Proton-Coupled Sugar and Amino Acid Transporters ...

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Annu. Rev. Plant Physiol. Plant Mol. BioI. 1993. 44:513-42

ANNUAL REVIEWS

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Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

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PROTON-COUPLED SUGAR AND AMINO ACID TRANSPORTERS IN PLANTS Daniel R. Bush Photosynthesis Research Unit, USDA Agricultural Research Service and Department of

Plant Biology, University of Illinois, 196 PABL, Urbana, Illinois 61801 KEYWORDS: symport, antiport, carrier, assimilate partitioning, membrane protein

CONTENTS INTRODUCTION ..................................................................................................................... 5 1 3

CONCEPTS AND DEFINITIONS ........................................................................................... 514 Active Transport and Diffusion.......................................................................... Pumps, Channels, and Carriers.........................................................................

SIS 516

BIOCHEMICAL DESCRIPTIONS........................................................................................ . .. 5 1 7 Isolated Membrane Vesicles and Imposed Proton Electrochemical Potentials................................................................................................... 517 Sucrose Transporters ......................................................................................... Glucose Transporters......................................................................................... Amino Acid Transporters ........................ ........ ... .............. ... ... ............ . .. .... ... ......

518 525 527

MOLECULAR CLONING ........................................................................................................ 530

Cloning Glucose Transporters ...........................................................................

530

Cloning a Sucrose Transporter .........................................................................

534

An Extended Family of Transport Proteins .......................................................

531

CRITICAL RESEARCH AREAS ............................................................................................. 535

Regulation..........................................................................................................

536

Transport Mechanisms.......................................................................................

536

Missing Links .......... ...... .. ........... ......... ................... .................................. ... .......

536

INTRODUCTION

/

Plants contain many heterotrophic tissue systems that are dependent upon sugar and amino acid import for normal growth and development. Indeed,

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every cell of the higher plant is at least transiently heterotrophic during early stages of differentiation. In general, oxidized forms of carbon and nitrogen are reductively assimilated in the photosynthetic tissues of the plant, and sucrose and amino acids are subsequently transported to the heterotrophic cells. The

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

impact of these import-dependent tissues on the overall economy of the plant is considerable. For example, as much as

80% of the carbon assimilated during

photosynthesis is exported from the leaf to satisfy the metabolic needs of the nonphotosynthetic cells

(64). A central feature of this resource-partitioning

step is phloem loading, and in many plants this process is dependent upon the function of an active sucrose carrier

(13, 40, 133). Thus, sugar transporters are

important contributors in both the systemic distribution of photoassimilate, as well as in the nutritional well being of the individual cell. Additional physio­ logical processes in which sugar and amino acid transport are essential activi­ ties include: seed filling, germination and seedling growth, filling and mobili­ zation in specialized storage organs, and secretion by nectaries. Clearly, these nutrient transport systems play fundamental roles in all aspects of plant biol­ ogy. The last review of sugar and amino acid transport in this series was a comprehensive survey of the literature that developed a good case for proton­ coupled symports in the plasma membrane of the plant cell (106). Much of the

data cited in that review was based on studies using intact cells and tissues. In their conclusion, the authors noted several problems associated with those experimental systems, such as intracellular compartmentation and diffusion in bulky organs, that limited the investigator's abi lity to study the molecular

details of sugar and amino acid transport. In the present review, I focus on recent advances using isolated membrane vesicles and molecular biological techniques that overcome many of the limitations associated with intact cells and tissues. These experimental approaches have provided significant insight into the transport properties, bioenergetics, and molecular structure of proton­ coupled sugar and amino acid transporters in the plant.

CONCEPTS AND DEFINITIONS Although basic concepts regarding membrane transport are familiar to all plant

biologists, some of the terminology used in this field can be confusing. This situation arises, in my opinion, because many of the descriptive terms used in membrane transport have evolved from two fundamentally distinct questions. Specifically, is transport across the membrane thermodynamically favorable, and secondly, what is the nature of the transport pathway? For example, diffusion describes the spontaneous movement of a molecule down its poten­ tial energy gradient. In the context of membrane transport, however, diffusion must also be defined with respect to the mechanism of translocation across the

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lipid bilayer (see below). In order to avoid potential ambiguity later on, I digress from the major focus of this review for a few moments to define some of the terms and concepts associated with sugar and amino acid transport. For a detailed discussion of membrane transport and bioenergetics per se, I call the

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

readers attention to excellent monographs by Harold (47) and Stein (124).

Active Transport and Diffusion The proton-coupled sugar and amino acid transport systems described here mediate active transport. Active transport is defined simply as net flux across a membrane against the potential energy gradient of the molecule in question. If the compound is uncharged, this represents flux against the chemical potential. If the molecule is charged, there is also an electrical component to consider, and thus it moves against its electrochemical potential. In all cases, active transport requires free energy to drive translocation. Active transport is further categorized as primary or secondary, depending on the transport mechanism.

PRIMARY ACTIVE TRANSPORT

Primary active transport describes translocation

that is linked directly to a chemical reaction, such as ATP hydrolysis. The proton-pumping ATPases are good examples of primary active transport sys­ tems (7, 8, 97).

SECONDARY ACTIVE TRANSPORT

Secondary active transport describes a pro­

cess that couples solute translocation against its electrochemical gradient to the flux of another molecular species that is moving down its electrochemical potential. Thus, a secondary active transporter links a thermodynamically unfa­ vorable transport reaction to a favorable reaction whose absolute magnitude is large enough to drive the flux of both molecules. In contrast to primary active transport, covalent bonds are not altered as a result of secondary active transport activity (47, 93, 94,). The proton-coupled sugar and amino acid transporters in plants are examples of secondary active transport systems that link substrate translocation across a membrane to the free energy available in a proton electrochemical potential difference.

SIMPLE DIFFUSION

Simple diffusion describes transport processes in which a

molecule moves passively across the membrane down its potential energy gradient. In simple diffusion, the rate at which a molecule passes through the bilayer is proportional to its lipid permeability and size (124).

FACILITATED DIFFUSION

In facilitated diffusion, the direction of net flux is

also down the potential energy gradient. In contrast to simple diffusion, how­ ever, the molecule moves across the membrane by a specific carrier or channel.

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Since the bilayer is a formidable barrier to most hydrophilic molecules, facili­

tated transport substantially increases their rate of translocation.

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Pumps, Channels, and Carriers Most transport activity associated with biological membranes is mediated by

specific, proteinaceous pathways. These membrane protein systems typically include multiple stretches of hydrophobic amino acid residues that form dis­

crete domains of membrane-spanning alpha helices and to a limited extent beta

strands (4, 25, 57, 70). Approximately 20 amino acids are required to span a

typical bilayer (57, 70, 84). The transmembrane helices pack together forming

a three dimensional structure that presents a hydrophobic exterior to the lipid

bilayer and a hydrophilic core, associated with the transport pathway, that is

readily accessible to the surrounding aqueous environments (119). It is import­

ant to note, however, that transport proteins may also contain substantial regions of structure that exist in solution outside the confines of the membrane.

Pumps are the primary active transport systems that couple translocation

directly to a chemical reaction. They can be single gene products, such as the

P-type ATPases (8, 97, 98), or large multimeric complexes composed of

several polypeptides. Both F-type and V-type ATPases fit in the later category (97,126a).

Channels catalyze facilitated diffusion. Although they usually exhibit some

level of selectivity, presumably through binding or steric exclusion, they are generally viewed as continuous aqueous pathways for substrate movement

across the membrane (17, 124). Because of the absence of a significant physi­

cal barrier, flux through a channel can be close to that predicted for diffusion

through water, and thus channel conductance is usually between 106 and 107

molecules per second per channel.

The term carrier is used to describe transport proteins that mediate either

facilitated diffusion or secondary active transport. This expression can be

misleading because it implies a freely mobile entity in the membrane that "carries" molecules from one side of the bilayer to the other. Although a few

antibiotic ionophores use this mechanism (47, 102), current evidence suggests

that ferrying or flipping across the membrane is energetically too costly to be a

general mechanism of membrane transport (47, 142). Likewise, carriers are

differentiated from channels in that they do not form continuous aqueous

pathways. Carriers appear to function with binding sites that have the capacity to face one side of the membrane or the other. This idea is best presented in the

alternating access model (47, 69, 127).

The simplest conceptualization of the alternating access model is that the

transport protein alternates between two conformational states in which the

substrate binding site is accessible on one side of the membrane and then the

other. In facilitated diffusion, binding affinity is the same in either conforma-

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Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

tional state. For secondary active transport,on the other hand,binding affinity is high on the uptake side of the membrane and lower on the discharge side, and of course the change in affinity is linked to the cotransported molecule

(69, 127). In either case, alternating access models envision comparatively small conformational changes inside the protein rather than macroscopic rota­ tion or translation of the carrier in the bilayer (127, 142). A consequence of 4 1 such interaction is that carrier conductances at 10 to 10 molecules per second per carrier are much lower than those observed for channels (47, 142). Carriers are also called uniports, symports, or antiports (93). These terms refer to facilitated diffusion (uniport) or secondary active transport in which flux is linked to the movement of a second molecular species in the same direction (symport) or the opposite direction (antiport). Carriers,pumps, and channels are also referred to as transporters and porters. Sound confusing? It can be. To quote from F. M. Harold (47), "Some porters are carriers, some channels: all pumps are carriers; there are barriers and leaks, wells and traps, sources and sinks, and each term means precisely what its author wants it to mean,neither more or less." Fortunately,in spite of our problems with precise terminology, the transporters always get it right!

BIOCHEMICAL DESCRIPTIONS Isolated Membrane Vesicles and Imposed Proton Electrochemical Potentials One of the most important factors contributing to recent advances in under­ standing sugar and amino acid transporters in the plant has been the successful development of isolated membrane vesicles as an effective experimental tool in plant transport biology (5, 13, 126). Purified membrane vesicles represent a powerful experimental system because the investigator can focus on specific transport processes while minimizing problems associated with intact cells and tissues (5, 11, 13,59, 96, 126). The major attributes of the membrane vesicle approach include: (a) the ability to study transport activity in a specific mem­ brane system (plasma membrane, tonoplast, endoplasmic reticulum, Golgi), (b) the elimination of posttransport compartrnentation and metabOlism, (c) controlled access to either surface of the membrane, and (d) the ability to manipulate intra- and extravesicular solution composition. Control of solution composition is a critical advantage because well defined transmembrane sub­ strate and ion concentration differences can be used to dissect the bioenerget­ ics of any given transport process. This is especially useful when examining proton-coupled transporters.

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Proton-coupled porters are secondary active transport systems that link substrate translocation across a membrane to the free energy available in a proton electrochemical potential difference (ilIlH+). This source of energy is a

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

function of both the proton concentration difference (ilpH) and the transmem­ brane electrical potential (il'P). Formally, illlH+ =

-2.3 RT ilpH + F il'P where

R is the gas constant, T is absolute temperature in Kelvin, and F is the Faraday constant. It is now generally accepted that AjlH+ across the plasma membrane of the plant cell is generated by a P-type, proton-pumping ATPase

(7, 5 0, 116,

126). The iljiH+ across the tonoplast is established by a V-type proton-pump­ ing ATPase and by a unique proton-pumping pyrophosphatase (PPase) (105,

126). Although the endogenous proton pumps can be used to generate the requisite iljiH+ for in vitro transport experiments

(5, 126), the utility of this

approach is limited by problems associated with membrane sidedness and the potential for changes in pump activity, for example resulting from chemical

modification, that indirectly alters proton coupled transport (11). An alterna­

tive approach is to energize the membrane vesicle with an imposed illlH+ (13,

14, 61, 85). This technique has the added advantage that the proton electro­

chemical potential can be applied in the form of a proton concentration differ­ ence (ilpH), a membrane potential difference (il'P), or a combination of the two. An imposed ilJl.H+ is often generated with pH and pK jumps (13). For imposed pH differences, the intravesicular solution composition of purified membrane vesicles is set at a predefined pH value. This is accomplished during membrane purification or with a series of freeze-thaw treatments (12). These vesicles are then diluted into an iso-osmotic transport solution that is set at a different pH. If the vesicles are not leaky to protons, i.e. no proton conductance pathways are open, then the dilution step imposes a iljlH+ of defined orientation (acid or basic inside). In many isolated membrane systems, such an imposed pH difference is stable for many minutes

( 1 1, 85). It should

be noted, however, that it has not been possible to isolate sealed membrane vesicles from every species tested

(D. R. Bush, unpublished). A similar exper­

imental approach is used to generate desired membrane potentials using potas­ sium gradients and valinomycin, a potassium-specific ionophore. For example, a negative il'P can be generated by diluting potassium-loaded membrane vesi­ cles into a potassium free transport solution. Valinomycin-mediated potassium diffusion out of the vesicles, in the absence of compensating charge flow,

generates a transmembrane electric potential, negative inside (12). The ability to manipulate independently ilpH and A'll can be very useful because it allows the investigator to analyze their respective contributions to proton-coupled transport.

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Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

Sucrose Transporters Sucrose i s the central carbon metabolite i n plants. I t i s the principle end product of photosynthetic carbon assimilation (125), and in assimilate parti­ tioning, it is the predominate form of reduced carbon transported to the hetero­ trophic cells (2, 48, 144). Even in those species that move other compounds as the major solute in the phloem (e.g. stachyose, raffinose, sorbitol, mannitol), sucrose is still a significant component of the translocation stream (145). In addition, carbon storage is frequently associated with sucrose accumulation in the vacuole. Spinach mesophyll cells, for example, store sucrose in the vacuole during the day for subsequent export in the night (34). Likewise, sugar cane and sugar beet are well known for long-term sucrose storage in the vacuole of specialized storage cells (48, 54).

Evidence for a proton-sucrose symport in the plasma membrane of the plant cell has accumulated since the late 1970s when Giaquinta (39) first suggested that such a carrier might be responsible for sucrose accumulation in the vascular tissue of the leaf (19, 40, 56, 68, 81, 82, 106 for review of earlier citations). In spite of widespread acceptance of this hypothesis, surprisingly little is known about this carrier. However, recent advances have provided new insight into the structure, transport properties, and molecular genetics of this essential transport system. The transport properties and bioenergetics of the proton-sucrose symport were recently described using plasma membrane vesicles (PMV) isolated from leaf tissue and imposed proton electrochemical potential differences (10, 11, 75). In these experiments, a pH jump was used to establish d�H+ in the necessary orientation to drive proton-sucrose cotransport into the purified PMV. Time-dependent sucrose accumulation was observed in both the pres­ ence and absence of the imposed ApH. However, the rate and extent of sucrose transport in the presence of the proton gradient greatly exceeded that observed in its absence (10, 11, 75). These results were interpreted as evidence for proton-coupled flux. Kalinin & Opritov (63) reached the same conclusion based on experiments monitoring changes in light scattering as a function of proton-coupled sucrose efflux from inside out PMV. Further evidence in sup­ port of secondary active transport was provided in complementary experi­ ments that showed ApH-dependent sucrose transport generates a substantial concentration gradient (10, 11, 73, 75). THE PROTON-SUCROSE SYMPORT

The rate of dpH-dependent sucrose transport saturated as a function of sucrose concentration (10, 11, 75, 140). This was consistent with carrier-medi­ ated translocation because the maximum rate of flux is limited by the number of active sites per unit membrane. The apparent Km for sucrose transport derived from these data was approximately 1 mM. Taken together, dpH-de-

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pendent transport and saturation kinetics provided unequivocal evidence in support of a proton-sucrose symport in the plant plasma membrane.

Substrate specificity The specificity of the proton-sucrose symport was exam­ ined in isolated PMV by testing various carbohydrates as potential competitive inhibitors. Even a tenfold excess of glucose, fructose, lactose, maltose, mannose, melibiose, or raffinose did not inhibit sucrose uptake (10, 11). This level of discrimination provided additional support for carrier-mediated transport. Hitz et al (53) initially studied substrate recognition in sucrose transport by examining the effect of 23 glycosides on sucrose uptake into protoplasts isolated from developing soybean cotyledons. The results of their survey sug­ gested that the hydroxyl groups of the fructose moiety of sucrose do not participate in substrate recognition. However, the fructosyl region of the mole­ cule does contain an essential hydrophobic surface that is required for binding. This latter conclusion was based upon the observation that phenyl glycosides were effective transport inhibitors. Additionally, the 3-, 4-, and 6-hydroxyls of glucose were shown to be important for binding, with the 3-hydroxyl abso­ lutely required. The authors noted that an essential hydrophobic surface and a cluster of important hydroxyl groups were also relevant to carbohydrate-pro­ tein interactions described for monoclonal antibodies and plant lectins. Inter­ estingly, hydrophobic interactions have also been implicated in other carbohy­ drate transport systems (43). Delrot et al (21) also examined substrate recogni­ tion using various sucrose derivatives and found similar results, although the 2-hydroxyl of glucose appeared to be more important for binding while the 6-hydroxyl had a lesser role than that observed by Hitz et al (53). A potential limitation of the work by Hitz et al (53) and Delrot et al (21) is that they were not able to measure independently the transport of their sucrose analogues. In addition, their experiments were performed with cell and tissue systems in which the identity and transport properties of the sucrose porter were not unequivocally established. Therefore, Hecht et al (49) reexamined the phenylglucopyranosides, developed for the study by Hitz et al (53), by investi­ gating their effect on the proton-sucrose symport in PMV isolated from sugar beet. An important contribution of this work was that they were able to document phenylglucoside transport and clearly show that these analogues were competing with sucrose for the same transport system. With the excep­ tion of a few subtle differences,their conclusions regarding substrate recogni­ tion were in close agreement with those of Hitz et al (53). Electrogenicity Since sucrose is a neutral compound, the cotransport of pro­ tons and sucrose into a membrane vesicle should be electrogenic. Several laboratories tested this prediction by examining the effect of membrane potential on proton-coupled sucrose transport (10, 12,63,75,140, 141). The results of

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

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those experiments showed that positive membrane potentials decreased and negative potentials increased transport activity. This is the expected result for an electrogenic transport system that moves positive charge into the isolated vesicle. Therefore, it was concluded that the sucrose symport system is electro­ genic. This conclusion is in agreement with earlier studies using electrophysio­ logical methods with intact cells (81, 82). The il/iH+ across a membrane is a function of both ilpH and il'P. An important concept associated with chemiosmotic theory (93, 94) suggests that these are thermodynamically equivalent and that the proton-sucrose symport should respond to either component of �jiH+. To test this idea, negative mem­ brane potentials were imposed in the absence of a proton concentration differ­ ence to drive sucrose accumulation. Although comparable rates of ilpH and il'P-driven sucrose transport were reported by Kalinin & Opritov (63) and Bush (12), other investigators reported lower rates of il'P-dependent flux (10, 140). This discrepancy is reconciled by the observation that the symport sys­ tem exhibits an acidic pH optimum (12, 141). Bush (12) examined .6.'P-driven

transport as a function of solution pH and demonstrated high rates of flux under acidic conditions only. Buckhout (10) and Williams et al (140) exam­ ined il'P-driven sucrose transport at basic pH values. Thus, low estimates of il'P-driven flux appear to be the result of suboptimal transport conditions rather than an inherent mechanistic difference in ilpH- versus il'P-driven sucrose accumulation.

pH-dependence The pH-dependence of the sucrose symport system has been examined in detail using both �'P- and �pH-driven flux in PMV isolated from sugar beet leaf tissue (12). The apparent Km for protons (0.7 J..LM ) derived from those experiments is significantly lower than that observed for sucrose. Williams et al (141) confmned this observation using il'P-driven flux in PMV isolated from castor bean cotyledons. This pH value for apparent proton binding may have important physiological consequences since the pH of the apoplastic space ranges from 5.0 (10 J.1M) to 6.0 (1 J..LM ) (99). In this rather acidic environment, proton availability is probably not a rate limiting step.

Stoichiometry The stoichiometry of the proton-sucrose symport is 1:1 (12, 120). This is an important parameter because it provides thermodynamic, as well as molecular, information about the transport mechanism. The experiments designed to quantify this relationship were based on the notion that proton-su­ crose cotransport would remove protons from a weakly buffered transport solution and that a sensitive pH electrode could be used to quantify proton depletion (12, 120). Bush (12) used a sucrose concentration difference, rather

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than LlpH, to drive the symport reaction. Independent quantification of both sucrose and proton flux was achieved using this experimental approach, and the stoichiometry was determined to be

1:1. This conclusion was consistent with (142) and with Lin's (82) estimate of proton-sucrose cotransport into soybean protoplasts. Slone & Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

the measured stoichiometries of other ion-sugar symports

Buckhout (120) confirmed these results in PMV in which they used Ll'P to drive symport-mediated proton and sucrose uptake. Moreover, they expanded this experimental approach by reexamining the kinetics, specificity, and inhibitor sensitivity of the symport as a function of proton transport. Significantly, their results (120, 121) paralleled earlier observations derived from sucrose transport data

(13). Together, these experiments also demonstrated the tight coupling

between proton and sucrose flux through the sucrose carrier.

Inhibitors

The proton-sucrose symport is inhibited by several compounds that

form covalent bonds with specific amino acid residues. These reagents are of interest because they are potential radiolabeled probes for identifying the symport protein and because group-specific chemical modification provides clues about which amino acid residues might play a role in the translocation process. Diethyl pyrocarbonate (DEPC) and p-chloromercuribenzenesulfonic acid (PCMBS) are potent inhibitors of the proton-sucrose symport, with 150 values of

30 and 750 �M, respectively (11, 120, 140). The imidazole ring of

histidine is the primary target of DEPC and the sulfhydryl group of cysteine is the primary target of PCMBS

(46). Sensitivity to PCMBS had been previously

observed in transport experiments examining sucrose uptake into isolated leaf discs (20, 38). N-ethylmaleimide (NEM) has also been shown to inhibit sucrose transport into isolated PMV

(75). However, labeling conditions appear to have

a significant influence on NEM activity because some laboratories have not been able to demonstrate transport inhibition (11,

140).

Time- and concentration-dependent inactivation of the sucrose symport by DEPC and PCMBS has been explored to gain insight into the location of the modified amino acid residues. DEPC inactivation of transport activity was significantly slowed in the presence of

7 roM sucrose (15). This observation

suggests the DEPC-modified histidine residue is at, or at least conformation­ ally linked to, the active site of the carrier. In these inactivation experiments, sucrose is not present inside the vesicles during exposure to the inhibitor. Therefore, substrate protection also suggests that the DEPC-modified histidine residue is accessible from the extracellular face of the plasma membrane where added sucrose can compete for binding. In support of that conclusion, the presence of

50 roM histidine on the inside of the vesicle as a DEPC R. Bush,

scavenger did not change the time course of inhibitor inactivation (D.

in preparation). It is noteworthy that a histidine residue has been implicated in a proposed charge-relay system that couples lactose transport to the proton

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

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motive force in the Lac permease of E. coli (60). PCMBS inactivation did not exhibit evidence of substrate protection (D. R. Bush, in preparation). The proton-sucrose symport is also inhibited by phlorizin, cytochalasin B, and forskolin (141; D. R. Bush, in preparation). These compounds are well known inhibitors of several glucose carriers (72, 117, 118, 131). Although the sucrose symport is not sensitive to glucose competition, these inhibitors were of interest because most of the porters found in a "superfamily" of sugar carriers recognize cytochalasin B and forskolin (16, 44; see below). In addi­ tion, a radiolabeled phenylazide derivative of forskolin is available as a pho­ toaffinity probe (136). In preliminary experiments, a 55-kDa plasma mem­ brane protein is specifically labeled by this reagent (D. R. Bush, unpublished). Sensitivity to these compounds suggests the active site and/or transport mech­ anism of the sucrose symport exhibits some similarity to other sugar carriers. Protein identification Recent attempts to identify the proton-sucrose symport have employed two different experimental approaches. Ritz and colleagues synthesized a photolyzable sucrose derivative, based on their analysis of sub­ strate binding (53), which specifically labeled a 62-kDa sucrose-binding protein in soybean cotyledons (108). In addition to binding the sucrose analogue, this protein appears in the plasma membrane at the same time as sucrose transport activity (108), and immunocytochemical localization of this protein in leaf tissue places it in the phloem (137). Although these data suggest the 62-kDa protein may be involved in active sucrose transport, a cDNA encoding the 62-kDa protein has been sequenced (W. D. Hitz, personal communication), and hydro­ pathy plots of the deduced amino acid sequence suggest it lacks the multiple transmembrane spans that appear to be a hallmark of integral membrane proteins involved in transport (3, 44, 119). Nevertheless, the sucrose-binding activity and immunocytochemical data regarding the 62-kDa protein can not be dismissed. In an enticing development, a recent abstract reports that antibodies directed against the 62-kDa protein inhibit sucrose transport into protoplasts isolated from developing Vicia faba seed (31). Perhaps the 62-kDa protein is part of a sucrose transport system that is related to the binding-protein-mediated transport systems initially characterized in Gram negative bacteria (52). These systems are composed of several polypeptides, including an extracellular protein that binds the substrate molecule and subsequently docks with a integral membrane protein complex that couples transport directly to ATP hydrolysis. In a related experimental approach, Gallet et al (33) identified a 42-kDa plasma membrane protein using differential incorporation of 14C_NEM in the presence or absence of 250 mM sucrose. Although eleven plasma membrane proteins were differentially labeled, the authors relied on a variety of indirect observations to focus on a 42-kDa protein as the sucrose transport system. In an accompanying paper, immunological evidence in support of that conclusion

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

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was presented, in which polyclonal antibodies against the 42-kDa protein decreased sucrose transport into isolated protoplasts by approximately 55% (74). More recently, it was shown that sucrose transport into isolated PMV was also inhibited by polyclonal antisera directed against the 42-kDa protein (32). The one cautionary note about these results is that the Western blots for this sera show that these antibodies cross-react with several proteins ranging in size from 38-kDa to 48-kDa (32, 79). Nevertheless, the NEM-binding protein is a good candidate as the sucrose symport. The first successful solubilization and reconstitution of the proton-sucrose symport was reported by Li et al (79). They used a mouse polyclonal serum directed against the 42-kDa protein to screen solubilized plasma membrane proteins eluted from a gel filtration column. The fraction exhibiting the highest cross-reactivity eluted in a broad peak at 120-kDa, which contained 23% of the total plasma membrane protein. This fraction was used to reconstitute ilpH-de­ pendent symport activity. Although the 42-kDa protein was present in the active fraction, SDS-denatured proteins derived from the gel filtration peak ranged in size from 30- to > 100-kDa, with a 280 nm absorbance maxima at approximately 130-kDa, 70-kDa, and < 20-kDa. Additionally, a silver stained SOS-PAGE of this fraction revealed many polypeptide bands (80). Even though these observations obscure the significance of the 42-kDa protein in the reconstituted system, this was an important contribution because success­ ful reconstitution is a critical first step towards biochemical identification of the symport protein. Sucrose is frequently a major component of the vacuolar solution, and in the specialized storage tissues of sugar cane and sugar beet, intracellular sucrose concentrations are measured in the hundreds of millimolar (48,87,90). In 1979 Doll et al (24) provided preliminary evidence for energy-dependent sucrose transport into isolated vacuoles based on a 40% stimulation in sucrose accumulation in the presence of MgATP. An accompa­ nying paper provided evidence for carrier mediated flux (138). Although our understanding of the bioenergetics of the tonoplast was still fragmentary when this work was published,these results pointed toward proton-coupled sucrose transport in which sucrose influx was linked to proton efflux from the vacuole. Convincing evidence for a proton-sucrose antiport was provided by Briskin et al using tonoplast membrane vesicles isolated from sugar beet tap roots (9). Results in support of antiport-driven sucrose transport included: (a) sucrose dissipation of an established transmembrane pH difference (acid inside), (b) 14 MgATP-dependent C-sucrose transport, (c) saturation kinetics with an ap­ parent Km of 12 mM, (d) decreased sucrose transport in the presence of a protonophore, and (e) sucrose-gradient dependent generation of a negative membrane potential. Each of these observations was consistent with an antiTHE PROTON-SUCROSE ANTIPORT

SUGAR & AMINO ACID PORTERS

525

port system in which sucrose transport into the vacuole is coupled to the efflux

of protons. Subsequent reports investigating sucrose transport into vacuoles and tonoplast membrane vesicles isolated from red beet (35,36) and Japanese artichoke tubers (65) have supported this conclusion. In spite of these promis­ Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

ing results, however, further progress in describing the proton-sucrose antiport has been hampered by problems in reproducing the initial observations (D. R.

Bush and D. P. Briskin, unpublished observations). Several laboratories have attempted without success to reproduce the sugar beet results, and in my opinion, the collective data suggests the sucrose antiport is an exceptionally labile transport system that is subject to rapid inactivation. This notion is consistent with recent results in red beet where ATP-dependent sucrose trans­ port was higher in tonoplast membrane vesicles isolated from purified vacu­ oles vs those isolated from tissue homogenates (36). Evidence for a proton-sucrose antiport in sugar cane tonoplast vesicles was presented recently by Getz et al (37). They reported increases in sucrose transport in the presence of ATP or

an

imposed ilpH (acid inside). Although

these data are consistent with antiport activity, this conclusion may be prema­ ture because the ATP- and LlpH-dependent increases in transport activity were comparatively small. Additionally, other published investigations of sucrose transport in sugar cane vacuoles and tonoplast vesicles have produced no evidence for proton-coupled sucrose translocation ( 100, 101, 13 9) Indeed, .

these reports provided convincing evidence for facilitated diffusion into the vacuole. A strong case for facilitated transport has also been made for sucrose uptake into vacuoles isolated from barley mesophyll cells (62). In light of these observations, Getz et al (37) speculated that an alternative explanation for their data might be that membrane energization per se results in conforma­ tional changes in tonoplast membrane proteins that unspecifically increases sucrose transport.

Glucose Transporters Although sucrose is the principle form of transported carbon in plants, glucose also plays a significant role in plant nutrition. This is especially true in hetero­ trophic tissues that hydrolyze sucrose in the apoplastic space before transport­ ing the hexose sugars into individual cells (54, 129). THE PROTON·GLUCOSE SYMPORT

No discussion of proton-glucose symport

activity in the higher plant would be complete without first acknowledging the seminal contributions of Komor and Tanner in describing the proton-coupled glucose (hexose) transport system in Chlorella (67, 69). Their work with the glucose symport in this single celled alga provided one of the earliest lines of experimental evidence in support of Mitchell's ideas regarding proton-coupled

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

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transport (93). Moreover, their discoveries contributed to the eventual recogni­ tion that the proton motive force is the fundamental currency of plant cell bioenergetics. In light of the impact of their research. it seems fitting that the Chiorella glucose transport system was the first proton-coupled symport cloned in an eukaryotic organism (113; see below). Evidence for a proton-glucose symport in the plasma membrane of the higher plant was initially provided by electrophysiological measurements (29, 30) and radiotracer studies with cultured cells and isolated protoplasts (42, 43, 83, 88, 123). Gogarten & Bentrupt's (42, 43) recent work with suspension cultures of Chenopodium rubrum was particularly interesting because they used a novel application of carrier-mediated exchange diffusion to examine the transport properties and substrate specificity of the proton-glucose symport. Their results suggest that the carrier binds protons and glucose in random order and that the active site contains a fixed negative charge. These conclu­ sions were derived from tracer experiments in which they were able to manip­ ulate both intra- and extracellular substrate concentrations while quantifying 14 [ C]3-0-methyl-D-glucose transport (42). 3-0-methyl-D-glucose (OMG) is an analogue of glucose that is frequently used in transport studies because it is not metabolized. Gogarten & Bentrup (43) also investigated the effect of various sugars and glucose derivatives on OMG flux across the plasma mem­ brane. From those results, they concluded that substrate recognition involves hydrogen bonding at C-l and C-3 as well as a critical hydrophobic interaction with the pyran ring (43). The first successful description of proton-glucose symport activity in puri­ fied PMV was reported recently by Tubbe & Buckhout (132). Previous studies with PMV preparations did not provide the requisite evidence supporting proton-coupled glucose transport. Rausch et al (104a) examined the effect of monoclonal antibodies directed against the mammalian Na+-glucose symport on glucose transport into an enriched PMV preparation from com. Although one of the antibodies stimulated glucose accumulation in a manner that was similar to its effect on the mamm alian system, the evidence for proton-glucose symport activity in this membrane preparation was not convincing. Verstappen et al (135) purified plasma membrane and tonoplast membrane vesicles from cultured tobacco cells. Their analysis of glucose transport in these two mem­ branes system provided good evidence for distinct carriers on each membrane. Unfortunately, the purified PMV did not hold imposed proton concentration differences, and consequently they were not able to investigate the bioenerget­ ics of the plasma membrane glucose carrier (135). Tubbe & Buckhout (132) described proton-coupled glucose transport in PMV isolated from sugar beet leaf tissue. This is the same membrane prepara­ tion previously used to investigate the proton-sucrose symport (10, 11). Glu­ cose transport was driven by either dpH or d'l', and the rate of dp:H+-depen-

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SUGAR & AMINO ACID PORTERS

527

dent transport was proportional to the size of the imposed gradient. In addition, both ilpH- and il,¥-dependent glucose uptake exhibited saturation kinetics with an apparent Km of 286 IlM. Substrate recognition was also examined by investigating the ability of several hexose analogues to inhibit glucose trans­ port and by quantifying the translocation of 2-deoxy-D-glucose, mannose, and 3-0-methyl-D-glucose. Their results were similar to those reported for Chenopodium (43), with the exception that xylose was not an effective inhibi­ tor. Based on this observation, Tubbe & Buckhout speCUlated that substrate interaction with the beet glucose (hexose) carrier is subtly different at the C-6 position from that observed in the carrier of Chenopodium cells. Williams et al (141) also reported ilpH-dependent glucose transport in beet PMV. Initial evidence for a proton-glucose anti­ port on the tonoplast is found in experiments with isolated vacuoles that demonstrate ATP-dependent stimulation in OMG transport (45). ATP-stimu­ lated transport was stereospecific, which suggests carrier-mediated flux, and inhibited by a proton ionophore. Further evidence in support of proton-glucose antiport activity was published by Thorn & Komor (128); using vacuoles isolated from sugar cane, they showed that depolarization of the vacuolar membrane potential (positive inside) and proton efflux were linked to OMG uptake. Rausch et al (104) provided additional evidence of proton-glucose antiport activity using tonoplast membrane vesicles isolated from Zea mays. These vesicles exhibited ATP-dependent OMG transport activity with an apparent Km of 110 IlM. In addition, antibodies directed against the vacuolar ATPase that inhibit proton pumping also decreased ATP-dependent glucose transport. Rausch (103) notes, however, that several attempts to demonstrate proton coupling in isolated tonoplast membrane vesicles have been unsuccessful. It remains to be shown if these experimental difficulties are due to the absence of a glucose antiporter or the result of unspecific damage to the carrier during membrane isolation.

THE PROTON-GLUCOSE ANTIPORT

Amino Acid Transporters In higher plants, inorganic nitrogen is typically absorbed from the soil solution as nitrate, transported to the leaf where it is reductively assimilated, and subsequently amino acids are exported to the heterotrophic cells (26). Al­ though there are exceptions to this generalized pathway of nitrogen acquisition (e.g. ammonium ions are sometimes the primary source of inorganic nitrogen, and in some plants nitrate is reductively assimilated in the roots), amino acids are the predominant form of nitrogen available to the heterotrophic tissues. Even in germinating seedlings, amino acids derived from seed storage protein

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are the primary source of reduced nitrogen translocated to the areas of new growth (114). Amino acids are transported into plant cells by proton-coupled symports. Some of the data in support of this conclusion include transient membrane depolarization (27, 28, 58, 109) and solution alkalization during the initial stages of amino acid transport (22, 91, 134, 143). These observations are consistent with proton-amino acid cotransport into the cell. Complementary experiments examining the kinetics and inhibitor sensitiv­ ity of amino acid uptake into intact cells provided evidence for carrier mediated transport (6, 7 1,106, 112, 122). More recently, isolated membrane vesicles have been used to study amino acid transport. Microsomal membrane vesicles isolated from zucchini hypocotyls were used in the first in vitro experiments demonstrating proton-amino acid symport activity (14). These membrane vesicles had been shown previously to hold imposed proton concentration differences for as long as one hour (85). The rate of ApH-dependent alanine transport in isolated vesicles was approxi­ mately ten fold higher than that observed in its absence. In addition, alanine was accumulated against a significant concentration gradient and saturation kinetics were observed. Together, these data provided direct evidence for proton-coupled amino acid transport (14).

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THE PROTON-AMINO ACID SYMPORTS

Since microsomal membrane vesicles were used to characterize transport activity (14), it was necessary to identify the membrane location of the alanine carrier. Therefore, the authors compared the distribution of alanine transport with known membrane markers on a sucrose density gradient. ApH-dependent alanine transport peaked at a density of 1.16 glee, which is typical of plasma membrane vesicles. In addition, it comigrated with plasma membrane ATPase activity, and it separated from tonoplast and endoplasmic reticulum enzyme markers. These results provided unequivocal evidence that the proton-alanine symport is located in the plasma membrane (14). After the initial demonstration of proton-coupled alanine transport, Li & Bush (76) used highly purified PMV isolated from sugar beet leaf tissue to investigate proton-amino acid symport activity in detail. In these experiments, ApH-dependent alanine, leucine, glutamine, glutamate, isoleucine, and argi­ nine transport was demonstrated. Alanine, leucine, glutamine, and glutamate exhibited saturable transport kinetics while isoleucine and arginine transport kinetics were biphasic. These results were consistent with carrier mediated flux. In support of that conclusion, transport activity was inhibited by chemical modification with DEPC. Symport-mediated alanine transport was electro­ genic, and both components of AjiH+' ApR and A'I', contributed to transport activity. Proton-coupled glutamine transport has also been demonstrated in

SUGAR & AMINO ACID PORTERS

529

PMV isolated from castor bean cotyledons and roots and from red beet roots

(140, 141). Since several amino acids exhibited proton-coupled transport activity, L i & Bush attempted to identify the number of amino acid carriers present in their

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

isolated membrane vesicles (76). To investigate this question, they selected six amino acids representing a range in charge and structural configurations for a broad survey of inter-amino acid transport competition. The rationale behind this experiment held that amino acids that are transported through the same carrier would inhibit one another' s flux across the membrane. Thus, the trans­ port of each test amino acid was examined in tum for evidence of competitive inhibition by each of the

20 common amino acids. The results of that survey

were consistent with four amino acid porters: an acidic amino acid symport, a basic amino acid symport, and two symports for the neutral amino acids. Although each symport system was relatively specific for a given group of amino acids, the authors noted that all four carriers exhibited some crossover specificity for amino acids in other groups (76). Previous estimates of the number of amino acid carriers in the higher plant ranged from one to three carriers (6, of Li

66, 92, 106, 1 12, 122). The unique feature & Bush's (76) results was their identification of two carriers for the

neutral amino acids. The neutral amino acid symports were initially resolved because of their differential affinity for isoleucine, valine, threonine, and pro­ line. In a subsequent study, the kinetics of amino acid transport competition among the neutral amino acids supported the presence of two carriers and, additionally, evidence for differential expression was presented

(77). Li &

Bush hypothesized that branching at the �-carbon in isoleucine, valine, and threonine results in steric interactions that block their access to one of the neutral symports. Li

& Bush (78) investigated the effect of several amino acid analogues on

neutral amino acid transport to identify structural features that are important in molecular recognition by neutral system I (isoleucine group porter) and neutral system

II (alanine and leucine group). D-isomers of alanine and isoleucine

were not effective transport antagonists of the L-isomers. These data are characteristic of stereospecificity and suggest that the positional relationship between the a.-amino and carboxyl groups is an important parameter in sub­ strate recognition. This conclusion was supported by the observation that �-alanine and analogues with methylation at the a.-carbon, at the carboxyl group, or at the a-amino group were not effective transport inhibitors. Specific binding reactions were also implicated in this study because substitution of the a.-amino group with a space-filling methyl or hydroxyl group eliminated bind­ ing activity. They conclUded from these observations that the binding site for the carboxyl end of the amino acid is a well defined space that is characterized by compact, asymmetric positional relationships and specific ligand interac-

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BUSH

tions (78). This conclusion is consistent with previous experiments examining a-aminoisobutyric acid transport in an aquatic liverwort

(28). In contrast,

analogues with various substitutions at the distal end of the amino acid were

potent antagonists. Although the molecular interactions associated with the

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distal portion of the amino acid were less restrictive, this component of the

enzyme-substrate complex was also important in substrate recognition because

the neutral amino acid symports were able to discriminate between specific neutral amino acids and exclude the acidic and basic amino acids (78).

VACUOLAR AMINO ACID TRANSPORT

Several recent studies of amino acid

transport into isolated vacuoles have demonstrated ATP-stimulated transport activity (23,4 1,5 5 , 87,89, 130). In the case of phenylalanine uptake, transport was stimulated by both MgATP and pyrophosphate, and accumulation was

reversed by proton ionophores

(55). These data link accumulation to the trans­

membrane pH gradient and are consistent with a proton-coupled antiport system.

In contrast, ATP-stimulated transport of several neutral and basic amino acids

was not coupled to the transmembrane �pH or �'P (23,4 1,89, 130). Indeed, in

these studies only M

l+-free ATP, or nOnhydrolyzable analogues, was capable

of stimulating amino acid accumulation. Although transport was apparently not

driven by il/lH\ ATP-stimulated flux was sensitive to transport competition (23,

89), and in some instances it was inhibited by chemical modification (89). These

results suggest at least one ATP-regulated, facilitated diffusion system is in­

volved in amino acid flux across the tonoplast membrane. In support of that conclusion, Thume

& Dietz ( 1 30) recently reported initial success in reconsti­

tuting ATP-regulated amino acid transport activity from solubilized tonoplast membranes.

MOLECULAR CLONING As in most areas of biology, recombinant DNA technology is contributing to

many important advances in describing the integral membrane proteins that mediate active transport processes. This is especially true for transport proteins

because the usefulness of traditional biochemical approaches is compromised by numerous experimental difficulties. For example, membrane proteins are generally of low abundance «

0.01 % total protein), they require a unique

hydrophobic environment to remain active, and they are difficult to track

during purification because most do not catalyze an easily monitored chemical

reaction. Therefore, with the exception of the proton pumps (7,97, 1 16, 126), very few transport proteins have been identified in plants. However, since the methods of gene cloning and sequencing have become relatively routine, many laboratories interested in transport biology are attempting to obtain new infor-

SUGAR & AMINO ACID PORTERS

53 1

mation about specific transport systems using "reverse" molecular genetics,

wherein they deduce the amino acid sequence of a specific transport protein

from the nucleotide sequence of a cloned cDNA or gene.

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Cloning Glucose Transporters The

Chlorella hexose transporter was the first proton-coupled symport cloned ( 1 13). Since this is an inducible transport system, Sauer & Tanner used a differential screening strategy to select potential clones encoding this carrier (1 1 3). They initially identified eight unrelated cDNAs in an eukaryotic organism

that were uniquely associated with induced cells. Each clone was sequenced and one of them exhibited significant homology with previously cloned glu­

cose porters. They used this partial clone to identify a full-length cDNA that

codes for a protein of

533 amino acids with a predicted molecular mass of 29%

57.4-kDa. The deduced amino acid sequence of the full length clone was

identical to the mammalian insulin-regulated glucose carrier and the yeast

SNF3 gene ( 1 8, 95) . Hydropathy plots of the deduced amino acid sequence

predicts 12 membrane-spanning domains with a relatively large hydrophilic sequence between domains six and seven. Sauer

& Tanner (1 1 3) concluded from these data that this cDNA codes for the Chlorella hexose carrier. Definitive proof that the Chlorella cDNA codes for the hexose carrier was provided when Sauer et al ( 1 1 0) successfully expressed the encoded protein in Schizosaccharomyces pombe. The algal cDNA was placed in a yeast expres­ sion vector behind the constitutive adh promotor and transformed into S. pombe. The transformed yeast cells exhibited concentrative OMG accumula­

tion, transport was inhibited by protonophores, and the apparent Kms for glucose,

OMG, and 6-deoxyglucose were the same as those described in Chlorella. In contrast, wild type cells and control transformants (transformed with insert-free vector) did not actively transport OMG,and their apparent Km values for glucose, OMG, and 6-deoxyglucose were approximately l Oa-fold higher than the cDNA transformants. These results provided unequivocal evi­

dence that the

Chlorella clone codes for the proton-hexose symport ( 1 10). ( 1 1 1 ) used the cDNA isolated from Chlorella as a probe to identify a genomic clone for a homologous gene in Arabidopsis thaliana. This gene codes for a protein containing 522 amino acids with a predicted molecu­ lar mass of 57.6-kDa. As with the Chlorella clone, the deduced amino acid sequence for the Arabidopsis gene (STP 1) exhibited significant homology with the sugar carriers previously described in mammals, yeast, and Es­ cherichia coli ( 1 8, 3, 95). To identify the substrate specificity of the Arabidop­ sis clone, STP1 was also expressed in S. pombe. Yeast cells expressing the STP1 protein exhibited active OMG accumulation, and the apparent Km for Sauer et al

glucose was l Oa-fold lower than control cells. Although these cells also trans­

ported fructose and galactose, they did not move sucrose. Two interesting

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BUSH

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I

4

,

7



,

10

11

12

Cytoplesm

Figure 1 Schematic diagram of the membrane orientation of a plant sugar carrier that is a member of the superfamily of porters. Membrane-spanning domains are represented as vertical rectangles. Some conserved sequences found in all members of the family, as well as those unique to the plant . carriers, are indicated (44, I l l : T.-I. Chiou & D. R. Bush, unpublished)

features of the Arabidopsis glucose carrier are that it does not contain an N-tenninal signal sequence, and like the Chiorella porter ( 1 1 3), it does not possess typical sites for N-glycosylation. Northern analysis showed that STP1 mRNA is highly expressed in leaf tissue.

An Extended Family a/ Transport Proteins

The genes for the Chiorella and Arabidopsis hexose carriers are part of a superfamily of porters that has been recently identified (3, 5 1 , 86). This group of related transporters was initially recognized when the sequences for the E. coli arabinose and xylose proton-coupled carriers were found to be homolo­ gous with the glucose porters from man and rat (86). Members of this family share significant regions of sequence identity, and their predicted membrane orientations fit the general motif of twelve membrane-spanning domains with a large hydrophilic region in the center of the protein (Figure 1 ; 44, 5 1). Griffith et al (44) recently reported the results of an exhaustive comparative analysis of the sequences for 34 carriers in this extended family in which they

SUGAR & AMINO ACID PORTERS

533

identified four subfamilies of related transporters. Interestingly, the largest subfamily contains predominantly sugar carriers found in a diverse group of prokaryotic and eukaryotic organisms. These sugar porters transport pentoses, hexoses, or disaccharides, and they usually recognize cytochalasin B and/or

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

forskolin. In addition, they mediate either secondary active transport or facili­ tated diffusion. Several highly conserved regions of sequence motif were identified in alignments of the four families

(44). Some of the conserved motifs are found

in every member of the superfamily, while others were only associated with specific groups of carriers. In the sugar family. for example. the sequences P-E-S-P-R-X-L and P-E-T-K-G-X-X-X-E are conserved

(65-97% per resi­

due) at the ends of the sixth and twelfth membrane-spanning domains. respec­ tively. An additional observation of note is that a conserved arginine or lysine residue is found in the beginning of the sixth transmembrane domain in the carriers that catalyze proton-coupled symport activity. but not in those porters that mediate facilitated diffusion (Figure

1).

The sequence comparisons b y Griffith e t a l provided convincing evidence that these transporters have evolved from a common ancestor

(44). In addition,

hydropathy predictions of membrane orientation and the significant degree of sequence identity and conserved motifs suggests the three-dimensional struc­ ture of carriers in this expansive group of integral membrane proteins is quite similar. This is a critical prediction because it implies that subtle differences in protein structure, rather than extreme changes in sequence and orientation, can have a profound impact on substrate specificity and transport coupling

(44).

Moreover, underlying this hypothesis is the notion that membrane transport at the molecular level uses subtle variation on a single mechanism to accomplish symport-, antiport-, and uniport-mediated transport. In support of that conclu­ sion, R. F. Gaber recently identified a mutant in a

Saccharomyces cerevisiae

facilitated glucose carrier that has transformed that porter into a glucose-potas­ sium cotransport system (submitted). The remarkable finding about this mu­ tant is that the acquired transport activity was introduced by single amino acid substitutions or additions in transmembrane domains 1,

4, 7, or 1 1 (See Figure 1 for numbering reference). Although molecular details of the glucose/potas­

sium coupling have yet to be worked out. Gaber has shown that potassium flux is obligatorily linked to glucose uptake. To test whether this is a general phenomenon. Gaber designed a selection strategy to generate a similar trans­ port phenotype in the facilitated galactose porter, and indeed a

Gal2 mutant

was isolated that catalyzes galactose-potassium cotransport as a result of a single amino acid change in transmembrane domain number

9 (personal com­

munication). Thus, a single amino acid change transforms a sugar carrier into an ion-sugar cotransport system. It should be noted also. that the notion of a

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

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BUSH

generalized mechanism is compatible with the alternating access model ( 127, 142). The extraordinary diversity in substrate specificity and transport properties found in this superfamily suggests that many carrier systems in the plant are likely derived from the same ancestral gene. Indeed, Sauer reports evidence for at least twelve related carriers in Arabidopsis thaliana (N. Sauer, personal communication). Likewise, in my laboratory we have used sequence informa­ tion from the superfamily to design degenerate primers for molecular cloning using the polymerase chain reaction. We have cloned four new members of this family from sugar beet leaf tissue (T.-J. Chiou & D. R. Bush, unpub­ lished). Together, these observations support the hypothesis that many plant transporters are members of this family of transport proteins.

Cloning a Sucrose Transporter The first successful cloning of a plant sucrose transporter was achieved by complementing an engineered yeast mutant with a plant cDNA library (107). Typically, Saccharomyces cerevisiae uses sucrose as a sole carbon source by secreting invertase and subsequently transporting the released hexoses into the cell. Alternatively, some strains are able to transport sucrose, perhaps through a maltose carrier. Riesmeier et al (107) obtained a yeast strain that is deficient in both invertase activity (suc-) and maltose transport (maLO). After confirming that these cells cannot grow on sucrose, they reintroduced cytosolic invertase or sucrose synthase as hydrolytic enzymes that would allow these mutants to metabolize sucrose, but only after it enters the cell. Thus, the rationale behind their cloning strategy was to transform these yeast mutants with a plant cDNA library constructed in a yeast expression vector and then to screen for trans­ formants that restore growth on sucrose. Growth on sucrose would presumably be the result of acquired transport activity or secretion of a plant invertase. With this approach, Riesmeier et al identified a clone from a spinach library that codes for a sucrose transporter (107). The transport properties of the spinach sucrose carrier expressed in yeast suggest it is a proton-sucrose symport ( 107). Transport activity was inhibited by protonophores, PCMBS, DEPC, and phenylglucoside. Additionally, the apparent Km for sucrose was 1.5 mM, and influx was dependent upon an acidic transport solution. Each of these characteristics is consistent with those re­ ported for the proton-sucrose symport characterized in isolated PMV (13; above). Taken together, these data provide convincing evidence that the spin­ ach cDNA clone, pS2 1, codes for a plant proton-sucrose symport. The spinach sucrose porter cDNA codes for a protein of 525 amino acids with a molecular mass of 55-kDa. The hydropathy plot predicts twelve mem­ brane-spanning domains with a central hydrophilic loop. In spite of the appar-

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SUGAR & AMINO ACID PORTERS

535

ATPa••

Glucose pH A 'J' pH

=



7.1 150

=

-

:::

... 5.5

mv

Figure 2

Schematic diagram highlighting the proton-coupled sugar and amino acid carriers found in the plant cell. Some facilitated diffusion systems are also indicated.

ent similarity to the superfamily of carriers, no sequence homologies to other mono- or disaccharide carriers, or any other protein, was reported. Conse­ quently, the authors concluded that the spinach sucrose carrier belongs to a unique class of sugar transport proteins

( 1 07). However, the conserved six

loop six motif suggests the sucrose carrier might be a distant member of the superfamily discussed above. Several laboratories have been developing expression cloning as a general

strategy for identifying sugar, amino acid, and inorganic ion transport systems

( 1 , 5a, 15, 107, 1 1 5 ) . When successful, not only does this approach allow for the rapid identification of low-abundance transport proteins and the genes encoding them, it gives the transport biologist a heterologous expression sys­ tem in which site-directed mutagenesis can be used to explore biophysical questions associated with porter structure and function. In addition, future experiments aimed at regulating the expression of these carriers in transgenic plants should considerably enhance our understanding of many complicated

physiological processes such as assimilate partitioning and nutrient acquisi­ tion.

536

BUSH

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:513-542. Downloaded from www.annualreviews.org Access provided by Bidhan Chandra Krishi Viswavidyalaya,Nadia,W.B. (BCKV) on 12/11/17. For personal use only.

CRITICAL RESEARCH AREAS The research results of the last ten years have contributed significantly to our understanding of sugar and amino acid transporters. We have moved beyond debating the existence of mediated flux and the nature of energy coupling to investigating the transport properties and bioenergetics of well resolved carrier systems (Figure 2). In addition, recent successes cloning sugar porters in plants are providing new insights into molecular structure and evolutionary relationships. As a result of these important advances, new questions are coming into focus. Below, I briefly discuss some of the critical research areas that should yield important contributions in the next decade of research activ­ ity. Regulation

There is considerable interest in the proton-coupled sugar and amino acid symports because of their manifold roles in assimilate partitioning. Now that individual carriers have been identified, we need to determine the levels of regulation that control transport activity. Current knowledge suggests these carriers can be regulated in a variety of ways, ranging from cell- and tissue­ specific expression to short-term biochemical modulation. Since it is likely that transport activity is tightly regulated in many complex physiological processes, this is an important area of future research. Transport Mechanisms

With newly acquired information about the transport properties and bioenergetics of plant sugar and amino acid carriers, there is now an opportu­ nity to address specific questions regarding transport mechanisms. In the case of the proton-coupled carriers, for example, what is the binding order for proton and substrate, how do protons move, are binding sites deep inside aqueous channels, and what amino acid residues participate in the transloca­ tion reaction? Some of these questions are accessible by kinetic analysis in isolated membrane vesicles. However, further insight into transport mecha­ nisms also requires more detailed information about the three-dimensional structure of these carriers in the bilayer. Missing Links

Three areas associated with sugar and amino acid transport merit further investigation. First, the putative sucrose efflux system in the mesophyll is likely an important regulator in controlling carbon flow out of the leaf, yet this system has not been identified or described. Likewise, detailed descriptions of the sugar and amino acid transporters in sink tissues and storage organs are needed to help solve recurring questions about the physiology of assimilate

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storage. Lastly, plasmodesmata may play an active role in carbon partitioning ( 1 33). Are they capable of mediating selective, concentrative transport or do they represent a different kind of "pore" in an expanded definition of the semipermeable membrane? PI�t transport biology is entering a new era of discovery as multidiscipli­ nary approaches are being focused on fundamental questions regarding im­ portant transport systems. Soon detailed knowledge about specific porters and the genes encoding them will contribute to new understanding about complex physiological processes that define the plant as a multicellular organism. This kind of integration will indeed denote a significant step forward. ACKNOWLEDGMENTS

Research in the author' s laboratory is supported by the Agricultural Research Service and by a grant from the Competitive Research Grants Office of the U.S. Department of Agriculture (90-37 1 30-5423).

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