2.1.8   Metabolite flux and organelle transporters

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Integration of photosynthetic metabolism between compartments and maintenance of discrete environments within organelles require controlled movement of substances across membranes. This control is provided by an array of highly specific transport proteins which span the lipid bilayer of membranes. These transporters act as gatekeepers determining which substances may enter or leave, how fast they may move, and whether their entry involves an exchange of metabolites or an input of energy.

(a)  Chloroplasts

Chloroplasts of higher plants are bounded by a double membrane known as the chloroplast envelope (Section 1.2). The two membranes of the envelope are separated by an average distance of only 6 nm, and between them is a small metabolic compartment known as the intermembrane space. Suspended within the chloroplast envelope there is a third, highly convoluted and protein-rich membrane system (thylakoid membranes) which delimits a separate metabolic compartment: the intrathylakoid space or lumen. The ground substance of chloroplasts (stroma) represents the third and largest compartment of chloroplasts (Section 2.1).

Some of the largest fluxes of metabolites in plants cross chloroplast membranes. In the intrathylakoid space, a continuous supply of water is required to sustain light-driven oxidation of water by photosystem II (PSII). The products of this process, protons and O2, must also be continuously released across the thylakoid membrane and beyond. CO2 is reduced within the stroma by the PCR cycle and incorporated into triose phosphates, starch, fatty acids, amino acids and terpenoids. These synthetic events are sustained by metabolite fluxes across the three membranes of the chloroplast. O2 is also reduced in the stroma in the oxygenase reaction catalysed by the enzyme ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco). One product of this reaction, 2-phosphoglycolate, is dephosphorylated and exported from stroma to cytoplasm where it is processed by reactions of the photorespiratory cycle. On balance, however, illuminated chloroplasts are O2 producers, and O2 must cross the chloroplast envelope to be used in mitochondrial electron transport or else return to the atmosphere.

Not all metabolites need cross chloroplast membranes by means of a transporter protein. Small uncharged molecules such as O2, H2O and CO2, as well as hydrophobic molecules such as lipids, can cross by simple diffusion. Some mono-carboxylic acids, such as acetate, can also cross the chloroplast membranes by diffusion. While simple diffusion of these molecules may occur at thylakoid membranes and across the inner membrane of chloroplast envelopes, facilitated diffusion is probably the predominant process mediating the flux of these and other metabolites across the outer membrane of those envelopes. This outer membrane contains a pore protein (a channel) which is non-specific and allows the passage of molecules up to about 3 nm in size (equivalent to a molecular weight of about 10 000). The inner membrane, in contrast, contains an array of specific translocators that control the flow of metabolites between cytosol and stroma.

Phosphate translocator

The most common transporter in the inner membrane is the phosphate/triose phosphate (3-phosphoglycerate) translocator, commonly known as the phosphate translocator (Figure 2.8). This antiport mediates the export of reduced carbon, in the form of triose phosphates, from the stroma into the intermembrane space, from which it can diffuse relatively freely into the cytosol. Most of the triose phosphates are used in synthetic reactions such as sucrose synthesis, but some are used in degradative reactions such as those of glycolysis and respiration. In C3 plants, the phosphate translocator catalyses a strict counter-exchange involving inorganic phosphate and phosphate molecules attached to the end of a three-carbon chain (e.g. triose phosphates, 3-PGA). During the day the main exchange process catalysed by the phosphate translocator is the export of triose phosphate for sucrose synthesis and the import of inorganic phosphate for ATP synthesis from ADP (C3 chloroplast in Figure 2.8).

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Figure 2.8 Phosphate translocators in plastids from different types of plants and tissues. In C3 chloroplasts, the main function of the phosphate translocator is in exporting triose phosphate (triose P) to the cytosol where it is used mainly for sucrose synthesis. During the day, triose phosphate is produced by the photosynthetic carbon reduction (PCR) cycle.

Chloroplasts from the leaves of C4 plants contain two forms of the phosphate translocator, one in the bundle sheath and the other in the mesophyll cells. The mesophyll translocator mediates phosphoenolpyruvate (PEP) export for use in CO2 fixation. In bundle sheath chloroplasts of certain C4 plants, 3-phosphoglycerate (3-PGA) is exported for reduction to triose phosphate in mesophyll cell chloroplasts.

The phosphate translocator in chloroplasts from plants with crassulacean acid metabolism (CAM) mediates PEP export in the daytime and triose P export at night. In contrast, the phosphate translocator in root plastids mediates the exchange of glucose-6-phosphate (G6P) and triose P. This exchange is important for the operation of the oxidative pentose phosphate pathway. G6P exchange for inorganic phosphate (Pi) is important in carbon import for starch synthesis. OAA = oxaloacetate. (Original drawing courtesy Ian Woodrow)

Different forms of the phosphate translocator are found in different types of plants and in different tissues within plants. In C4 plants there are two different forms of the phosphate translocator which differ from the C3 translocator in their ability to transport PEP. One form of the C4 translocator is located in bundle sheath cell chloroplasts and the other one in mesophyll cell chloroplasts. Both forms transport triose phosphates, 3-PGA, inorganic phosphate and PEP, but the ability of the bundle sheath translocator to transport 3-PGA and PEP is lower than that of the mesophyll translocator. This difference reflects the major metabolic fluxes sustained by the two forms of the translocator.

In mesophyll cells of C4 plants, PEP, which is synthesised in the stroma from imported pyruvate, is exported in exchange for inorganic phosphate to the cytosol where it is used by PEP carboxylase to fix CO2 (C4 mesophyll chloroplast in Figure 2.8). In the bundle sheath chloroplasts of certain C4 plants (e.g. maize), relatively rapid 3-PGA–triose phosphate exchange is catalysed by the phosphate translocator. These chloroplasts lack PSII and thus the ability to reduce phosphoglycerate to triose phosphate. Phosphoglycerate is exported to the mesophyll chloroplasts for reduction and returned to the bundle sheath in the form of triose phosphate, which is used largely for the PCR cycle.

Plants with crassulacean acid metabolism (CAM) contain a chloroplast phosphate translocator which, similar to that of C4 plants, has a relatively high ability to transport PEP. During the day, the main flux sustained by the phosphate translocator is the exchange of 3-PGA and PEP. PEP is synthesised in the stroma and exported to the cytosol where it is converted into 3-PGA. The 3-PGA is, in turn, taken up by the chloroplast and used for starch synthesis (CAM chloroplast in Figure 2.8). The carbon source for PEP synthesis is malate which, after release from the vacuole, is decarboxylated and converted into pyruvate. The pyruvate enters the chloroplast, by means of a specific H+/pyruvate or Na+/pyruvate symport, where it is converted into PEP. A similar pyruvate translocator has been found in chloroplasts of C4 mesophyll cells, some C3 plants and the bundle sheath cells of some C4 plants.

The phosphate translocator also catalyses triose phosphate–inorganic phosphate exchange in CAM plants. At night starch is degraded to triose phosphates, which are exported from chloroplasts for production of PEP (CAM chloroplast in Figure 2.8). PEP is the substrate for PEP carboxylase, an enzyme catalysing CO2 fixation to form oxaloacetate. This metabolite is then converted into malate which is stored in the vacuole for use during subsequent daytime (Figure 2.7).

Despite little research on the phosphate translocators of other types of plastids, different forms are known to occur in root plastids, amyloplasts and chromoplasts. The main function of root plastids is the reduction of nitrite for which energy is supplied by the oxidative pentose phosphate pathway. Because these plastids do not have the enzyme fructose-1,6-bisphosphatase, operation of the oxidative pentose phosphate pathway requires the exchange of stromal triose phosphate for cytosolic glucose-6-phosphate (see root plastid in Figure 2.8). Accordingly, this root plastid translocator has been shown to have a relatively high ability for transporting glucose-6-phosphate.

Dicarboxylate translocators

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Figure 2.9 Dicarboxylate translocators on the inner membrane of chloroplasts have at least two important metabolic functions. A malate/oxaloacetate (OAA) translocator facilitates the export of reducing equivalents to the cytosol. NADPH is one of the products of the photosynthetic electron transport chain in chloroplasts. Two dicarboxylate translocators are involved in ammonia assimilation during photorespiration. In this process, ammonia incorporated into glutamate (Glu), the production of which is sustained by 2-oxoglutarate (2-OG) import from the cytosol. Glutamine (Gln) and 2-oxoglutarate are substrates in a reaction that produces two molecules of glutamate. Of these, one is used for subsequent ammonia assimilation and is exported in exchange for malate. (Original drawing courtesy lan Woodrow).

At least two translocators mediate the exchange of a range of dicarboxylates across the inner membrane of chloroplasts. They include malate, oxaloacetate, 2-oxoglutarate, aspartate, succinate, glutamate and glutamine (Figure 2.9). These translocators play a key role in two major metabolic processes, namely (1) the photorespiratory nitrogen cycle, and (2) export of reducing equivalents from chloroplasts. During photorespiration (Figure 2.10) ammonia is released by mitochondria and diffuses into chloroplasts (Figure 2.9) where it is incorporated into glutamate. Production of glutamate, which is catalysed by glutamine synthetase and glutamate synthase, requires a supply of 2-oxoglutarate from the stroma. Exported glutamate is not exchanged directly for 2-oxoglutarate. Rather, glutamate is exported in exchange for malate by the dicarboxylate translocator, and the malate is in turn exported in exchange for 2-oxoglutarate by a specific translocator (Figure 2.9) which cannot transport glutamate. There is therefore no net malate flux across the chloroplast envelope during this process.

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Figure 2.10 The photorespiratory carbon oxidation (PCO) cycle involves movement of metabolites between chloroplasts, peroxisomes and mitochondria. Transport of glycerate and glycolate across the inner membrane of chloroplasts may involve separate translocators as shown here, or it may involve a single translocator that exchanges two glycolate molecules for one molecule of glycerate. Transport of metabolites across the peroxisomal membrane most likely occurs through unspecific channel proteins, similar to those in the outer membranes of mitochondria and chloroplasts. These outer membranes are not included in this diagram. Mitochondria take up two molecules of glycine and release one molecule of serine. A specific translocator most probably mediates the exchange of these amino acids. (Original drawing courtesy lan Woodrow)

A second dicarboxylate translocator in the inner membrane of chloroplasts mediates a specific oxaloacetate–malate exchange (Figure 2.9). Export of malate, subsequent oxidation to oxaloacetate in the cytosol, and subsequent import of oxaloacetate into chloroplasts for reconversion to malate facilitates a supply of reducing equivalents to the cytosol and peroxisomes. A malate/oxaloacetate translocator is also present in C4 plants where it mediates the uptake of oxaloacetate formed by PEP carboxylase into mesophyll chloroplasts where it is reduced to malate. This is an important part of the C4 cycle.

Glycolate/glycerate translocator

In C3 plants, the rate of oxygenation catalysed by Rubisco, and thus the rate of photorespiration, is generally about one-third the rate of carboxylation. Phosphoglycolate production in the stroma is thus one of the largest metabolic fluxes in plants (Figure 2.10) and such flux is greatly facilitated by the close proximity of chloroplasts, peroxisomes and mitochondria. Indeed, these organelles are often contiguous (Figure 2.11).

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Figure 2.11 A transmission electron micrograph showing close juxtaposition of chloroplast (C), mitochondrion (M) and peroxisome (P) in a mesophyll cell of an immature leaf of bean (Phaseolus vulgaris). This group of organelles is held within a granular cytoplasmic matrix adjacent to a cell wall (CW) and includes a partial view of a small vacuole (V). Scale bar = 1 µm (Electron micrograph courtesy Stuart Craig and Celia Miller)

Phosphoglycolate is dephosphorylated in the stroma and then exported to the cytoplasm by a specific translocator. For every two glycolate molecules exported, one molecule each of CO2 and glycerate are produced. The glycerate is transported into the chloroplast where it is phosphorylated (to produce 3-PGA) and used in the PCR cycle. Biochemical details of the exchange of two glycolate molecules for one glycerate molecule are not yet resolved. Either a single translocator mediates exchange of two glycolate molecules for one molecule of glycerate and one hydroxyl ion, or else separate glycolate and glycerate translocators exist (Figure 2.10).

ATP/ADP translocator

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Figure 2.12 ATP synthesis in mitochondria requires a continuous supply of ADP and inorganic phosphate (H2PO4-). ADP and phosphate are imported in exchange for ATP and an hydroxyl ion, respectively. These exchange processes, which are mediated by separate translocators, are favoured by the electrochemical potential difference across the inner membrane (indicated by the series of positive and negative charges). This difference results from proton translocation by the electron transport chain. (Original drawing courtesy lan Woodrow)

The inner membrane of a chloroplast envelope contains a translocator which catalyses the exchange of ATP and ADP (Figure 2.12). Chloroplasts are thus analogous to mitochondria, but differ in that ATP import is favoured. This translocator is especially active in developing chloroplasts of young leaves, providing ATP while photosynthetic capacity is still developing.

(b)  Peroxisomes

Higher plants contain at least three specialised classes of peroxisomes which are defined according to their main enzyme content and metabolic function. The first class is glyoxisomes and these organelles are present in post-germinative seedlings and senescent organs. Their prime function is metabolism of storage lipids by means of a glyoxylate cycle. The second class of peroxisomes occurs in root nodules of some nitrogen-fixing legumes. These organelles contain ureate oxidase which is involved in synthesis of ureides which represent the main nitrogenous compound exported from root nodules. The thirdclass of peroxisomes is found in photosynthetically active tissues such as leaves and is an integral part of the photorespiratory cycle (Figures 2.10, 2.11). In addition to these three classes, there is a range of unspecialised peroxisomes isolated from a range of tissues including tubers, roots, fruits, petals and shoots that contain catalase and low levels of glycolate oxidase (general features of all classes of peroxisome).

Unlike chloroplasts and mitochondria, peroxisomes are encapsulated by only a single membrane (Figure 2.11). This membrane contains proteins, but no specific translocator has yet been identified. The membrane may contain a non-specific channel, similar to the one identified in rat liver peroxisomes and those in the outer membranes of chloroplasts and mitochondria. Such channels would provide a facilitated diffusion for exchange of metabolites between cytosol and peroxisomal compartments.

Metabolites in leaf peroxisomes are further compartmented by the process of metabolic channelling where enzymes of photorespiratory metabolism are organised into multi-enzyme complexes. In such systems, intermediates are not necessarily released into the aqueous phase of the peroxisome but ‘passed’ between active sites of different enzymes. This type of compartmentation protects a cell from toxic intermediates such as hydrogen peroxide and glyoxylate.

(c)  Mitochondria

All mitochondria house two metabolic compartments (Figure 2.22): first, an intermembrane space between outer and inner membranes of the mitochondrial envelope and, second, a matrix, held inside the highly convoluted inner membrane. As with the chloroplast envelope, outer and inner mitochondrial membranes differ in composition. The outer membrane is composed largely of lipids, whereas the inner membrane is composed of roughly equal proportions of proteins and lipids. These proteins comprise an electron transport system plus an array of specific translocators that regulate flow of metabolites to and from the metabolic pathways of the matrix. The outer membrane does not contain such an array of translocators; it contains a channel-forming porine protein which is nonspecific and allows entry of molecules of up to about 2.5 nm in size (equivalent to a molecular weight of about 7000 kDa).

Not all metabolites require a translocator in order to cross the inner membrane of mitochondria. Small uncharged molecules such as CO2, NH3 and H2O diffuse into and out of the mitochondria at relatively high rates. Moreover, certain amino acids and monocarboxylates can cross the inner membrane in their neutral form.

Enzymes catalysing reactions of the tricarboxylic acid (TCA) cycle (Figure 2.23) reside within the mitochondrial matrix. This cycle, which requires a supply of pyruvate and malate from the cytosol, provides reducing equivalents to the electron transport chain for ATP synthesis and numerous substrates for the biosynthetic reactions in the rest of the cytoplasm. Enzymes in the mitochondrial matrix also feature in other major metabolic pathways including photorespiration, lipid metabolism and amino acid synthesis. As outlined below, the scale of these various metabolic functions and therefore the importance of respective metabolite translocators will vary according to organ function and growing conditions.

Amino acid translocators

In photosynthetic tissues and especially in C3 plants, one of the largest metabolic fluxes sustained by mitochondrial metabolite translocators is that of photorespiration. As outlined above (Figure 2.10; see also Section 2.3), this process involves chloroplasts, peroxisomes and mitochondria. During these processes, referred to collectively as photorespiration, mitochondria take up two glycine molecules from the peroxisomes and release one molecule each of serine, CO2 and ammonia. A specific glycine translocator appears to catalyse exchange of two glycine molecules for one serine molecule and an hydroxyl ion. Neither CO2 nor ammonia requires a translocator to cross the inner membrane.

While several other amino acids have been shown to enter mitochondria by diffusion across both membranes, there are a number of other specific translocator proteins on the inner membrane. These proteins transport glutamate, aspartate and proline. The exchange of amino acids across the inner membrane is important for protein synthesis and turnover within the mitochondria, for the interconversion of amino acids, and possibly for the exchange of reducing equivalents with the cytoplasm. There is, however, little evidence that amino acids are imported into mitochondria as substrates for the TCA cycle. One exception to this may involve proline import. Proline accumulates in plant cells when they are subject to especially water and cold stress. When the stress is removed, proline is rapidly metabolised in the mitochondria where it is ultimately converted to 2-oxoglutarate, a TCA cycle intermediate.

Adenine nucleotide and phosphate transport

A principal function of mitochondria is production of ATP. The enzyme responsible, F1-ATPase, is located on the inner surface of the inner membrane (Figure 2.24). ATP synthesis therefore requires a continuous flow of ADP and phosphate across the inner membrane. ADP import and ATP export is catalysed by a specific translocator that exchanges ATP for ADP. The rate of this exchange is enhanced by a proton gradient across the inner membrane that favours export of the more negatively charged ATP molecule (Figure 2.12). Inorganic phosphate is transported separately, but the translocator for this metabolite is also located on the inner membrane and appears to catalyse the exchange of phosphate with an hydroxyl ion. As with ATP export, phosphate import is favoured by a proton gradient that exists across the inner membrane (Figure 2.12).

Pyruvate transport

The TCA cycle in the mitochondrial matrix generally requires a supply of pyruvate to generate reductants required for electron transport and ATP synthesis. Pyruvate, generated by glycolysis in the cytosol, is transported across the inner membrane by a specific translocator that catalyses an exchange of pyruvate (one negative charge) with an hydroxyl ion (one negative charge), thus maintaining electrical neutrality. Uncharged pyruvic acid can also diffuse across the inner membrane at appreciable rates. However, pyruvic acid has a relatively low pKa and consequently passive diffusion of this substance is probably not important under physiological conditions. Inside the matrix, pyruvate is oxidatively decarboxylated by the multienzyme pyruvate dehydrogenase complex to yield NADH, CO2 and, especially important, acetyl-CoA which then enters the TCA cycle.

Dicarboxylate translocators

Pyruvate is not and cannot be the only TCA cycle substrate (Figure 2.23). In almost all tissues, acetyl-CoA entering the TCA cycle is not oxidised completely to CO2. Rather, a significant proportion of the carbon (in the form of various intermediates) is removed from the cycle for biosynthetic reactions. Because the TCA cycle cannot catalyse the net synthesis of any intermediate, depletion of intermediates by the biosynthetic pathways must be balanced by an import of at least one intermediate. In most tissues, malate is the intermediate imported to replenish the TCA cycle.

Oxaloacetate and aspartate can also be imported to varying degrees. Once in the matrix, malate may be oxidised by the TCA cycle or converted to pyruvate or a combination of both processes. Malate is produced in the cytoplasm in the degradation of proteins or from PEP, an end-product of glycolysis.

Malate import is mediated by several translocators: first, a dicarboxylate translocator which exchanges malate for inorganic phosphate, malonate or succinate; second, a 2-oxoglutarate transporter which exchanges 2-oxoglutarate for malate, malonate, succinate and oxaloacetate, but not inorganic phosphate; third, a uniport protein that facilitates malate import. Because of a requirement for charge compensation (malate carries two negative charges), malate translocation by this uniport is probably coupled to oxaloacetate transport in the opposite direction by another uniport. By operating in synchrony, these two uniports could therefore act as an effective malate/oxaloacetate exchange translocator.

Malate/oxaloacetate exchange is especially important in green tissue where it plays a key role in sustaining photorespiration. Imported oxaloacetate is reduced using the NADH produced during glycine oxidation. Malate, the product of this reduction, is then exported to the peroxisome where its oxidation is linked to the reduction of β-hydroxypyruvate. A malate/oxaloacetate translocator therefore mediates export of reducing equivalents from the matrix.

Tricarboxylate translocators

There is evidence for several translocators capable of catalysing citrate transport across the inner membrane of mitochondria, but only one tricarboxylate translocator has been isolated and characterised. This translocator exchanges citrate and malate. However, there is a difference in charge between these two compounds. At physiological pH, citrate has three negative charges whereas malate has only two. A separate proton translocation by the electron transport chain is therefore required to restore charge balance.

Citrate is synthesised exclusively in mitochondria (Figure 2.23) and is exported to the cytosol for conversion to 2-oxo-glutarate in reactions catalysed by aconitase and NADP-iso-citrate dehydrogenase. A continuous supply of 2-oxoglutarate is required primarily for ammonia assimilation by chloroplasts in a reaction catalysed by glutamine synthetase. Citrate export and subsequent use in ammonia fixation is not needed to
sustain the relatively high rate of ammonia production associated with photorespiration. For this process, 2-oxoglutarate is produced in the peroxisomes as a product of photorespiration and imported into the chloroplast for ammonia assimilation. Photorespiration does not, therefore, result in an overall release of nitrogen.

(d) Vacuolar malate translocators and CAM photosynthesis

Malate transport from cytosol to vacuole across the tonoplast is mediated by at least two types of channel. One channel is involved in malate uptake. This channel opens when the pH gradient across the tonoplast is sufficient to allow for uptake of the negatively charged malate ion against a concentration gradient. This pH gradient constitutes an energy source for malate uptake and is generated by both ATP-dependent and pyrophosphate-dependent proton pumps which translocate protons from cytosol to vacuole. A second channel mediates malate efflux and also opens and closes in response to changes in pH gradient across the tonoplast. Malate efflux, however, generally occurs down a concentration gradient.

Some of the largest fluxes of malate into and out of vacuoles have been measured in CAM plants where CO2 is fixed at night via PEP carboxylase (Figure 2.7). Fixed CO2 is incorporated into malate, which is then transported across the tonoplast and stored at a relatively high concentration in vacuoles. During the daytime, malate is released from vacuole to cytosol where it is decarboxylated. CO2 released in this process is then fixed by the PCR cycle in chloroplasts.

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