Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator

Abstract

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...

Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization

“…Recently, a putative glucose transporter has been described for the chloroplast envelope membrane that operates with an unexpectedly high V max value (Weber et al 2000). These results suggest that starch hydrolysis is the principle degradative pathway.…”

Molecular cloning, functional characterization and expression analysis of a novel monosaccharide transporter gene OsMST6 from rice (Oryza sativa L.)

“…1 Comparison of amino acid sequences of MST proteins from rice and other organisms. The predicted protein sequence of OsMST6 is compared with those of the previously characterized MSTs OsMST1-5 and 8 from rice (Toyofuku et al 2000;Ngampanya et al 2003;Mamun et al 2006;Wang et al 2007), SopGlcT from spinach (Weber et al 2000), and HsGLUT1 from human (Mueckler et al 1985). Black boxes indicate identical amino acid residues, and gray boxes represent amino acid residues with similarity of more than 50%.…”

Expression profiling of starch metabolism-related plastidic translocator genes in rice

“…Based on previous reports on starch metabolism-related plastidic translocators in maize and Arabidopsis (i.e., Arabidopsis TPT, Knappe et al 2003a;maize TPT, Fischer et al 1994; Arabidopsis GPT, Knappe et al 2003b;maize GPT, Kammerer et al 1998;Arabidopsis PPT1, Fischer et al 1997;Arabidopsis PPT2, Knappe et al 2003a;maize PPT, Fischer et al 1997;maize BT1, Sullivan et al 1991;Arabidopsis NTT1, Neuhaus et al 1997; Arabidopsis NTT2, Mohlmann et al 1997; Arabidopsis and maize pGlcT, Weber et al 2000; Arabidopsis MT1, Niittyla et al 2004) candidates for plastidic translocator genes in rice were first searched in the rice full-length cDNA (KOME) database (Kikuchi et al 2003; http://cdna01.dna.affrc.go.jp/cDNA/), and the Gramene database (Ware et al 2002; http:// www.gramene.org/). Subsequently, the protein databases Rice Membrane Protein Library (RMPL; http:// www.cbs.umn.edu/rice/), and the ARAMEMNON (http://aramemnon.botanik.uni-koeln.de/) were accessed for confirmation.…”