Proc. NatL Acad. Sci. USA Vol. 80, pp. 1290-1294, March 1983 Botany

An Arabidopsis thaliana mutant defective in dicarboxylate transport (/malate shuttle/glutamine transport) S. C. SOMERVILLE*t AND W. L. OGREN*t *Department of Agronomy, University of Illinois, Urbana, Illinois 61801; and tU. S. Department of Agriculture, Agricultural Research Service, Urbana, Illinois 61801 Communicated by Harry Beevers, November 8, 1982

ABSTRACT Reactions of the photorespiratory pathway of C3 ically to CO2 at rates that significantly reduce net CO2 assim- plants are found in three subcellular organelles. Transport pro- ilation (9), and ammoniaaccumulates totoxiclevels (8). Because cesses are, therefore, particularly important for maintaining the glutamate is synthesized in the chloroplast (7) and consumed uninterrupted flow of carbon through this pathway. We describe in the peroxisome, the transfer ofglutamate and 2-oxoglutarate here the isolation and characterization of a photorespiratory mu- between these two organelles is a necessary component ofpho- tant of Arabidopsis thaliana defective in chloroplast dicarboxylate torespiratory nitrogen metabolism. transport. Genetic analysis indicates the defect is due to a simple, Transport systems operating at the peroxisome-bounding recessive, nuclear mutation. Glutamine and inorganic phosphate been characterized. However, studies with transport are unaffected by the mutation. Thus, in contrast to pre- membrane have not vious reports for pea and spinach, glutamine uptake by Arabi- isolated have revealed the presence of a trans- dopsis chloroplasts is mediated by a transporter distinct from the porter, designated the dicarboxylate transporter, which cata- dicarboxylate transporter. Both the inviability and the disruption lyzes the counter-exchange of several dicarboxylic acids across of amino-group metabolism of the mutant under photorespiratory the chloroplast inner membrane (10). These compounds include conditions suggest that the primary function of the dicarboxylate malate, 2-oxoglutarate, aspartate, and glutamate (10). Gluta- transporter in vivo is the transfer of 2-oxoglutarate and glutamate mine is also a reported substrate for this carrier (11, 12). There- across the chloroplast envelope in conjunction with photorespira- fore, the chloroplast dicarboxylate transporter is implicated as tory nitrogen metabolism. The role commonly ascribed to this an important component of the photorespiratory pathway. Be- transporter, conducting malate-aspartate exchanges for the in- cause this transporter is also capable of effecting malate-as- direct export of reducing equivalents from the chloroplast, ap- partate exchanges, ithas been ascribed the role ofmediatingthe pears to be a minor one. indirect export of reducing equivalents to the cytoplasm (13, 14). The constituent reactions of the photorespiratory pathway of The isolation and characterization of a photorespiratory mu- higher plants occur in three organelles, the chloroplast, the mi- tant defective in chloroplast dicarboxylate transport is reported tochondrion, and the peroxisome. Thus, transport processes must here. Biochemical and physiological analyses of the mutant have intervene at several steps of the pathway (Fig. 1) (1, 2). To the proven useful in determining the specificity of this transporter extent that the transport of substrates, products, or cofactors is and in ascertaining its primary in vivo role. limiting, these transport processes may be expected to exert a strong regulatory influence on photorespiratory metabolism. The photorespiratory pathway is initiated by the oxygenation MATERIALS AND METHODS of ribulose bisphosphate by the bifunctional enzyme ribulose- Plant Material andCulture. Both the mutant line CS156, the bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (3, 4). 02 subject of this study, and the previously described line CS113, and CO2 act as competitive substrates for this enzyme, and the a glutamate synthase-deficient mutant (8), were recovered in a degree to which carbon is diverted from the Calvin cycle to the screen for mutants of Arabidopsis thaliana (L.) Heynh. (race photorespiratory pathway is a function of the ratio of these two Columbia) with defects in photorespiratory metabolism (6). The gases in the atmosphere (5). Experimentally, the flux of carbon basis of the mutant selection procedure is that strains with de- through the pathway can be suppressed without adverse effect fects in the photorespiratory pathway cannot survive at atmo- byplacingplants in an atmosphere enriched in CO2 or enhanced spheric levels of CO2 and 02 but grow normally at 1% CO2, when by transferring plants to an atmosphere enriched in 02(6). Three- photorespiration is suppressed. quarters of the carbon entering the pathway is returned to the Plants were grown according to described methods and con- poolof Calvin-cycle intermediates as phosphoglycerate. The re- ditions (15). For most of this study, a line descended from a maining carbon is lost as CO2 at the glycine decarboxylase step backcross of CS156 to the wild type was used. Experiments were in the mitochondrion. conducted with plants at the rosette stage of development (3- Tightly integrated with the photorespiratory carbon cycle is 4 wk from seeding). Procedures for making genetic crosses and the photorespiratory nitrogen cycle in which ammonia released measuring gas exchange have been described (15). during glycine deamination is refixed by the sequential action Labeling Studies. Plants were labeled with 14CO2 for 10 min, ofglutamine synthetase and glutamate synthase (Fig. 1) (7). The and the distribution of label among products was determined resultant glutamate supports both photorespiratory ammonia by ion-exchange and thin-layer chromatography (15-17). refixation (8) and glyoxylate amination (9). In the absence of Glutamate Synthase Assays. Glutamate synthase was as- adequate glutamate pools, glyoxylate is oxidized nonenzymat- sayed in crude extracts of leaf material after centrifugation at The publication costs of this article were defrayed in partby page charge Abbreviation: Chl, chlorophyll. payment. This article must therefore be hereby marked "advertise- tPresent address: MSU-DOE Plant Research Laboratory, Michigan State ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Univ., East Lansing, MI 48824. 1290 Downloaded by guest on September 29, 2021 Botany: Somerville and Ogren Proc. Natl. Acad. Sci. USA 80 (1983) 1291

Krebs m.M to inhibit C02-dependent 02 evolution (22). Cycy The silicone oil layer filter centrifugation technique was used to measure the transport of radiolabeled compounds into freshly CHHLOROPLAST TP isolated, intact chloroplasts (23). The standard assay medium APG contained, in addition to the standard components, 10-30 tig ADP of Chl and 1-3 ,uCi (3.7-11.1 x 104 Bq) of 3H20. Glutamine L3-dPGA I,-diPGA transport was determined at pH 7.9. The silicone oil layer con- sisted of AR200/AR20 60:40 (wt/wt) (Wacker Chemie, SWS Silicones, Adrian, MI). Chloroplast volumes were determined

TP PGA with [14C]sorhitol in parallel experiments under the same con- C02 ditions as the. transport assays (23). The average sorbitol-im- Sucros permeable space was 38 ,ul/mg of Chl and 44 p.l/mg of Chl for P-Glycolate wild-type and mutant chloroplasts, respectively: Transport as- I______I______says, commonly 3-, 5-, or 10-sec duration, were performed in I the dark at 50C. For some experiments, chloroplasts were pre- Mdote tMabte Glycerote GlyWcote loaded with the compound to be assayed for uptake by adding a small volume of stock solution to freshly prepared chloroplasts to give a final concentration of 20 mM. After 15 min on ice in OAA OAA OHPyruvate Qlyoxyote the dark, the chloroplasts were collected by centrifugation (270 '4 >342: X g at 40C for 40 sec) and resuspended in medium lacking the -2064 A amg,hb compound. Apparent Km and Vm.x values were estimated from -Asp Sor Gly Asp Scatchard plots of the data presented in the text. Chl was de- PEROXISOE T I termined spectrophotometrically in ethanol (24). J L

I x^A RESULTS Ser * Gly Mutant Isolation and Genetic Analysis. In an atmosphere that ,NWCOH suppressed photorespiration (1% C02/99% air), the mutant line THF T NH3 CS156 was capable of normal growth and development. How- ever, in standard atmospheric conditions (N2 containing 0.03% MITOCHONDRION CO2 and 21% 02), the mutant became yellow and lost vigor within NAM NOD 3-4 days. The F1 plants from a CS156 x wild-type cross were healthy in standard atmospheres, suggesting the mutant line carried a recessive, nuclear mutation. In a derivative F2 pop- FIG. 1. Schematicpresentation of thephotorespiratory carbon and ulation, 196 plants exhibited the wild-type phenotype and 55 nitrogen cycles. C1-THF, N5,N'0-methylenetetrahydrofolate; DT, di- carboxylate transporter; OAA, oxaloacetate; 20G, 2-oxoglutarate; PT, were yellow after 4 days in a normal atmosphere. Thus, the mu- phosphate translocator; PGA, phosphoglycerate; RuBP, ribulose bis- tation in line CS 156 responsible for the growth requirement for phosphate; THF, tetrahydrofolate; TP, triose phosphate. high CO2 was inherited as a simple, recessive, nuclear mutation (X2 = 1.276; P > 0.25). The locus defined by this mutant was 30,000 x g and desalting by Sephadex G-25 column chroma- designated dct (dicarboxylate transport). The F1 plants from a tography (8). Protein was determined by a dye-binding assay cross of CS 156 with CS 113, a glutamate synthase-deficient line (18) with bovine serum albumin as the standard. (8), were normal in appearance and capable of sustained growth Ammonia and Amino Acid Determinations. Plants were in a normal gas regime. Because the mutation in CS156 com- equilibrated. in darkness for 25 min and then illuminated (300 plemented that in CS113, the dct locus was genetically distin- microeinsteins m-2se&- of photosynthetically active radiation) guished from the gluS locus. for various lengths of time in an open gas exchange system main- Gas Exchange Analyses. CO2 fixation was measured on in- tained at 25TC and 70% relative humidity. The photorespiratory dividual plants in nonphotorespiratory (Fig. 2A) and photores- gas regime was nitrogen containing 357 1.l of CO2 liter' and piratory (Fig. 2B) gas regimes. The photosynthesis rate of the 49% 02. Methods for quantitating ammonia and amino acid lev- mutant exceeded that of the wild type in an atmosphere of low els in leaf tissue have been described (8). Amino acid values were 02 concentration (Fig. 2A). In contrast, photosynthesis was im- normalized by using the amount of phenylalanine per mg of paired in the mutant in atmospheres that promoted photores- chlorophyll (Chl) as a basis to correct for losses that occurred piration. In the photorespiratory gas regime of nitrogen con- during the preparation of some samples. taining.49% 02 and 357 A.l of C02 liter', the final rate of CO2 Chloroplast Isolation and Transport Assays. Chloroplasts were -fixation in CS156.was reduced to 27% of the rate in wild type prepared from Percoll gradient-purified protoplasts (19) and used (Fig. 2B). CO2 evolution into C02-free 50% 02/50% N2, a mea- immediately. The intactness of the chloroplast preparations was sure of photorespiration, also was reduced in mutant plants (data determined by the ferricyanide test (20). not presented). Thus, the lesion in mutant CS156 disrupted Oxoglutarate + glutamine-dependent 02 evolution by iso- photorespiratory metabolism specifically and had no detectable lated chioroplasts was measured in a Hansatech 02 electrode at effect on photosynthesis and growth in nonphotorespiratory en- 25°C at a light intensity of 1,000 microeinsteins m 2sec- of vironments. photosynthetically active radiation at the surface of the cuvette Metabolite Distribution. A defect in photorespiratory me- (21). Chloroplasts (20-40 ,ug of Chl) were added to the standard tabolism was evident in the mutant from the altered distribution assay medium [300 mM sorbitol/28 mM HepesrKOH, pH 7.6/ pattern of metabolites labeled with '4Co2 for 10 min in a pho- 10 mM NaHCO3/2.5 mM EDTA/0.15 mM potassium phos- torespiratory gas regime (Table 1). Notably, the accumulation phate/0. 1%. fatty acid-free bovine serum albumin/300 units of of 14C label in the basic fraction was reduced and that found in catalase per ml (19)]. D,L-Glyceraldehyde was added to 6-10 the acid-i fraction was increased in mutant plants compared to Downloaded by guest on September 29, 2021 1292 Botany: Somerville and Ogren Proc. Natl. Acad. Sci.-, USA 80 (1983) In consideration of the difficulties associated with equating 30 the distribution of radioactive label. and'mass flow of metabo- lites, we undertook a quantitative analysis of amino acid pools in the leafunderphotorespiratory conditions. The results of this 20 experiment confirmed that the mutant harbored a defect in glutamate metabolism (Fig. 3)'. Upon illumination, glutamate levels in the mutant declined, suggesting that utilization ofthis 10 amino acid. greatly exceeded its synthesis (Fig. 3A)'. Glutamine 0 levels in the mutant declined slightly and then stabilized at a

cq relatively high level (Fig. 3B). Lack of depletion of glutamine pools and accumulation of '4C label in 2-oxoglutarate (Table 1) implied that the mutant was not able to convert these two com- C, 0 pounds to glutamate. Ammonia accumulated to abnormally high bo levels in the mutant plants (Fig. 3C). A similar result was ob- ; 20 served in the gluS. mutant, CS113, which.was unable to refix B photorespiratory ammonia by the /glu- tamate synthase reactions (8). The-metabolism of glycine (Fig.

0~ = 10 3D) and that ofserine (Fig. 3E), both intermediates ofthe pho- ¢I e--l' torespiratory pathway, were also abnormal in the mutant line. These are considered secondary effects resulting from the dis------0 ruption of photorespiratory metabolism.

8.0 0 A 0 20 40 60 80 Time, min 4.0 FIG. 2. NetCO2fixationbywild!typeandmutantArabidopsisplants under nonphotorespiratory (A) and photorespiratory (B) conditions. The 00 gas regime in A was nitrogen containing 352 gl of C02 liter-' and 2% 0 - 02 and inB was nitrogen containing 357 AI of C02 liter-' and 49% 02. 8.0 -o B. -* Response of wild type; ---, response of mutant CS156;-; dark conditions; m, light conditions. The response shown represents the av- erage of two experiments. 4.0 wild-type plants. Labeling of all major constituents of the basic was fraction depressed. The increase of 14C label in the acid-i 0 fraction was primarily due to an increased labeling of 2-oxoglu- o 0 tarate. In these respects the labeling pattern of the dct mutant closely resembled that of a glutamate synthase-deficient line, CS113 (Table 1), implicating a defect in glutamate synthesis as 0 the cause for the high CO2 requirement of the dct mutant. a0.2 0 0 Table 1. Percentage distribution of the products of Q 0/- 14c0 assimilation* 4a) Distribution (%) by strain D A~~D Fraction Wild type CS156 CS113 2 2.0. Basic 41.0 17.2 16.3 Glutamate 1.5 0.7 0.5 Glycine 21.2 9.0 10.0 1.0 Serine 5.8 3.6 2.2

Acid-i 6.3 29.8 22.6 j Malate 3.6 2.4 2.6 0 Glycerate 1.0, 0 0 E 2-Oxoglutarate 0. 14.0 8.7 0 Acid-2 24.0 27.3 33.5 Acid-3 8.9 11.3 13.1 1.0 Neutral 14.0 11.0 10.8 Insoluble 4.6 5.1 6.2 Recovery 100.8 101.7 102.5 0 10 20 30' * Values given are the percentage of total 14C incorporated into leaves. Time, min. Plants were labeled with 14C02 at 5-15 min from the onset of, illu- mination. The photorespiratory gas regime was as described, andthe FIG. 3. Amino acid and ammonia levels in leaves of wild-type (o) conditions during labeling were 25TC, 70% relative humidity, and 300 and mutant CS156 (o)Arabidopsis plants under photorespiratory con- microeinsteins m 2sec' of photosynthetically active radiation. Each ditions. (A) Glutamate. (B) Glutamine. (C) Ammonia. (D) Glycine. (E) value is the mean of three determinations. Serine. Downloaded by guest on September 29, 2021 Botany: Somerville-and Ogren Proc. Natl. Acad. Sci. USA 80 (1983) 1293

Enzyme Analyses. Crude leaf extracts of mutant plants showed A /- B C 63% ofwild-type levels offerredoxin-dependent glutamate syn- 120 thase activity (data not presented). However, previous analyses of gluS mutants showed that F1 (wild-type x gluS) plants with 50% of wild-type levels of glutamate synthase activity were _.40H 80 phenotypically normal (8). Thus, the observed reduction of en- zyme activity in CS 156 was not sufficient to account for the high 40 CO2 growth requirement in the mutant. Also, as noted above, 0- the mutation in CS156 was not allelic to the gluS mutation in /00 S-4 CS113. 0 T. fI Transport Assays. Physiological studies of metabolite pools -a) 1.0 2.0 D 1.0 2.0 0 1.0 2.0 3.0 strongly indicated glutamate metabolism was disrupted in the dct mutant, but in vitro enzyme assays did not verify this con- E F clusion. To examine this inconsistency, glutamate synthase was measured in isolated, intact chloroplasts as 2-oxoglutarate + glutamine-dependent 02 evolution (21). At low external con- centrations of 2-oxoglutarate, no 2-oxoglutarate + glutamine- !8 o'e dependent 02 evolution by mutant chloroplasts was detected 0~ (Table 2). However, when the 2-oxoglutarate concentration was 0 0 raised to 100 mM, chloroplasts from both the mutant and wild 0 1.0 2.0 0 4.0 8.0 0 0.4 0.8 1.2 type exhibited glutamate synthase activity. Thus, it appeared External concentration, mM that the enzyme was present and active in chloroplasts from the dct mutant, confirming conclusions from in vitro enzyme as- FIG. 4. The uptake of several compounds into the sorbitol-im- permeable space of chloroplasts from wild-type (e) and mutant CS156 says. On the basis of these experiments, it was considered prob- (0) plants. All experiments were conducted at 5YC. Chloroplasts used able that chloroplasts from the mutant were substantially less in these experiments were >94% intact. The compounds assayed for permeable to 2-oxoglutarate than were wild-type chloroplasts. uptake were 2-oxoglutarate (A), aspartate (B), glutamate (C), malate The uptake of 2-oxoglutarate and several other compounds (D), glutamine (E), and inorganic phosphate (F). For experiments pre- by isolated chloroplasts was measured directly by using the sil- sented in A, B, C, and D, chloroplasts were preloaded with the com- icone oil filter centrifugation technique (23). For wild-type pound to be assayed for transport. chloroplasts, the uptake of four dicarboxylates, glutamine, and inorganic phosphate became saturated at high external sub- iments, chloroplasts were incubated in high concentrations of substrate prior to measuring uptake to ensure that the internal strate concentrations, a characteristic of facilitated transport (Fig. 4). To better characterize the transporters in wild-type Arabi- supply of dicarboxylate did not limit the uptake of exogenously supplied compounds by the counterexchange transporter. Pre- dopsis chloroplasts, kinetic parameters describing the concen- loading wild-type chloroplasts with malate, and tration dependence of transport rates for these compounds were 2-oxoglutarate, glutamate stimulated malate, 2-oxoglutarate, and glutamate up- determined (Table 3). Apparent Km values were slightly higher take, respectively, =2-fold. This enhancement of transport rate than those reported for spinach (10, 25) and pea (12). Values of cannot be attributed to significant carryover of substrate from Vma, for the four dicarboxylates and glutamine were consider- ably higher (10, 12). For chloroplasts from the mutant, the the preloading to the assay step. The amount of carryover was transport of malate, 2-oxoglutarate, aspartate, and glutamate calculated to be 31-90 nmol (equivalent to a 0.010 to 0.028 mM increase in the substrate concentration in the assay was severely reduced. However, the uptake of glutamine was mixture) by comparing the rate or aspartate at ex- indistinguishable from the wild type, suggesting that this amino of glutamate uptake low ternal concentrations not stimulated with rate acid was transported by a carrier distinct from the dicarboxylate by preloading the when a transport rate transporter (Fig. 4E). The-uptake of inorganic phosphate was expected linear relationship between and unaffected by the lesion at the dct locus in the mutant (Fig. 4F). concentration is assumed. In addition to enhancing the rate of to a re- Similar results were obtained at 250C. Thus, it is unlikely that uptake and, hence, the observed Vmax, preloading led mM the loss ofdicarboxylate transport activity was an artifact of low- duction by 0.5 of the apparent Km of 2-oxoglutarate and temperature effects on the mutant chloroplast envelope. Also, malate for wild-type chloroplasts. Uptake of 2-oxoglutarate and the normal functioning of two carriers in the envelope of the glutamate by mutant chloroplasts was unaffected by the pre- step. in mutant indicated that the lesion was specific for dicarboxylate loading However, the absence of preloading, malate transport and did notaffect thegeneral architecture of the chlo- uptake by the mutant could not be detected. A low rate of as- .roplast envelope. partate uptake, which did not saturate at 2.0 mM, was observed when the preloading step was omitted. With preloading, a re- The chloroplast dicarboxylate transporter is reported to op- duced level of aspartate transport was erate by a some exper- apparently facilitated counterexchange mechanism (10). For measured (Fig. 4B).

Table 2. Glutamine + 2-oxoglutarate-dependent 02 evolution by isolated chloroplasts Table 3. Kinetic constants of uptake of several compounds by wild-type chloroplasts 02 evolution, Amol/mg of Chl per hr VmLax, PMo0/mg Substrate Km x 103 of Chl per hr Substrate, mM Wild type CS156 Glutamine 2-Oxoglutarate strain mutant Malate 1.3 130 2-Oxoglutarate 0.5 176 5 3 4.77 0 Aspartate 0.5 111 5 100 4.00 10.60 Glutamate 2.7 150 Glutamine 2.4 42 The percentage chloroplast intactness was 88% for wild type and 76% for CS156. Inorganic phosphate 0.5 90 Downloaded by guest on September 29, 2021 1294 Botany: Somerville and Ogren PSProc. Natl. Acad. Sci. USA 80'(1983) DISCUSSION and aspartate uptake by chloroplasts of the dctmutant may rep- Mutant line CS156 carries a recessive nuclear mutation re- resent transport activity by this second carrier. Aspartate (10) sponsible for the specific loss of chloroplast dicarboxylate trans- and 2-oxoglutarate (M. 0. Proudlove and D. A. Thurman, per- port.activity. Thus, although.the dct mutant showed substantial sonal communication) are recognized by more than one chlo- in vitro glutamate synthase activity, in vivo analyses indicated roplast carrier. In each case, the second transporter was de- that the enzyme was essentially inoperative because of sub- scribed as having a relatively low activity. strate limitation (Table 2 and Fig. 3). Mutants harboring defective transporters generally have been The lethality of the dct mutant in atmospheres promoting identified as being resistant to an exogenously supplied toxic photorespiration can be explained by the in vivo loss of gluta- substance. For this reason, transport processes operating across mate synthase function. This in turn restricts glyoxylate ami- the plasma membrane of simple organisms have been the most nation and photorespiratory ammonia refixation (Fig. 3C) by amenable to analysis with. mutants. The dot mutant of Arabi- limiting the supply of glutamate (Fig. 3A). If glyoxylate is not dopsis demonstrates that intracellular transport in eukaryotes transaminated, it does not accumulate but undergoes nonen' also can be subject- to mutant analysis. zymatic oxidation, resulting in, a greatly enhanced loss of re- cently fixed carbon. As a result, Calvin-cycle intermediates are 1. Chollet, R. & Ogren, W. L. (1975) Bot. Rev. 41, 137-179. depleted, leading to adecline in netCO2fixation (Fig. 2B). Sim- 2. Tolbert, N. E. (1979) in Encyclopedia of Plant Physiology, New Series, eds. Gibbs, M. & Latzko, E. (Springer, New York), Vol. ilar explanations have been offered for the high CO2 growth re- 6, pp. 338-352. quirement of four other classes of mutants with defects in pho- 3. Bowes, G., Ogren, W. L. & Hageman, R. H. (1971) Biochem. torespiratory nitrogen metabolism (8, 9, 26, 27).- These results Biophys. Res. Commun. 45,716-722: emphasize the interdependence of the photorespiratory carbon 4. Lorimer, G. H. (1981) Annu. Rev. Plant Physiol. 32, 349-383. and nitrogen cycles. 5. Laing, W. A., Ogren, W. L. & Hagemnan, R, H. (1974) PlantPhysiol, The chloroplast dicarboxylate transporter is generally con- 54, 678-685. 6. Somerville, C. R. & Ogren, W. L. (1979) Nature (London) 280, sidered an essential component of a malate shuttle for trans- 833-836. porting reducing equivalents across the chloroplast inner mem- 7. Keys, A. J., Bird, I. F., Cornelius, M. J., Lea, P. J., Wallsgrove brane (13, 14). Because mutant CS156 was recovered in a screen R. M. & Miflin, B. J. (1978) Nature (London) 275, 741-743. for photorespiratory mutants and was capable of normal pho- 8. Somerville, C. R, & Ogren, W. L. (1980) Nature (London) 286, tosynthesis (Fig. 2A) and growth in nonphotorespiratory re- 257-259. gimes, the major role of the chloroplast dicarboxylate trans- 9. Somerville, C. R. & Ogren, W. L. (1981) Plant Physiol 67, 666- 671. porter in vivo must be as a component of the photorespiratory 10. Lehner, K. & Heldt, H. W. (1978) Biochim. Biophys. Acta 501, nitrogen cycle. Conversely, the proposed role for this trans- 531-544. porter as a part of the chloroplast malate shuttle appears to be 11. Gimmler, H., Schafer, G., Kraminer, H. & Heber, U. (1974) Planta of minor physiological importance. The chloroplast phosphate 120, 47-61. translocator is the only other known route for indirectly ex- 12. Barber, D. J. & Thurman, D. A. (1978) Plant Cell Environ. 1, 297- porting reducing equivalents from the chloroplast at significant 303. 13. Heber, U. (1974) Annu. Rev. Plant Physiol. 25, 393-421. rates (14, 25). However, both ATP and NAD(P)H are exported 14. Heldt, H. W. (1976) in The Intact Chloroplast, ed. Barber, J. in an obligate manner by this transporter. It seems likely that (Elsevier/North-Holland, Amsterdam), pp. 215-234. some mechanism must exist for modulating the cytosolic levels 15. Somerville, C. R. & Ogren, W. L. (1982) in Methods in Chloro- of ATP and NADH. The low level of dicarboxylate transport plast Biology, eds. Edelman, M., Hallick, R. B. & Chua, N. H. activity observed in the dct mutant may be adequate to meet (Elsevier, Amsterdam), pp. 129-138. this 16. Cossins, E. A. & Sinha, S. K. (1966) Biochem. J. 101, 542-549. requirement. 17. Block, R. J., Durrum, E. L. & Zweig, G. (1955) A Manual of Pa- The dct mutant has been useful for determining the speci- per Chromatography and Paper Electrophoresis (Academic, New ficity of the dicarboxylate transporter. Previous studies based York), p. 168. on back-exchange (11) and competition (12) experiments sug- 18. Spector, T. (1978) Anal. Biochem. 86, 142-146. gested glutamine was transported across the chloroplast enve- 19. Somerville, C. R., Somerville, S. C. & Ogren, W. L. (1981) Plant lope by the dicarboxylate transporter. This appears to be in- Sci. Lett. 21, 89-96. with the observation that is 20. Lilley, R. M., Fitzgerald, M. P., Rientis, K. G. & Walker, D. A. consistent glutamine transport (1975) New Phytol. 75, 1-10. unaffected by the lesion at the dct locus in the mutant. Clearly 21. Anderson, J. W. & Done, J. (1977) Plant Physiol 60, 354-359. a separate transporter for glutamine, distinct from the dicar- 22. Stokes, D. M. & Walker, D. A. (1972) Biochem. J. 128, 1147-1157. boxylate carrier, must occur in the chloroplast envelope. The 23. Heldt, H. W. (1980) Methods Enzymol 69, 604-613. discrepancy between previous and present results regarding the 24. Wintermans, J. F. G. M, & Demots, A. (1965) Biochim. Biophys. specificity of the dicarboxylate carrier can be resolved if two Acta 109, 448-453. exist in the en. 25. Fliege, R., Flugge, U. I., Werden, K. & Heldt, H. W. (1978) carriers with overlapping specificity chloroplast Biochim. Biophys. Acta 502, 232-247. velope. The carrier defined by the dct mutant recognizes di- 26. Somerville, C. R. & Ogren, W. L. (1980) Proc. Natl Acad. Sci. USA carboxylates but not glutamine (Fig. 4), whereas the second car- 77, 2684-2687. rier presumably transports dicarboxylates at a low rate and 27. Somerville, C. R. & Ogren, W. L. (1982) Biochem. J. 202, 373- glutamine. The low level of malate, 2-oxoglutarate, glutamate, 380. Downloaded by guest on September 29, 2021