Search

Abstract: The common precursor for isoprenoid biosynthesis in plants, isopentenyl diphosphate (IPP), is synthesized by two pathways, the cytosolic mevalonate pathway and the plastidic 1‐deoxy‐D‐xylulose 5‐phosphate/methylerythritol phosphate (DOXP/MEP) pathway. The DOXP/MEP pathway leads to the formation of various phosphorylated intermediates, including DOXP, 4‐hydroxy‐3‐methylbutenyl diphosphate (HMBPP), and finally IPP. There is ample evidence for metabolic cross‐talk between the two biosynthetic pathways. The present study addresses the question whether isoprenoid intermediates could be exchanged between both compartments by members of the plastidic phosphate translocator (PT) family that all mediate a counter‐exchange between inorganic phosphate and various phosphorylated compounds. Transport experiments using intact chloroplasts, liposomes containing reconstituted envelope membrane proteins or recombinant PT proteins showed that HMBPP is not exchanged between the cytosol and the chloroplasts and that the transport of DOXP is preferentially mediated by the recently discovered plastidic transporter for pentose phosphates, the xylulose 5‐phosphate translocator. Evidence is presented that transport of IPP does not proceed via the plastidic PTs although IPP transport is strictly dependent on various phosphorylated compounds on the opposite side of the membrane. These phosphorylated transcompounds are, in part, also used as counter‐substrates by the plastidic PTs but appear to only transactivate IPP transport without being transported.

In Arabidopsis thaliana, the Toc34 receptor component of the chloroplast import machinery is encoded by two independent but highly homologous genes, atToc33 and atToc34. We have isolated a T-DNA insertion mutant of atToc33 which is characterized by a pale phenotype, due to reductions in the levels of photosynthetic pigments, and alterations in protein composition. The latter involve not only chloroplast proteins but also some cytosolic polypeptides, including 14-3-3 proteins which, among other functions, have been proposed to be cytosolic targeting factors for nucleus-encoded chloroplast proteins. Within the chloroplast, many, though not all, proteins of the photosynthetic apparatus, as well as proteins not directly involved in photosynthesis, are found in significantly reduced amounts in the mutant. However, the accumulation of other chloroplast proteins is unaffected. This suggests that the atToc33 receptor is responsible for the import of a specific subset of nucleus-encoded chloroplast proteins. Supporting evidence for this conclusion was obtained by antisense repression of the atToc34 gene in the atToc33 mutant, which results in an exacerbation of the phenotype.

The nucleotide sequence of several cDNA clones coding for the phosphate translocator from spinach chloroplasts has been determined. The cDNA clones were selected from a lambda gt10 library prepared from poly(A)+ mRNA of spinach leaves using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the isolated translocator protein. A 1439 bp insert of one of the clones codes for the entire 404 amino acid residues of the precursor protein corresponding to a mol. wt of 44,234. The full‐length clone includes 21 bp at the transcribed non‐coding 5′ region with the ribosome initiation sequence ACAATGG, a 1212 bp coding region and 199 bp at the non‐coding 3′ region excluding the poly(A) tail which starts 17 bp downstream from a putative polyadenylation signal, AATAAT. According to secondary structure predictions the mature part of the chloroplast phosphate translocator exhibits high hydrophobicity and consists of at least seven membrane‐spanning segments. Using plasmid‐programmed wheat germ lysate the precursor protein was synthesized in vitro and could be imported into spinach chloroplasts where it is inserted into the inner envelope membrane.

The polypeptides of relative molecular masses (Mr) 22,000, 29,000, and 36,000 represent three major constituents of the chloroplast envelope of spinach (Spinacia oleracea L.) leaves. The Mr 22,000 polypeptide has been localized in the outer membrane, whereas the two other peptides have been attributed to the inner envelope membrane (Joyard et al., 1983). The Mr 29,000 polypeptide has been identified as the "phosphate translocator" (Flügge and Heldt, 1979). In this investigation, we studied the three envelope polypeptides by means of immunocytochemistry. Using indirect immunofluorescence, all three polypeptides were visualized in cryostat sections of formaldehyde-fixed leaf tissue. They were found in both palisade and spongy parenchyma cells and in guard cells, as indicated by a strong fluorescence in the chloroplast periphery. In contrast, fluorescein isothiocyanate or protein A-gold labeling of isolated fixed chloroplasts resulted only in visualization of the Mr 22,000 polypeptide, a constituent of the outer membrane. We further studied the morphological distribution and frequency of this peptide by electron microscopic evaluation of platinum-carbon replicas after freeze-etching or label-fracture and of ultra-thin sections. By use of these three methods, the polypeptide was found to be randomly distributed in the outer envelope membrane and easily accessible to the immunomarker. Average marker density, as obtained by freeze-etching and label-fracture, was approximately 130 gold particles per square micron.

The spinach triose phosphate/phosphate translocator and the 37-kDa protein are both integral components of the chloroplast inner envelope membrane. They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein and the CF0II-subunit of the thylakoid ATP synthase, respectively. In addition, the transit peptide of the CF0II-subunit that contains signals for the transport across both the envelope and the thylakoid membrane was fused to the mature parts of both envelope membrane proteins. The chimeric proteins were imported into isolated spinach chloroplasts, and the intraorganellar routing of the proteins was analyzed. The results obtained show that the N-terminal extensions of both envelope membrane proteins possess a stroma-targeting function only and that the information for the integration into the envelope membrane is contained in the mature parts of the proteins. At least part of the integration signal is provided by hydrophobic domains in the mature sequences since the removal of such a hydrophobic segment from the 37-kDa protein leads to missorting of the protein to the stroma and the thylakoid membrane.

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 fission 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 histidine-tagged protein possess substrate specificities identical to those of the authentic chloroplast protein, providing definitive evidence 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.

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C4 plant, transports inorganic phosphate and phosphorylated C3 compounds in which the phosphate group is linked to the C3 atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C3 plant, spinach. In contrast to the phosphate translocator from C3-mesophyll chloroplasts, that of C4-mesophyll chloroplasts catalyzes in addition the transport of C3 compounds where the phosphate group is attached to the C2 atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5'-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [3H]-H2DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.

The salts of several weak acids have been used to render the envelope permeable to protons. In order to investigate the role of stromal pH changes in the light regulation of CO2 fixation, formate, octanoate, nitrite, and glyoxylate have been tried as tools to reverse the light-dependent alkalization of the stroma. For this purpose, the decrease of the stromal pH in illuminated spinach chloroplasts, as caused by the addition of these substances or by instantaneous lowering of the pH in the medium, has been compared with the corresponding decrease of CO2 fixation and the change of stromal metabolite levels. It appears from out data that formate and octanoate are suited best to obtain a specific inhibition of CO2 fixation by lowering the stromal pH. The measurement of the corresponding metabolite levels indicates that this inhibition is primarily due to an inhibition of fructose- and sedoheptulose bisphosphatase. It is concluded that these two enzymes are important regulatory steps for the light control of CO2 fixation.

The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH2-terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.

A chloroplast outer envelope membrane protein was cloned and sequenced and from the sequence it was possible to deduce a polypeptide of 6.7 kDa. It has only one membrane-spanning region; the C terminus extends into the cytosol, whereas the N terminus is exposed to the space between the two envelope membranes. The protein was synthesized in an in vitro transcription-translation system to study its routing into isolated chloroplasts. The import studies revealed that the 6.7-kDa protein followed a different and heretofore undescribed translocation pathway in the respect that (i) it does not have a cleavable transit sequence, (ii) it does not require ATP hydrolysis for import, and (iii) protease-sensitive components that are responsible for recognition of precursor proteins destined for the inside of the chloroplasts are not involved in routing the 6.7-kDa polypeptide to the outer chloroplast envelope.

The nucleotide sequence of several cDNA clones coding for the phosphate translocator from spinach chloroplasts has been determined. The cDNA clones were selected from a lambda gt10 library prepared from poly(A)+ mRNA of spinach leaves using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the isolated translocator protein. A 1439 bp insert of one of the clones codes for the entire 404 amino acid residues of the precursor protein corresponding to a mol. wt of 44,234. The full‐length clone includes 21 bp at the transcribed non‐coding 5′ region with the ribosome initiation sequence ACAATGG, a 1212 bp coding region and 199 bp at the non‐coding 3′ region excluding the poly(A) tail which starts 17 bp downstream from a putative polyadenylation signal, AATAAT. According to secondary structure predictions the mature part of the chloroplast phosphate translocator exhibits high hydrophobicity and consists of at least seven membrane‐spanning segments. Using plasmid‐programmed wheat germ lysate the precursor protein was synthesized in vitro and could be imported into spinach chloroplasts where it is inserted into the inner envelope membrane.

The export of primary photosynthesis products from chloroplasts into the cytoplasm is mediated by the triose phosphate translocator. The transporter is an integral membrane protein localized at the inner envelope of chloroplasts. In order to study the expression of the major chloroplast envelope protein gene E29, which is assumed to function as the translocator, we have isolated corresponding cDNA clones from potato. A full-length clone was sequenced and shown to be highly homologous to the E29 gene from spinach. Expression on the RNA level is restricted to green tissues, is light dependent and cannot be induced by sucrose in darkness. The presence of a single-copy gene argues for the existence of different translocator systems responsible for import and export of carbohydrates in chloroplasts and amyloplasts.

The major chloroplast envelope membrane protein E29 is central for the communication between chloroplasts and cytosol. It has been identified as the triose phosphate translocator (TPT) exporting the primary products of the Calvin cycle (i.e., triose phosphates and 3-phosphoglycerate) out of the chloroplast in a strict counter exchange for Pi. To study the in vivo role of the TPT, transgenic potato plants were constructed that have a reduced expression of the TPT at both the RNA and protein level due to antisense inhibition. Chloroplasts isolated from these plants show a 20-30% reduction with respect to their ability to import Pi. The reduced TPT activity leads to a reduction of maximal photosynthesis by 40-60%, to a change in carbon partitioning into starch at the expense of sucrose and amino acids, and to an increase of the leaf starch content by a factor of approximately 3. At early developmental stages the inhibited plants are retarded in growth compared to the wild type.