc@ Current
Biology
1991, 3:695-701
tion of the proton-motive force. The H+ pump, which has been shown to be present in at least some cell types, directly generates ASH and AY from ATP hydrolysis. The proton-motive force is the energy source for many solute transport systems in animal cells. Among these, the peptide transporter can be considered as the prototype of H+ coupled solute transport systems of the animal cell plasma membrane. This transporter is of major physiological importance because of its participation in the absorption of an important group of nutrients in the small intestine and the kidney. It uses both the chemical and the electrical components of the proton-motive force as its energy source, and the progress made in understanding the molecular characteristics of this transporter has been more impressive than that made with other H+ coupled solute transport systems [4-S]. The last 2 years have seen dramatic breakthroughs in different aspects of this transporter. Significant progress has also been made in understanding other H+ coupled transport systems. The purpose of the current review is to highlight these recent advances in the area of H+ coupled solute transport systems in animal cells.
The peptide
transporter
Direct evidence for the coupling of the proton-motive force to active transport of peptides in the small intestine
Although a number of studies have unequivocally demonstrated proton-motive-force-dependent, active transport of small peptides in brush-border membrane vesicles derived from the kidney, the results with intestinal brushborder membrane vesicles have been conflicting. The stimulation of peptide transport in these vesicles by an inwardly directed H+ gradient (APH) has been shown in many studies. Similarly, the electrogenic nature of the H+ -dependent peptide transport has also been demonstrated in different laboratories. However, the overshoot Ltd ISSN 0955-0674
695
6%
mmbrane
permeability
Na+
Scheme’
2K+
Na+-K+
Energy
transduction
3Na’
pump
ATP + ApNa + A’?
ADP
H’
4 N
Nat
Hi
ApNa
H’
exchanger
tf
ApH
phenomenon, which is indicative of active transport against a concentration gradient in membrane vesicles, has been observed in some studies but not in others. (Readers are referred to [9] for a detailed analysis of available information on this particular aspect of peptide transport.) The absence of uphill transport in such studies might have resulted from an inability to maintain the experimentally imposed proton-motive force in isolated membrane vesicles. Two recent reports, however, by Dantzig and Bergin [lo**] and Calonge et al. [ll**], have provided unequivocal evidence for the energization of the intestinal peptide transporter by the proton-motive force. Dantzlg and Bergin used the Caco-2 ceU line as a model for studying the characteristics of the intestinal peptide transporter. In these cells, uptake of cephalexin, a @lactarn antibiotic known to be transported via the peptide transporter, was Na + -independent and exhibited an acidic extracellular pH optimum. A cellmedium ratio greater than one was observed in a Na+ -free buffer with an extracellular pH of 6.0. The uptake was shown to be dependent on metabolic energy because poisons such as oligomycin, azide and dinitrophenol signiUcantly inhibited it. Protonophores, which collapse a transmembrane H+ gradient, also caused marked inhibition of the uptake. These data provide direct evidence for the energization of the peptide transporter by an inwardly directed ttansmembrane H+ gradient in Caco-2 cells. Although the role of a tr-ansmembrane potential difference in the catalytic function of the transporter was not investigated in this study, as the experiments were conducted on intact cells it is most likely that the electrical component of the proton-motive force also contributed to the energization of the peptide transporter. Calonge et al. [ ll**] demonstrated uphill transport of a dipeptide in the small intestine with the proton-motive force as the energy source using ATP-depleted chicken intestinal cells, which behave like large membrane vesicles. This experimental system has numerous advan-
H+-pump
ATP
+
ApH
+ A’+’
Fig. 1. Transport systems which generate ApH+. AY is the electrical component of the electrochemical gradient. ApH and ApNa represent the chemical component.
tages over isolated intestinal brush-border membrane vesicles. The chicken intestinal cells have a large intracellular volume and are not subject to the possible alterations that normally occur during the isolation of brushborder membrane vesicles, such as cell lysis, divalent cation aggregation and resealing. In these ATP-depleted cells, uptake of Gly-Sar was Na+-independent, but stimulated by an inwardly directed H+ gradient. Generation of an inside-negative membrane potential also increased Gly-Sar uptake. Although concentrative uptake was not evident in the presence of either the chemical component (A~H) or the electrical component (AW of the protonmotive force, when both components were simultaneously present there was clear evidence for a transient uphill accumulation of Gly-Sar. This study provided direct evidence for the energization of the intestinal peptide transporter by the proton-motive force. The same investigators had previously demonstrated in intact chicken enterocytes that Gly-Sar uptake was active, partially dependent on Na+ , showed an acidic extracellular pH optimum and was sensitive to inhibition by amiloride, an inhibitor of the Na+-H+ exchanger [ 121. This can be taken as strong evidence for the role of the Na+-H+ exchanger in the generation of the transmembrane H+ gradient across the intestinal brush-border membrane in metabolically active, intact enterocytes. At the present time, it appears that the idea of the protonmotive force being the energy source for intestinal peptide transport has not been widely accepted among investigators in the area of intestinal absorption. This apparent skepticism is evident when one peruses the currently available textbooks on gastrointestinal physiology. Many of these textbooks still either state that intestinal peptide transport is energized by an electrochemical Na+ gradient or seem to have taken a somewhat ‘middle of the road position’ stating that the mechanism of peptide transport is controversial. It is our hope that the two reports men-
Proton-coupled
solute transport
tioned above will enhance the acceptance of the protonmotive force as the driving force for the intestinal absorption of small peptides and remove the skepticism that currently exists in the minds of the textbook writers.
Tripeptides
as substrates
for the peptide
transporter
In the past, most studies on peptide transport used only dipeptides as substrates and any conclusion drawn about whether the peptide transporter recognizes longer peptides as substrates was based merely upon competition experiments. However, because these competition experiments were all done under conditions in which peptidases associated with the brush-border membrane can potentially hydrolyze the test peptides to generate dipeptides, the possibility exists that the observed competition in these experiments was not real. Non-availability of radioactive tripeptides and hydrolysis of most tripeptides by brush-border peptidases are the primary reasons for the lack of direct studies on tripeptide transport. Recently, however, four papers have appeared in the literature that directly demonstrate intact transport of tripeptides and its energization by a proton-motive force. In 1989, Wilson et al. [ 131 reported on the characteristics of the transport of an intact tripeptide, GlyGly-Pro, in isolated intestinal brush-border membrane vesicles. In this report, hydrolysis of the test peptide was prevented by differential removal of the membrane-bound peptidases by papain digestion. Uptake of intact Gly-Gly-Pro could be demonstrated in these peptidase-depleted membrane vesicles. One year later, our laboratory reported similar results in renal brush-border membrane vesicles with Phe-Pro-Ala as the tripeptide substrate, though in this study hydrolysis of the peptide was prevented by irreversibly inhibiting dipeptidyl peptidase N, the enzyme responsible for breakdown of X-ProY type peptides [ 141. More recently, we took another approach which enabled us to investigate directly the transport of peptides containing three or more amino acids without interference from peptide hydrolysis [ 151. In this approach, a novel rat strain (Japanese Fisher 344) [ 161, which exhibits a genetic deficiency of dipeptidyl peptidase N was used. Because of the absence of the enzyme, purified renal brush-border membrane vesicles were unable to hydrolyze Phe-Pro-Ala. With these membrane vesicles, we could demonstrate that the tripeptide was transported into the vesicles in intact form [ 17**]. Uptake of the tripeptide was found to be Na+-independent, but was stimulated by an inwardly directed H+ gradient. Transient accumulation of the peptide against a concentration gradient was evident in the presence of the H+ gradient. The H+ -gradient-dependent uptake was electrogenie. This was the first demonstration of H+ -gradientdriven tripeptide uptake. Availability of this unique system for studying tripeptide transport made it possible for further investigations to delineate the competitive interaction between dipeptides and tripeptides with the peptide transporter [ 180~1. These studies have unambiguously demonstrated that a common transporter is responsible for the uptake of di- and tripeptides containing neutral amino acids.
E
in the animal
cell plasma
membrane
Ganapathy and Leibach
Because the Japanese Fisher 344 rats lack dipeptidyi peptidase N not only in the kidney but also in the small intestine, this particular rat strain will become very useful in future studies for investigating the characteristics of tripeptide transport in the small intestine. Moreover, uptake of longer peptides containing four or five ammo acids can also be studied in intestinal and renal brushborder membrane vesicles isolated from this rat strain without interference from hydrolysis by carefully selecting peptides of particular amino acid composition and sequences. Studies along these lines are currently underway in our laboratory.
The chemical
nature
of the peptide
transporter
One of the primary goals of studies on a transport system is to analyze the chemical nature of the proteins responsible for the transport function in order to understand the chemical basis of the transport mechanism. This can be achieved by purifying the membrane proteins responsible for the transport function to homogeneity and/or cloning the genes coding for these proteins. Significant progress has been made in the last year in this area with respect to the intestinal peptide transporter. Kramer et al. [ 19.01 have apparently purified the intestinal peptide transporter. The molecular weight of the transport protein is 127 kD. The evidence for its identity with the peptide transporter is as follows: the protein is preferentially labelled by photoreactive derivatives of dipeptides and j3-lactam antibiotics; the photolabelling is specifically inhibited by substrates of the transporter; and the polyclonal antibodies raised against this protein are able to inhibit the catalytic function of the peptide transporter in isolated intestinal brush-border membrane vesicles and also block the photoaffinity labelling of the protein. However, the final and unequivocal proof for the identity of this protein with the peptide transporter will be to demonstrate that this protein, when incorporated into art&ial membrane vesicles, does indeed carry out a peptide transport function. But this evidence is yet to come. Kramer et al [20*] have also demonstrated that the 127kD protein is not part of a brush-border peptic&e. The protein could be physically separated from the major peptidases of the brush-border membrane, namely aminopeptidase N and dipeptidyl peptidase N. These Sndings are interesting because it has often been hypothesized in the past that the peptic&es of the intestinal and renal brush-border membranes may indeed catalyze the transmembrane movement of amino acids and/or peptides. The study by Kramer et al [20*] disproves this hypothesis. Of relevance to this particular point are our own observations that the renal brush-border membrane vesicles isolated from the Japanese Fisher 344 rats do not possess the dipeptidyl peptidase N protein, but exhibit normal peptide transport activity [15]. We have recently succeeded in expressing the intestinal peptide transporter in Xen0pu.r laev& oocytes in a functional form [21**]. Following microinjection of rabbit intestinal poly(A) + mRNA, expression of an exogenous
697
698
Membrane
permeability
peptide transporter in the oocytes can be demonstrated by measuring Gly-Sar uptake. The expressed transporter exhibits characteristics similar to those of the transporter studied in rabbit intestinal brush-border membrane vesicles. The transporter is speciiic for peptides and its activity is stimulated by an inwardly directed H+ gradient. This oocyte system can become a useful assay technique for the expression cloning of the intestinal peptide transporter. Although the brush border membranes isolated from the small intestine and the kidney possess peptide transport activities with similar properties, there is accumulating evidence that the proteins responsible for the transport function in these membranes may not be identical. The labelling pattern with the photoreactive derivatives of transporter substrates in intestinal and renal brush-border membranes are markedly different [ 22,231. The two transport systems also differ in their inhibition by various amino-acid-group-specilic reagents [ 23-261.
Other
interesting
aspects of peptide
transport
It is widely accepted that while a majority of proteindigestion end products enter the enterocytes across the brush-border membrane in the form of small peptides, they exit across the basolateral membrane predominantly as free amino acids. This happens because of the efficient intracellular hydrolysis of peptides by cytosolic peptidases. However, there is substantial evidence in intact tissue preparations that a small, but significant, amount of intact peptides do cross the basolateral membrane. This suggests that the basolateral membrane may possess mechanisms for peptide transport. But, until recently, there was no direct study on the characteristics of peptide transport across the basolateral membrane. The report by Dyer et al [27*] is the first to investigate basolateral membrane peptide transport. The basolateral membrane vesicles used in this study were highly enriched in ouabain-sensitive, K+ -activated phosphatase with no detectable contamination from brush-border membranes as assessed by cross-reaction with antibodies against the sodium-glucose symporter. Uptake of Gly-Pro in these vesicles was carrier-mediated and was stimulated by an inwardly directed H+ gradient. This stimulation was inhibited by protonophores.The results of the study thus indicate that there is a peptide transporter in the enterocyte basolateral membrane. It is very likely that this transporter plays a role in the exit of intact peptides from the cells across the basolateral membrane. Another recent paper on peptide transport, by Bird and Lloyd [28**], deserves attention as it provides evidence for the presence of a peptide transporter in the lysosomal membrane. Lysosomes participate in the breakdown of cellular proteins which enter this organelle by processes involving endocytosis and pinocytosis. There is no definite information available on the nature and fate of end products of lysosomal protein digestion. We speculated almost a decade ago that, similar to protein digestion and absorption in the small intestine, breakdown of proteins within the lysosomes may not be complete and
that the end products may in fact consist of free amino acids as well as small peptides which exit the lysosomes via specific transporters located in the lysosomal membrane [29]. This idea was very diIferent from the generally held view at that time that free amino acids were the primary end products of lysosomal protein digestion and that these end products diRused out of lysosomes to be used by the cell, Between that time and now, numerous papers have appeared in the literature demonstrating carriers for a variety of amino acids in the lysosomal membrane. In fact, the different amino acid transport systems now known to be present in the lysosomal membrane are as many and diverse as those of the plasma membrane. Although handling of dipeptides by lysosomes has been investigated in the past, the existence of a specific transporter for these compounds was never invoked. The recent discovery of transport systems not only for amino acids but also for compounds such as glucose and sialic acid has led Bird and Lloyd [28**] to reinvestigate the lysosomal handling of dipeptides. Using an ‘osmotic protection’ method, these investigators analyzed the passage of dipeptides across the lysosomal membrane. It was concluded from the study that lysosomes possess a transport mechanism which accepts L-dipeptides as substrates but excludes D-dipeptides. It is very likely that this transporter catalyzes extrusion of dipeptides arising from intra-lysosomal protein breakdown. The involvement of H+ in the catalytic activity of the lysosomal dipeptide transporter has not been investigated in this study. However, it is tempting to speculate that, because the intenor of the lysosome has an acidic environment with a H+gradient across the lysosomal membrane in the direction of lysosome-to-cytoplasm, the H+ gradient is the most logical driving force for active extrusion of dipeptides from the lysosome.
Other
H+-coupled
solute transport
systems
Evidence has been presented in recent years that the system ~-amino acid transporter, which is Na+ -independent, is indeed energized by the proton-motive force in certain animal cells [30]. This system operates via the amino acid-H+ symport mechanism and uses both the chemical and the electrical components of the proton-motive force as the energy source. This transport system has recently been reconstituted in proteoliposomes and the reconstituted transporter exhibits H+ dependence [31]. The lactate transporter is another system which uses a transmembrane H+ gradient as the driving force. The lactateH+ symport (or phenomenologically indistinguishable lactate-hydroxyl exchange) is electroneutral and hence is not inlluenced by transmembrane electrical potentials. Those cells which actively produce lactic acid possess this transport system. The transporter most probably plays a role in the protection of the cell from potential acid-loading as a result of accumulation of lactic acid. The presence of this transporter has been documented in erythrocytes, cardiac muscle, skeletal muscle, small intestine and placenta [32-341. Intestinal absorption of folate is also
Proton-coupled
X
solute
transport
H’
X-
in the animal
cell plasma
H+
membrane
Canapathy and Leibach
X-
OH
X
H’
X-
OH-
Xc
H+
Scheme
Mechanism
Example
Substrate-H’
symport
Substrate-H’
Peptide transporter System r-amino acid transporter
symport
Lactate
ApH + A't'
Driving force
2. Transport
systems
which
use
transporter,
Substrate-OH-
folate
antiport
Substrate-H+
transporter
Organic
APH
1 Fig.
or
antiport
cation
transporter
APH -L
ApH+.
H+ -gradient dependent, and the mechanism of transport is either folate-hydroxyl exchange or its phenomenological equivalent, folate-H+ symport [35]. Another interesting system which is coupled to a transmembrane H+ gradient is the organic cation-H + antiporter [ 361. This system is electrically silent and has been described in the kidney, intestine and placenta. The mechanistic aspects of the transport systems discussed above are illustrated in Fig. 2. Ln addition to the above-mentioned transport systems which exclusively use the proton-motive force components as the driving force, there are other systems in arimal cells which accept either Na+ or H+ as the co-substrate. This includes the glucose transporter in the intestinal brush-border membrane [37], and various amino acid transport systems in the renal brush-border membrane [38,39].
Conclusions The plasma membrane of animal cells possesses necessary energy-transducing mechanisms to convert metabolic energy into the proton-motive force. It has been established beyond doubt in recent years that many solute transport systems in this membrane are energized by either the chemical component alone (ApHI or by a combination of the chemical and electrical components (ASH + AY) of the proton-motive force (Fig. 2). The direction of solute movement via these H+ -coupled transport systems is dictated by the direction of the electrochemical H+ gradient as well as the solute gradient
across the plasma membrane under normal physiological conditions. The net charge on the solute molecule appears to play a crucial role in determining the operational mechanism of the transport system. The substrates of the peptide transporter and the system ~-amino acid transporter normally exist as zwitterions at physiological pH. These transport systems operate via the solute-H + symport mechanism. They serve to transport the solutes into the cell against a concentration gradient and derive energy from the H+ gradient as well as the membrane potential. The substrates of the lactate transporter and the folate transporter are anions at physiological pH. Both transport systems are energized by a transmembrane H+ gradient, but are not influenced by the membrane potential. The electrically silent nature of these systems is the result of either solute-H+ symport or solute-OH-antiport. These two operational mechanisms, however, are phenomenologically indistinguishable. The lactate transporter can operate to move lactate either into or out of the cell, depending upon the intracellular and extracellular concentrations of lactate. The tissues such as erythrocyte, muscle and placenta, which generate lactate from cellular metabolism, transport lactate out of the cell via this transport system as a means of eliminating the potentialfy harmful acid load. On the other hand, under the conditions in which the cellular production of lactate is low, circulating lactate can be transported into these tissues via the same transporter for use. The folate transporter primarily functions to supply the cells with folate. The intestinal folate transporter is responsible for the absorption of dietary folate. The substrates of the organic cation transporter are positively charged at physiological pH. The transporter in most
699
cases functions to actively eliminate organ? cations from the cell via substrate-H+ antiport. Many compounds, exogenous (mostly xenobiotics) as well as endogenous, act as substrates for the system. The transporter derives its energy from a transmembrane H+ gradient. Because the system is electroneutral, its activity is not inlluenced by a membrane potential. In summary, the old dogma that the sodium-motive force has completely replaced the proton-motive force as the energy source for active transport of organic solutes in animal cells is no longer tenable. Prior to the discovery of the existence of energy-transducing mechanisms in the animal cell plasma membrane to generate the protonmotive force, there appeared to be no reason to question the idea of Na+ being the sole coupling ion in solute transport in animal cells. With the identification of the Na+-H+ exchanger and the H+ pump and with the im pressive number of H+-coupled solute transport systems which are now known to be present in the animal cell plasma membrane, there is enough reason to believe that H+ is also an important coupling ion for solute transport in animal cells.
man Intestinal Ceil Line, Caco-2. &o&m 1027:211-217. This report demonstrates of cephalexin, a substrate Caco-2 cell line. 11.
..
and recommended
12.
13.
14.
reading
Papers of special interest, published within the annual period of review, have been highlighted as:
. ..
of interest of outstanding
1.
KABACK HR: Molecular Biology of Membrane Transport From Membrane to Molecule to Mechanism. Hurug~ Lect 1989, 83:77-105.
2.
16.
interest
CRANE RK: The Gradient Hypothesis and Other Models of Carrier-mediated Active Transport. Rev Pbysiol Birxbem Phmacol
1977,
78:9’+159.
3.
Stculrlc~n, VP: Membrane-Linked Energy Transductions. Bioenergetic Functions of Sodium: H+ is not Unique as a Coupling Ion. Eurj Biccbem 1985, 151:199-208.
4.
GANAPATHYV, IXIFiACH FH: Is Intestinal Peptide Trans. port Energized by a Proton Gradient? Am J Pbysiof 1985, 249:G153G160.
5.
Jpn
J Plysiol
1985,
35:179191.
6.
GAN~PATHYV, LEtBACHFH: Carrier Mediated Reabsorption of SmaII Peptides in Renai Proximal Tubule. Am J PLysio/ 1986, 251:F94>F953.
7.
GANMATHY
V, MNAMOTO Y, LE~BACHFH: Driving Force for Peptide Transport in Mammaiian Kkirtey and Intestine. ConOib InJkdn
0.
9.
10.
..
l%er
Clin Nutr
1987,
17:54-&L
GANAPATHYV, Mxv~o’ro Y, IEIBACHFH: Properties and Phys iologicai Functions of the Mammalian RenaI Peptide Transport System. Mu Bbsci 1987, 65:91-98. Mmmaws DM: Protein Absorption. Development and Present State of the Subject. 1991, New York, NY: Wiley Iiss. D,wtzt~ AH, BERGH L Uptake of the Cephalosporin. Ccpbalexitt, by a Mpeptide Transporter Carrier In the Hu-
1990,
uphill transport transporter, in the
CALONGEML ILUNDAINA, BOLLJFER J: Giycyl-tSarcosIne Transport by ATPdepleted Isolated Enterocytes from Chicks. Am J Pbyciol 1990, 259zG775G780.
ONCE ML ILLINDAINA, BOLLIFERJ: Ionic Dependence of Glycylsarcosine Uptake by Isolated Chicken Enterocytes. / Cell P@siol 1989, 138:579-585. WIIX)N D, Bm J& RAMAs\vAhtvK: Characteristics of Tripeptide Transport in Human JejunaI Brush-border Membrane Vesicles. Biocbim Biophvs Acta 1989, 986123-129. TIRLIPPATHIC, KLI~ L P, GANAPATHYV, LEIBACHRI: Evidence for Tripeptide-H+ Cotransport in Rabbit Renai Brush Border Membrane Vesicles. Eiocbem J 1990, 268:27-33. T~RLIPPATHIC, MNAMOTO Y, GANAPATHY V, ROE~EL RA, Wurrroao GM, ~XIBACH FH: Hydroiysis and Transport of Proline-containing Peptides in Renal Brush-border Membrane Vesicles born Dipeptidyl Peptidase IV-positive and Dipeptidyl Peptidase IV-negative Rat Strains. J Biol &em 1990, 265:1476-1483. WATANABEY, KOJ~MAT, FUJ~MOTO Y: Deficiency of Membranebound Dipeptidyl Aminopeptidase IV in a Certain Rat Strain.
17. ..
Experientiu
1987. 43:4C+401.
T~RUPPATHIC, GANAPATHYV, &BACH FH: Evidence for Tripeptide-Proton Symport in Renal Brush Border Membrane Vesicles. Studies in a Novel Rat Strain with a Genetic Absence of Dipeptidyl Peptidase IV. J Biol Cbem 1990,
265:2048-2053. This report provides the first direct Aidence for the coupling of protonmotive force and tripeptide transport in the kidney. This study ‘was conducted in a unique rat strain (Japanese Fisher 344) which does not possess dipeptidyl peptidase IV activity. Because of the absence of the enzyme, tripeptides of the X-Pro-Y type remain unhydrotyzed during incubation with renal brush border membrane vesicles making it possible to investigate the properties of intact tripeptide transport. 18.
HOSHI T: Proton-Coupled Transport of Organic Solutes in AnitnaI CeU Membranes and Its Relation to Na+ Transport.
Acta
These investigators used a unique experimental system to demonstrate the energization of the intestinal peptide transporter by the protonmotive force. When chicken enterocytes were depleted of their ATP content, they behaved like large membrane vesicles and were suitable for uptake measurements. In these vesicles, the uptake of a dipeptide, Gly-Sar, was found to be Na+-independent. H+.dependent and electrogenic. The uptake was concentrative when the chemical (inwardly directed H+ gradient) and the electrical (inside-negative membrane potential) components of the proton-motive force were simultaneously present.
15.
References
the H+-gradient-dependent for the intestinal peptide
Biqdys
..
TIRUPPATH~C, GANAPAIHY V, IXIBACH FH: Kinetic Evidence for a Common Transporter for GlycyIsarcosine and Phenylalanylprolykakanine in Renal Brush Border Membrane Vesicles. J Biol &em 1990, 265:1487&14874.
In this investigation, interaction between a dipeptide, Gly-Sar, and a tripepride, Phe-Pro-Ala, during their transport in renal brush border membrane vesicles isolated from Japanese Fisher 344 rats wds studied. These rats do not possess dipeptidyl peptic&e N and, therefore, uptake of intact Phe-Pro-Ala could be studied. The classic A-B-C test was employed to determine whether a common transporter is involved in the transport of ci- and tripeptides. The results of the study suggest that transport of neutral di- and tripeptides across the renal brush-border membrane occurs via a common transporter. 19.
..
KRAMERW, Gtrrwnt U, GIRBIG F, IEIPE I: Intestinal Absorption of Dipeptides and j%Lactam Antibiotics. II. Puriftcation of the BindIng Protein for Dipeptides and B-Lactam Antibiotics from Rabbit SmaR Intestinal Brush Border Membranes. Bicdim BiqLys Acka 1990, 1030~50-59.
This is the first report to claim puritication of the intestinal peptide transporter. The purified protein has a molecular weight of 127 kD and is preferentially labelled with phototeacdve derivatives of the substrates of the peptide transporter. Po!yclonal antibodies raised against this pro-
Proton-coupled
solute
transport
tein are able to block the catalytic function of the peptide transporter in intestinal brush border membrane vesicles. 20. .
KRAMERW, DECHENTC, GIRFJIGF, GUTJAHRU, NEUBAUERH: intestinal Uptake of Dipeptldes and p-Lactam Antibiotics. 1. The Intestinal Uptake *tern for Dipeptides and p-Lactam Antibiotics is not Part of a Brush Border Membrane Peptidase. Biocbim BiqLys Acka 1990 1030:41-@. This study documents the physical separation of the putative dipeptide transport protein of the intestinal brush-border membrane from two of the major brush-border peptidases, aminopeptidase N and dipeptidyi pepridase Iv. 21. ..
MNMOTO Y, THOMP.%~NYG, HOWARD EF, GANAPATHYV, hIBACH FH: Functional Expression of the Intestinal PeptideProton Cotransponer in Xenopus laevis Oocytes. J Biol Cbem 1990, 265~4742-4745. This paper describes functional expression of the intestinal peptide transporter in Xenq~us oocytes following injection of poly(A)+ mRh!A isolated from rabbit intestinal mucosal cells. The expressed transporter exhibits the substrate specilicity and the H+.dependence chatacteristics similar to the native rabbit intestinal peptide transporter. 22.
KRAMERW, II~~E I, PE’IZOLDTE, G~RBIGF: Characterization of the Transport System for /3-Lactam Antibiotics and Dipep tides in Rat Renal Brush Border Membrane Vesicles by PhotoalTinity Labeling. Biocbim Biq2ys Acfu 1988, 939:167-172.
23.
KRAMERW, DURCKHE~MER W, G~IG F, GUTJAHRU, LEIPE I, OEKONOMOPU~~~RI InlIuence of Amino Acid Side-chain Modification on the Uptake System for p-Lactam Antibiotics and Dipeptides t%om Rabbit Small Intestine. Biochim Sic&s Acka 1990, 1028:174-182. MNAMO’T’OY, GANAPA-IHYV, LEIBACHFH: Identification of Histidy1 and Thiol Groups at the Active site of Rabbit Renal Dipeptide Transporter. J Biol &em 1986, 261:16133-16140.
24.
25.
IQ70 M, MAEGAWAH, OKANO T, INUI KI, Horn R: Effect of Various Chemical Modifiers on H+ Coupled Transport of Cepharadine via Dipeptide Carriers in Rabbit lntestinal Brush Border Membranes: Role of Hlstidine Residues. J Phmacol .!3$1 k 1989, 251:74+749.
26.
MNA~%OTOY, TIRUPPA’IHIC, GANAPATHYV, LEBACH FH: Involvement of Thiol Groups in the Function of the Dipep tide/Proton Cotranspott System in Rabbit Renal BrushBorder Membrane Vesicles. Biocbim Biqpbys Acta 1989, 978:2>31.
27. .
DYER J, BEECHJZYRB, GORVEL JP, Shlrm RT, WINSTON R, SHIRAZI-BEECH!ZY SP: Giycyl-L-Proline Transport in Rabbit Enterocyte Basolateral Membrane Vesicles. Biocbem J 1990, 269:56>571. Until now, there has been no direct study on peptide transport in isolated intestinal basolateral membrane vesicles. This paper is the first to demonstrate the presence of a peptide transporter in this membrane. Interestingly, this carrier also exhibits the H+-dependence characteristic, as does the brush border membrane peptide transporter. 28 ..
Bou, SJ, ~YD JB: Evidence for a Dipe.ptide Porter in the Lysosomal Membrane. Ebchim Bic@ys Acfu 1990, 1024267-270.
in the animal
cell plasma
membrane
Ganapathy
and Leibach
This study is, in a way, a reinvestigation of the earlier observations on the msomal handling of dipeptides. The presence of a transport mechanism for dipeptides was never invoked in earlier SNdieS. In contrast, experimental data from the present Study provide a strong argument in favor of the existence of a specific transport mechanism for dipeptides in the lysosomal membrane. GANAPATHY V, PA~HUZYDH, FONTELESMC, LEIBACHFH: Pepti29. dases and Peptide Transport in Mammalian Kidney. Cunti Nephrol 1984, 42:10-18. M~LIMOTO Y, SATO K, OH-YASHKIT, MOHRIT: Leucine-Proton 30. Cotransport System in Chang Liver Cell. J Bid C&m 1986, 261:4549-4554. 31. M~UMOTO Y, MOHRI T: Reconstitution of the L-LeucineH+ Cotransponer of the Plasma Membrane from Chang Liver Cells into Proteoliposomes. Biocbim Biqdys Acta 1991, 1061:171-174. TIRUPPATHIC, B~LKO~E’IZ DF, GANAPATHYV, MNAMOTO Y, 32. IIIEIACH FH: A Proton Gradient, not a Sodium Gradient, is the Driving Force for Active Transport of Lactate in Rabbit Intestinal Brush-Border Membrane Vesicles. Bkxbem J 1988, 256:21‘+233. B~ow-rz DF, LEIEZACH FH, MN~E~HVB, GANA~ATHYV: A Pro33. ton Gradient is the Driving Force for Uphill Transport of Lactate in Human Placental Brush-Border Membrane Vesicles. J Biol Gem 1988, 263:1382%13830. HALESTRAP AP, POOLERC, CRANMERSL: Mechanisms and Reg 34. ulation of Lactate, Pyruvate and Ketone Bcdy Transport Across the Plasma Membrane of Mammalian Cells and their Metabolic Consequences. Biocbem Sot Truns 1990, 18:1132-1135. 35. SCHRONCM, WASHINGTONC JR, BLAZER BL: The Transmembrane pH Gradient Drives Uphill Folate Transport in Rabbit Jejunum. Direct Evidence for Folate/Hydroxyl Exchange in Brush Border Membrane Vesicles. J Clin Invest 1985, 76:203%2033. Ross CR, HOWHAN PD: Transport of Organic Anions and 36. Cations in Isolated Renal Plasma Membranes. Annu Reu Pbannacol Toxicol 1983, 23:65-85. HOSHIT, TAKLKVAN, ABE M, TAJIMAk Hydrogen Ion-coupled 37. Transport of ~-Glucose by Pblorizin-sensitive Sugar Carrier in Intestinal Brush-border Membranes. Biocbim BiqXys Acta 1986, 861:483-Q%. ROIGAARD-PETERSEN H, JACOBSENC, SHEIKH Ml: H+-L-Proline 38. Cotransport by Vesicles from Pars Convoluta of Rabbit Proximal Tubule. Am J P&siof 1987, 253:F15-F20. RAJENDRAN V-M. BARRYJA, KLEINMANJG, RAMASWAMYK: Pro39. ton Gradient-dependent Transport of Glycine in Rabbit Renal Brush-border Membrane Vesicles. J Biol &em 1987, 262314974-14977.
V Ganapathy and FH Leibach, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912, USA
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