SummaryThe Arabidopsis mutant dicarboxylate transport (dct ) is one of the classic mutants in the photorespiratory pathway. It requires high CO 2 levels for survival. Physiologic and biochemical characterization of dct indicated that dct is de®cient in the transport of dicarboxylates across the chloroplast envelope membrane. Hence, re-assimilation of ammonia generated by the photorespiratory cycle is blocked. However, the defective gene in dct has not been identi®ed at the molecular level. Here, we report on the molecular characterization of the defective gene in dct, on the complementation of the mutant phenotype with a wild-type cDNA, and on the functional characterization of the gene product, DiT2, in a recombinant reconstituted system. Furthermore, we provide the kinetic constants of recombinant DiT1 and DiT2, and we discuss these data with respect to their functions in ammonia assimilation. Moreover, an analysis of the transcript levels of DiT1 and DiT2 in different C 3 -and C 4 -type plant species is presented, and we demonstrate that the substrate speci®city of DiT2 from the C 4 -plant Flaveria bidentis is similar to its counterpart from C 3 plants.

The subcellular localization of hexokinase activities in plant cells has been a matter of debate for a long time. We have isolated a hexokinase cDNA fragment from glucose-fed spinach leaves using a differential display reverse transcription-PCR approach. The corresponding cDNA was expressed in Escherichia coli and an antiserum, raised against the recombinant protein, was used in subcellular localization studies. The spinach hexokinase could be localized primarily to the outer envelope membrane of chloroplasts where it is inserted via its N-terminal membrane anchor. We suggest that the chloroplast envelope hexokinase is involved in the energization of glucose export from plastids rather than in the sugar-sensing pathway of the plant cell.z 1999 Federation of European Biochemical Societies.

During photosynthesis, part of the fixed carbon is directed into the synthesis of transitory starch, which serves as an intermediate carbon storage facility in chloroplasts. This transitory starch is mobilized during the night. Increasing evidence indicates that the main route of starch breakdown proceeds by way of hydrolytic enzymes and results in glucose formation. This pathway requires a glucose translocator to mediate the export of glucose from the chloroplasts. We have reexamined the kinetic properties of the plastidic glucose translocator and, using a differential labeling procedure, have identified the glucose translocator as a component of the inner envelope membrane. Peptide sequence information derived from this protein was used to isolate cDNA clones encoding a putative plastidic glucose translocator from spinach, potato, tobacco, Arabidopsis, and maize. We also present the molecular characterization of a candidate for a hexose transporter of the plastid envelope membrane. This transporter, initially characterized more than 20 years ago, is closely related to the mammalian glucose transporter GLUT family and differs from all other plant hexose transporters that have been characterized to date.

During photosynthesis, part of the fixed carbon is directed into the synthesis of transitory starch, which serves as an intermediate carbon storage facility in chloroplasts. This transitory starch is mobilized during the night. Increasing evidence indicates that the main route of starch breakdown proceeds by way of hydrolytic enzymes and results in glucose formation. This pathway requires a glucose translocator to mediate the export of glucose from the chloroplasts. We have reexamined the kinetic properties of the plastidic glucose translocator and, using a differential labeling procedure, have identified the glucose translocator as a component of the inner envelope membrane. Peptide sequence information derived from this protein was used to isolate cDNA clones encoding a putative plastidic glucose translocator from spinach, potato, tobacco, Arabidopsis, and maize. We also present the molecular characterization of a candidate for a hexose transporter of the plastid envelope membrane. This transporter, initially characterized more than 20 years ago, is closely related to the mammalian glucose transporter GLUT family and differs from all other plant hexose transporters that have been characterized to date. INTRODUCTIONIn plants, carbon fixed during the day is exported from the chloroplasts in the form of triose phosphate (trioseP), which is converted in the cytosol to sucrose. Sucrose often serves as the predominant photoassimilate being allocated to sink tissues. The export of trioseP from the chloroplasts is mediated by the trioseP/3-phosphoglycerate/phosphate translocator (TPT; Fliege et al., 1978; Flügge et al., 1989). Rather than being exported, a considerable amount of the fixed carbon is maintained within the chloroplasts and is involved in the biosynthesis of transitory starch, which could amount to approximately one-half of the carbon assimilated by photosynthesis during the day. During the next dark period, transitory starch is mobilized to sustain a continuous supply of carbon (i.e., sucrose) for export to growing sinks as well as for energy metabolism in leaves. Mutants lacking the ability to synthesize (Caspar et al., 1985; Hanson and McHale, 1988;Huber and Hanson, 1992; Geiger et al., 1995) or degrade transitory starch (Zeeman et al., 1998a(Zeeman et al., , 1998bCaspar et al., 1991) show reduced growth under conditions in which photosynthesis is restricted.Starch degradation could follow either the phosphorolytic pathway, yielding trioseP, or the amylolytic pathway, leading to free sugars, glucose (Glc), and maltose. There is evidence that the dominant pathway for the degradation of transitory starch is the amylolytic one. First, trioseP, the end product of the phosphorolytic pathway, must be exported from the chloroplasts and subsequently be converted to hexose phosphate (hexoseP) in the cytosol. This reaction is controlled by the regulatory metabolite fructose 2,6-bisphosphate, which is a strong inhibitor of the cytosolic fructosebisphosphate phosphatase (Stitt, 1990). During the transition from...