Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only ‫ف‬ 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates. INTRODUCTIONDuring C 3 photosynthesis, energy from solar radiation is used for the formation of phosphorylated C3 sugar phosphates, triose phosphates (trioseP), and 3-phosphoglycerate (3-PGA); these products are exported from the chloroplasts into the cytosol via the trioseP/3-PGA/phosphate translocator (TPT). In the mature leaves of most plants, the exported photosynthates are then used in the formation of sucrose, which is allocated via the phloem to the heterotrophic plant organs, such as young leaves, roots, seeds, fruits, or tubers. In these sink tissues, sucrose serves as a source of carbon and energy and is first cleaved by the action of invertases or sucrose synthase; the products of these reactions are converted into hexose phosphates.Nongreen plastids of heterotrophic tissues are carbohydrate-importing organelles and, in the case of amyloplasts of storage tissues, the site of starch synthesis. Because these plastids are normally not able to generate hexose phosphates from C3 compounds due to the absence of fructose 1,6-bisphosphatase activity (Entwistle and ap Rees, 1988), they rely on the import of cytosolically generated hexose phosphates as the source of carbon for starch biosynthesis and, in addition, for the oxidative pentose phosphate pathway. The results of transport measurements with intact organelles or reconstituted tissues from different plants suggest that thi...

We have purified a plastidic phosphate transport protein from maize endosperm membranes and cloned and sequenced the corresponding cDNAs from maize endosperm, maize roots, cauliflower buds, tobacco leaves, and Arabidopsis leaves. All of these cDNAs exhibit high homology to each other but only approximately 30% identity to the known chloroplast triose phosphate/phosphate translocators. The corresponding genes are expressed in both photosynthetically active tissues and in nongreen tissues, although transcripts were more abundant in nongreen tissues. Expression of the coding region in transformed yeast cells and subsequent transport measurements of the purified recombinant translocator showed that the protein mediates transport of inorganic phosphate in exchange with C3 compounds phosphorylated at C-atom 2, particularly phosphoenolpyruvate, which is required inside the plastids for the synthesis of, for example, aromatic amino acids. This plastidic phosphate transporter is thus different in structure and function from the known triose phosphate/phosphate translocator. We propose that plastids contain various phosphate translocators with overlapping substrate specificities to ensure an efficient supply of plastids with a single substrate, even in the presence of other phosphorylated metabolites.

Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only approximately 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates.

We have purified a plastidic phosphate transport protein f r o m maize endosperm membranes and cloned and sequenced the corresponding cDNAs from maize endosperm, , maize roots, cauliflower buds, tobacco leaves, and Arabidopsis leaves. All of these cDNAs exhibit high homology t a e a c h other but only -30% identity to the known chloroplast triose phosphate/phosphate translocators. The corresporrding genes are expressed in both photosynthetically active tissues and in nongreen tissues, although transcripts were more abundant in nongreen tissues. Expression of the coding region in transformed yeast cells and subsequent transport measurements of the purified recombinant translocator showed that the protein mediates transport of inorganic phosphate in exchange with C3 compounds phosphorylated at C-atom 2, particularly phosphoenolpyruvate, which is required inside the plastids for the synthesis of, for example, aromatic amino acids. This plastidic phosphate transporter is thus different in structure and function from the known triose phosphate/phosphate translocator. We prsp,ose that plastids contain various phosphate translocators with overlapping substrate specificities to ensure an effici6nt supplyiof plastids with a single substrate, even in the presence of other phosphorylated metabolites.

The mature part of the chloroplast triose phosphate-phosphate translocator was cloned into the yeast expression vector pEVP11. This construct was used to transform cells from both Saccharomyces cerevisiae and the rmsion yeast Schizosaccharomyces pombe. The chloroplast translocator protein was functionally expressed in the transformed yeast cells and represented about 1-2% of the Sch. pombe cell membrane protein. It was localized to mitochondrial membranes and/or membranes of the rough endoplasmic reticulum. In order to purify the recombinant translocator protein, a sequence encoding a C-terminal tag of six histidine residues was introduced into the corresponding cDNA. The expressed histidine-tagged translocator protein was purified from the transformed yeast cells under nondenaturing conditions to apparent homogeneity by a single-step affinity chromatography using a Ni2+ nitrilotriacetic acid resin. Both the expressed triose phosphate translocator and the recombinant histidinetagged protein possess substrate specificities identical to those of the authentic chloroplast protein, providing definitive evidOnce for its identity as the triose phosphate translocator and further disproving its assignment as the receptor for chloroplast protein import. The yeast expression system in combination with the Ni2+ nitrilotriacetic acid chromatography thus provides a valuable tool for the production of purified membrane proteins in a functional state.During photosynthetic CO2 fixation, the fixed carbon (in the form of triose phosphates and 3-phosphoglycerate) is exported from the chloroplasts into the cytosol, where it is converted into other substances such as sucrose and amino acids. This transport is mediated by the Frankfurt) and the yeast expression vector pEVP11 was kindly provided by N. Sauer (University of Regensburg). All other chemicals were of the highest purity available.Cloning Procedures. The DNA encoding only the mature part of the spinach translocator protein was generated by PCR from the full-length cDNA clone encoding the entire precursor protein (2). As a sense primer, a synthetic oligonucleotide corresponding to the beginning of the mature part of the cTPT with an additional ATG codon was used (P1), and the second oligonucleotide was a (reverse) T7 primer. The resulting DNA fragment was first cloned into EcoRV-cut pBSC to yield clone pBSC-mPtra. This vector was digested with Sal I, the Sal I ends were filled in, and the Sal I-filled/BamHI fragment was then ligated into the Sac I-filled/BamHI-cut yeast expression vector pEVPll containing the S. cerevisiae LEU2+ gene (8) 2155The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The primary sequences of the chloroplast triose phosphate/phosphate translocator precursor proteins from C4-plants (maize mesophyll cells and Flaveria trinervia) and from the C3-type Flaveria pringlei were determined. The mature parts of these translocators possess 83-94% identical amino acid residues. The C4-translocator protein can be correctly targeted to C3-type chloroplasts and inserted into the envelope membrane. Expression of the mature parts of these chloroplast translocators (cTPT) in transformed yeast cells and subsequent reconstitution of the functional proteins reveals the difference between the recombinant translocator proteins from the two cell types with respect to the transport of phosphoenolpyruvate. Comparison of the cTPT sequences from F. pringlei and F. trinervia in combination with computer-aided molecular modelling of the substrate translocation pore leads to the suggestion, that only minor exchanges of amino acid residues between the C3- and C4-translocator proteins are sufficient to extend their substrate specificities to recognize also phosphoenolpyruvate.

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Oberflächenverbundstoffe (im Sprachgebrauch: Kunstleder), die heute standardmäßig als Polstermate-rial im Interieur oder Automobilbereich verwendet werden, bestehen meist aus Polyurethan oder PVC, vereinzelt auch aus einem Mischverbau beider Polymere. Kunstleder ist ein Mehrschichtverbundwerkstoff, bestehend aus mehreren Polymerschichten, die sukzessive per Umkehrstreichverfahren als viskose Masse auf ein Substrat wie beispielsweise Papier aufgetragen werden. Im darauffolgenden Heizkanal vernetzt beziehungsweise geliert die Polymermasse auf einem textilen Träger, der dem Konstrukt die entsprechenden mechanischen Festigkeiten als auch seine Haptik verleiht. Ein entsprechender Lack bestimmt die finale Haptik, aber auch das Abrasionsverhalten und die Anschmutzbarkeit. Eine Herausforderung bei der Herstellung von Kunstleder ist es, die Atmungsaktivität zu gewährleisten.