Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the ß-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks.

Metabolite transporters in plastids.

Communication between plastids and the surrounding cytosol occurs via the plastidic envelope membrane. Recent findings show that the outer membrane is not as freely permeable to low molecular weight solutes as previously thought, but contains different channel-like proteins that act as selectivity filters. The inner envelope membrane contains a variety of metabolite transporters that mediate the exchange of metabolites between both compartments. Two new classes of phosphate antiporters were recently described that are different in structure and function from the known triose phosphate/phosphate translocator from chloroplasts. In addition, a cDNA coding for an ATP/ADP antiporter from plastids was isolated that shows similarities to a bacterial adenylate translocator.

Reaction mechanism and asymmetric orientation of the reconstituted chloroplast phosphate translocator.

Proteoliposomes loaded with varying levels of internal substrates were used in bisubstrate initial velocity studies to gain insight into the transport mechanism of the reconstituted chloroplast phosphate translocator. The kinetic response to trans substrates clearly indicated that the one-to-one exchange mediated by this translocator proceeds via a ping-pong type, and excluded a sequential type of reaction mechanism. It is also shown that reconstitution of the protein leads to an unidirectional orientation of the protein within the liposomes being orientated right-side-out with respect to chloroplasts. Different transport affinities were observed on either side of the membrane and only the outward-facing transport site of the translocator is able to bind inhibitors i.e. pyridoxal 5'-phosphate (PLP) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).

Purification and functional reconstitution of the 2-oxoglutarate/malate translocator from spinach chloroplasts.

The chloroplast 2-oxoglutarate/malate translocator was solubilized from envelope membranes by the detergent n-dodecyl beta-D-maltoside and purified to apparent homogeneity by anion-exchange chromatography followed by gel permeation chromatography. During the purification procedure, the activity of the translocator was monitored by functional reconstitution into phospholipid vesicles. The purified translocator protein has an apparent molecular mass of about 45,000 as revealed by SDS-PAGE. Based on the specific reconstituted transport activity, the purification was about 31-fold with an overall yield of identical to 50%. The substrate specificity of the purified translocator closely resembles that described for the native transport system in intact chloroplasts.

Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts.

The uptake of phosphate and phosphorylated compounds into the chloroplast stroma has been studied by silicone layer filtering centrifugation. 1. Inorganic phosphate, 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde phosphate are transported across the envelope leading to an accumulation in the chloroplast stroma. This uptake proceeds by a counter exchange with phosphate and phosphorylated compounds present there. 2. The transport shows saturation characteristics allowing the determination of Km and V. 3. The phosphorylated compounds transported act as competitive inhibitors of the transport. From measurements of the Km and Ki the specificity of the transport is described. 4. The transport of inorganic phosphate and 3-phosphoglycerate is inhibited by p-chloromercuriphenyl sulfonate, pyridoxal 5'-phosphate and trinitrobenzene sulfonate. 5. The activation energy of phosphate transport as determined from the temperature dependence is evaluated to be 16 kcal (0--12 degrees C). 6. It is concluded that inorganic phosphate, 3-phosphoglycerate, dihydroxy-acetone phosphate and glyceraldehyde phosphate are transported by the same carrier, which has been nominated phosphate translocator. 7. Simultaneous measurements of the proton concentration in the medium and the transport into the chloroplasts show that the transfer of 3-phosphoglycerate involves a transfer of a proton into the same direction. 8. Measurements of the pH dependence of the transport indicate that all substances including 3-phosphoglycerate are transported by the phosphate translocator as divalent anions. 9. The physiological function of the phosphate translocator is discussed.

ADP/ATP Translocator from Pea Root Plastids (Comparison with Translocators from Spinach Chloroplasts and Pea Leaf Mitochondria).

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.

Transport of isoprenoid intermediates across chloroplast envelope membranes.

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 trans compounds are, in part, also used as counter-substrates by the plastidic PTs but appear to only trans activate IPP transport without being transported.

Polypeptides of the chloroplast envelope membranes as visualized by immunochemical techniques.

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 role of plastidial glucose-6-phosphate/phosphate translocators in vegetative tissues of Arabidopsis thaliana mutants impaired in starch biosynthesis.

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.

On the translocation of proteins across the chloroplast envelope.

Most of the chloroplast proteins are coded for in the nucleus and are synthesized in the cytosol from where they are subsequently transported into the different chloroplast compartments. The structural properties of the N-terminal extensions (transit peptides) of these nuclear-coded precursor proteins are discussed as well as the energy requirements for their translocation and the involvement of receptor proteins and that of other (ATP-dependent) factors.

Phosphorylation of a 51-kDa envelope membrane polypeptide involved in protein translocation into chloroplasts.

In this report we demonstrate that a 51-kDa outer-envelope membrane protein (P51) is involved in protein translocation into chloroplasts. Furthermore it is shown that phosphorylation of P51 is functionally related to the process of binding and/or importing precursor proteins into chloroplasts. Several lines of evidence have been obtained supporting this suggestion. First, protein import into chloroplasts was inhibited by the membrane-impermeable agent pyridoxal 5'-phosphate, which has been shown to react with a component of the protein-import apparatus. Phosphorylation of envelope membrane polypeptides using [gamma-32P]ATP in the presence of pyridoxal 5'-phosphate resulted in an increased incorporation of 32P radiolabel into a 51-kDa membrane polypeptide (P51). A close correlation between the inhibition of protein import and the increase in the phosphorylation state of P51, both as a function of PLP concentration, was observed. Second, binding of purified precursor proteins to chloroplasts resulted in a specific increase in the phosphorylation state of P51. This effect was not exerted by the mature form of the precursor protein lacking the presequence. Third, internally generated ATP was able to compete specifically with externally added [gamma-32P]ATP for the phosphorylation of P51. Fourth, digestion of the outer-envelope membrane with low amounts of thermolysin resulted in a loss of protein import activity, which was associated with the removal of the phosphorylation site of P51. Phosphorylation of P51 proceeds with an apparent Km (ATP) of about 5 microM, which is much lower than the ATP concentration required for the protein translocation itself. We suggest that two different ATP-dependent processes are involved in protein translocation into chloroplasts. P51 represent presumably a regulatory component of the protein-import apparatus or the protein receptor itself.

A rapid method for measuring organelle-specific substrate transport in homogenates from plant tissues.

A rapid method is described for measuring organelle-specific metabolite transport systems in crude homogenates from plants. The tissues were homogenized in liquid nitrogen, extracted with buffer and reconstituted into artificial membranes. The method allowed demonstration of the known different substrate specificities of chloroplast triose phosphate/phosphate translocators from C3- and C4-plants, of the triose phosphate/phosphate translocator from non-green tissue, and of the dicarboxylate translocator. It thus by-passes the necessity to isolate intact plant organelles and, in addition, only a low amount of tissue material is required for transport measurements.

Energy dependence of protein translocation into chloroplasts.

The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP-dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast.

5-Oxoprolinase from rat kidney, I. Assay, purification, and determination of kinetic parameters.

A new assay for the determination of 5-oxoprolinase activity is described. The enzyme 5-oxoprolinase was purified from rat kidney 285-fold to apparent homogeneity, as judged by analytical disc electrophoresis and discontinous polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The specific activity of the preparation was 122 mU/mg of protein. A complete initial rate kinetic analysis of the forward reaction catalyzed by 5-oxoprolinase was carried out using 5-oxo-L-proline and MgATP2theta as substrates. The computer-fitted double reciprocal plots showed intersecting patterns indicating a sequential mechanism. The data were fitted by weighted linear regression analysis using the complete equation for bisubstrate reactions. The limiting Michaelis constants for 5-oxoproline and MgATP2theta were calculated to be 31.6 +/- 2.3 muM and 172.7 +/- 11.5muM, respectively. The maximum forward rate is 1.2 +/- 0.02 mumol X min-1; the turnover number 7.0 min-1.

A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.

A rapid method based on a defined methanol-chloroform-water mixture for the quantitative precipitation of soluble as well as hydrophobic proteins from dilute solutions (e.g., column chromatography effluents) has been developed. The effectiveness of this method is not affected by the presence of detergents, lipids, salt, buffers, and beta-mercaptoethanol.

Diversity of specificity and function of phosphate translocators in various plastids.

This report gives a comparison of the specificity of phosphate translocators in various plastids. Whereas the phosphate translocator of the C(3) plant spinach mediates a counter exchange between inorganic phosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate, the phosphate translocators in chloroplasts from C(4) and CAM plants transport phosphoenolpyruvate in addition to the above mentioned metabolites. In plastids from pea roots the phosphate translocator also transports glucose 6-phosphate. This diversity of phosphate translocators is discussed in view of the special functions of the various plastids.

Sequence analysis and protein import studies of an outer chloroplast envelope polypeptide.

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.