Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.     

Hydrodynamic properties of the Triton X-100-solubilized chloroplast phosphate translocator

The molecular weight of the phosphate translocator isolated from spinach envelope membranes was measured in the nonionic detergent Triton X-100. The Stoke's radius of the protein-detergent complex was estimated by gel filtration. The partial specific volume was estimated by equilibrium centrifugation and by differential sedimentation in sucrose gradients containing H₂O and ²H₂O and the sedimentation coefficient was estimated from the same centrifugation experiments. The phosphate translocator-Triton X-100 complex has an apparent molecular weight of 177 500. Its high partial specific volume (0.86 cm³/g) suggests that bound detergent contributes significantly to the mass. Correcting for the bound detergent (1.9 g/g protein), a molecular weight of 61 000 for the protein moiety of the complex was calculated. These results suggest that the isolated phosphate translocator exists as a dimer. The shape of the dimer is described as a prolate ellipsoid of revolution with semiaxes calculated to be 6.59 and 1.59 nm in length.     

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).     

The phosphate translocator of the chloroplast envelope. Isolation of the carrier protein and reconstitution of transport

This report describes the solubilization and purification of the phosphate translocator of spinach chloroplasts and the reconstitution of its activity by incorporation into liposomes. (1) Prior to the isolation, the carrier is specifically labelled by treatment with 2,4,6-trinitrobenzenesulfonic acid and NaB[³H]H₄. (2) After preextraction of purified envelope membranes with Brij 58 for removing other loosely bound membrane proteins, the phosphate translocator is extracted with Triton X-100. After passing the resulting extract over a DEAE-Sepharose column followed by sucrose density gradient ultracentrifugation, the translocator protein is purified to apparent homogeneity. The 5–6-fold purification thus obtained concurs with earlier findings that the phosphate translocator protein represents 15–20% of the envelope membrane protein. This highly purified protein is suitable for studies of the hydrodynamic parameters of the translocator. (3) Since the exposure to detergents affects the activity of the translocator protein, alternatively, a rapid batch procedure for the purification of the translocator protein employing hydroxyapatite is used, yielding within 15 min the phosphate translocator protein of about 70% purity. (4) After incorporation of this protein fraction into liposomes, a specific transport of phosphate into these liposomes is observed, which van be terminated by inhibitor stop with pyridoxal 5′-phosphate. This uptake is only observed when the liposomes have been preloaded with phosphate or 3-phosphoglycerate, but not with 2-phosphoglycerate. Thus, like in intact chloroplasts, also the reconstituted transport facilitates an obligatory and specific counter exchange of anions. The apparent K_m for the transport of phosphate by this reconstituted system is about 0.8 mM, which is comparable to the corresponding value in intact chloroplasts. The calculated turnover of 150–300 min⁻¹ (20°C) accounts for 3–6% of the original activity.     

The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-alpha-dipalmitoylglycerophosphoethanolamine (Pam2 GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator. The temperature dependence of the anisotropy decay showed that above 25 degrees C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (eta 1). Extrapolation to eta 1 = 0 Pa.s yielded a correlation time of phi = 20 +/- 5 ns, t = 30 degrees C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr = 2.0 x 10(9) rad2.s-1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was eta 2 approximately 12 mPa.s. The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of eta 1 = 1 mPa.s yielded a value of phi r approximately 450 ns corresponding to Dr = 8.8 x 10(7) rad2.s-1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross-sectional area of the protein part embedded in the membrane was calculated to be approximately 9 nm2. This cross-sectional area is large enough to include at most 14 membrane-spanning helices. Our results also indicated that at lipid/protein molar ratios greater than or equal to 1.5 x 10(4): 1 aggregation occurred in the model membranes below 30 degrees C. However, above 30 degrees C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.     

Analysis of the role of the phosphate translocator and external metabolites in steady-state chloroplast photosynthesis

The role of the phosphate translocator and the importance of the extrachloroplastic concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in steady-state photosynthesis is examined with a kinetic model. The steady-state stromal concentrations of these compounds are calculated as a function of the rate of the various partial reactions of photosynthesis, at various external concentrations which span those likely to occur in vivo. It is shown how the net transport requirements of the various reactions necessitate different adjustments in the stromal concentrations of these compounds, away from the equilibrium values expected in the absence of metabolism. Under most circumstances, the high exchange capacity of the phosphate translocator relative to the transport requirements of CO₂ fixation limits the extent of these displacements, but conditions when the phosphate translocator is limiting photosynthesis are observed and discussed. The model provides a basis for a more quantitative understanding of the role of the phosphate translocator and the external concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in photosynthesis.     

Regulation of the reconstituted chloroplast phosphate translocator by an H+ gradient

This report describes the influence of ΔpH on the transport of phosphate, triose phosphate and 3-phosphoglycerate catalyzed by the phosphate translocator in a reconstituted system. The H⁺ gradient across the liposome membrane is adjusted by the addition of external buffer solution and maintained for several minutes. The following results are obtained: (1) An inward directed H⁺ gradient leads to an increase of 3-phosphoglycerate transport and to a decrease of phosphate and triose phosphate transport. (2) An H⁺ gradient in the opposite direction results in a restriction of 3-phosphoglycerate influx whereas the influx of phosphate and triose phosphate is enhanced. (3) The magnitude of the pH effect depends on the internal substrate. Compared to the homoexchange mode, the effect of applied ΔpH is more pronounced in the heteroexchange mode. (4) Transport of phosphate and 3-phosphoglycerate is influenced by ΔpH in a different manner. In the case of phosphate and triose phosphate transport the observed effects are associated with changes in the apparent K_m values whereas in the case of 3-phosphoglycerate transport the application of a pH gradient is linked to a change of V_max. (5) In competition experiments with both substrates in the external medium, ΔpH influences the effect of phosphate as a competitive inhibitor of 3-phosphoglycerate transport whereas the effect of 3-phosphoglycerate on phosphate transport is not affected by a pH gradient. (6) The measured apparent K_m and V_max values under the influence of ΔpH can be used for the calculation of substrate fluxes across the envelope during illumination. It can be demonstrated that the increase of stromal pH in the light gives rise to a considerable change in the ratio of the substrates transported. Under conditions without pH gradient, the species transported out is mainly 3-phosphoglycerate and the species transported in is mainly triose phosphate. These fluxes are reversed when a pH gradient is applied (light conditions).     

The N-terminal hydrophobic region of the mature phosphate translocator is sufficient for targeting to the chloroplast inner envelope membrane.

To locate the sequence required for directing the phosphate translocator to the chloroplast inner envelope membrane, a series of chimeric proteins constituting parts of the phosphate translocator and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, which is normally located in the stroma, has been produced. Reciprocal exchanges of the presequences and mature sequences of the phosphate translocator and the small subunit indicated that the phosphate translocator presequence contains stromal targeting information and that the mature protein is responsible for inner envelope membrane targeting. Chimeric proteins containing the N-terminal 46 amino acid residues of the phosphate translocator were directed to the inner envelope membrane. Subdivision of this region into its composite hydrophilic and hydrophobic regions showed that the hydrophobic region alone, which consists of amino acid residues 24 to 45, was able to direct the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to the inner envelope membrane.     

Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants

The introduction of an antisense DNA into transgenic potato (Solanum tuberosum L.) plants decreased the expression of the chloroplast triose-phosphate translocator and lowered its activity by 20–30%. With plants propagated from tubers, the effect of the transformation on photosynthetic metabolism was analysed by measuring photosynthesis, the formation of leaf starch, and the total and subcellular metabolite contents in leaves. Although the transformants, in contrast to those propagated from cell cultures, did not differ from the wild-type plants in respect to rates of photosynthesis, plant appearance, growth and tuber production, their photosynthetic metabolism was found to be severely affected. The results show that the decrease in activity of the triose-phosphate translocator in the transformants caused a fourfold increase in the level of 3-phosphoglycerate and a corresponding decrease in inorganic phosphate in the stromal compartment, resulting in a large increase in the synthesis of starch. Whereas during a 12-h day period wild-type plants deposited 43% of their CO₂ assimilate into starch, this value rose to 61–89% in the transformants. In contrast to the wild-type plants, where the rate of assimilate export from the leaves during the night period was about 75% of that during the day, the export rate from leaves of transformants appeared to be much higher during the night than during the day. As the mobilisation of starch occurs in part hydrolytically, resulting in the formation of glucose, the triose-phosphate translocator loses its exclusive function in the export of carbohydrates from the chloroplasts when the photoassimilates are temporarily deposited as starch. It appears that by directing the CO₂ assimilates mainly into starch, the transformants compensate for the deficiency in triose-phosphate translocator activity in such a way that the productivity of the plants is not affected by the transformation.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - 3-PGA 3-phosphoglycerate - Rubisco ribulose,1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - trioseP triose phosphate - WT wild type The able technical assistance of Mrs. K. Wildenberger and Mrs. A. Großpietsch is gratefully acknowledged. This work has been supported by the Bundesminister für Forschung und Technologie.     

Expression of genes encoding the tobacco chloroplast phosphate translocator is not light-regulated and is repressed by sucrose

A cDNA encoding the complete precursor of the phosphate translocator of the chloroplast inner envelope membrane has been isolated from a tobacco leaf (Nicotiana tabacum cv. Samsun) gt 11 library. The tobacco cDNA is 1546 by in length and encodes a precursor protein of 401 amino acid residues with a deduced molecular weight of 43705. A putative processing site between Ala-73 and Ala-74 of the precursor protein is suggested by comparison with the N-terminal sequences of the pea and spinach proteins. Removal of the transit peptide produces the mature protein of 328 amino acid residues with a molecular weight of 36038. Southern blot analysis suggests there is probably one copy of the phosphate translocator gene in the pea haploid genome and two copies in the tobacco haploid genome, one derived from each ancestral parental genome. Messenger RNAs essentially equivalent in size to the cDNAs (approx. 1.6 kb) were detected in extracts of all organs examined from tobacco and pea, including leaves, stems, sepals, petals, seed-pods, tendrils and roots. An immunochemically related protein of a similar size to the phosphate translocator was detected in the equivalent pea organs. The levels of both mRNA and protein in non-photosynthetic organs were lower than those in photosynthetic organs. Tobacco phosphate translocator mRNA was present at high levels in etiolated tissue and did not increase significantly after 24 h illumination. Germination and growth of tobacco seedlings in the presence of sucrose caused a 3.3-fold decrease in the level of the phoshate translocator mRNA.     

The adenine nucleotide translocator in apoptosis

Alteration of mitochondrial membrane permeability is a central mechanism leading invariably to cell death, which results, at least in part, from the opening of the permeability transition pore complex (PTPC). Indeed, extended PTPC opening is sufficient to trigger an increase in mitochondrial membrane permeability and apoptosis. Among the various PTPC components, the adenine nucleotide translocator (ANT) appears to act as a bi-functional protein which, on the one hand, contributes to a crucial step of aerobic energy metabolism, the ADP/ATP translocation, and on the other hand, can be converted into a pro-apoptotic pore under the control of onco- and anti-oncoproteins from the Bax/Bcl-2 family. In this review, we will discuss recent advances in the cooperation between ANT and Bax/Bcl-2 family members, the multiplicity of agents affecting ANT pore function and the putative role of ANT isoforms in apoptosis control.     

Phosphate translocator and adenylate translocator in chromoplast membranes

It is shown by the criteria of saturation kinetics, specificity, and inhibition experiments that chromoplast membranes from the daffodil flower contain a phosphate translocator for the counter-exchange of phosphate, and 3-phosphoglycerate, as well as phosphoenolpyruvate; they also contain an adenylate translocator. This is the first report on the occurrence of these translocators in non-green plastids. Both translocators exhibit certain dissimilar properties when compared to the corresponding systems of chloroplasts. The transport rates of both translocators are sufficient to allow a prominent fatty acid synthesis in isolated chromoplasts when C₃ intermediates of the glycolytic pathway or adenine nucleotides are used as energy sources.     

Compartmentation of labeled fixation products in intact mesophyll protoplasts from Avena sativa L. after in-situ inhibition of the chloroplast phosphate translocator

Leaf mesophyll protoplasts of oat (Avena sativa L.) were allowed to fix ¹⁴C-labeled bicarbonate in the absence or presence of pyridoxal phosphate (PLP), a specific inhibitor of the phosphate translocator of the inner envelope membrane of chloroplasts. The incubation was terminated by a method of rapid integrated protoplast homogenization and fractionation, and compartmented levels of label contained in sugars, phosphate esters, amino acids and organic acids were determined. The results show that the addition of PLP to a suspension of intact protoplasts causes an accumulation of phosphate esters in the chloroplasts stroma for up to 2.5 min of incubation, with a corresponding decrease in the cytosol. Prolonged treatment of protoplasts with PLP in the light resulted in a decrease of starch-associated label, combined with higher levels of labeled sugars in the cytosol, indicating a switch from phosphorolytic to hydrolytic starch degradation. Together with the determination of pool sizes of triose phosphates and of inorganic phosphate, the results demonstrate that the method employed is an important tool in investigating processes of intracellular regulation. They are discussed with respect to the permeability and possible side reactions of PLP, as well as in the light of reports on PLP action on isolated chloroplasts.Abbreviations P_i orthophosphate - PLP pyridoxal 5-phosphate - TP triosephosphate     

The biosynthesis of the mitochondrial ADP, ATP translocator.

The biosynthesis of the ADP,ATP carrier was studied in mitochondria of Neurospora crassa. The carrier was isolated as the carboxyatractylate-protein complex and characterized in dodecylsulphate/polyacrylamide gel electrophoresis to have a Mr = 33 000. Applying the inhibitors chloramphenicol for the intramitochondrial translation and cycloheximide for extramitochondrial translation, the site of synthesis of this polypeptide was found to be extramitochondrially located.     

The ultrastructure of the chloroplast lamellae

Summary The present study has shown that the thylakoid membrane consists of a central layer, probably lipid, covered on both sides with protein particles. The thickness of the middle layer reaches 40 Å, and the diameter of the attached globules 60 Å. The particles are partially embedded to a depth of about 20 Å in the central layer. If these globules are removed, the lipid layer appears perforated, indicating that the protein molecules are in direct contact through the lipid layer. On the outer side of the thylakoid membrane the particles are grouped in fours, forming a multienzyme complex of 120 Å diameter and 60 Å thickness. No such aggregates have been observed on the inner side.In frozen plastids a tripartite membrane at the periphery of a granum has a thickness of 120 Å, whereas the two membranes of adjacent thylakoids are together only 200 Å thick. This indicates that the particles attached to the two neighbouring membranes are interlocked and form a rigid layer.In comparison with the freeze-etched preparations the chemically fixed plastids show considerably thinner lamellae. They appear as unit membranes, consisting of a central layer 35 Å thick and two dense strata, each of 20 Å. This discrepancy cannot be explained simply by shrinkage in fixation and dehydration. It is proposed that fixation causes uncoiling of the protein molecules. In consequence, thin protein films are formed on each side of the lipid layer, and each appears as a dark band after osmium- or permanganate fixation. This new model of the thylakoid membrane is compared with other recent schemes.

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Zusammenfassung In der vorliegenden Untersuchung wird gezeigt, daß die Thylakoidmembran aus einer zentralen, wahrscheinlich lipidhaltigen Schicht besteht, die beidseitig mit Proteinpartikeln bedeckt ist. Die Dicke dieser Mittelschicht beträgt 40 Å und der Durchmesser der darauf liegenden Kügelchen 60 Å. Sowohl die innern wie die äußern Partikel sind ungefähr 20 Å tief in der zentralen Schicht eingebettet. Werden sie beim Herstellen des Gefrierabdruckes herausgerissen, so erscheint die Lipoidlamelle perforiert. Das zeigt, daß die Enzyme durch diese Schicht hindurch in direktem Kontakt stehen. Auf der Außenseite der Thylakoidmembran sind vier Partikel zu einem Multi-Enzym-Komplex von 120 Å Durchmesser und 60 Å Dicke vereinigt. Auf der Innenseite konnten solche Zusammenlagerungen nicht beobachtet werden. Die Dicke einer dreischichtigen Membran, wie sie z. B. an der Außenseite eines Granumstapels zu beobachten ist, beträgt 120 Å. Die Doppelmembranen benachbarter Thylakoide (200 Å dick) sind durch die auf der Oberfläche vorhandenen Partikel eng verzahnt.Ein Vergleich der Bilder von Plastiden nach der Gefrierätzung und nach einer gewöhnlichen Fixierung ergibt, daß die Lamellen durch die Fixierung und Entwässerung dünner werden. Sie zeigen die Struktur einer Einheitsmembran bestehend aus einer 35 Å dicken Zentralschicht und den beiden anliegenden 20 Å dicken osmiophilen Lamellen. Die unterschiedliche Lamellendicke (Gefriergetrocknet: 120 Å, fixiert: 75 Å) kann nicht nur auf die bei der Fixierung und Entwässerung eintretende Schrumpfung zurückgeführt werden. Es ist wahrscheinlich, daß die Fixierung eine Entknäuelung der Proteinmoleküle bewirkt. Der dadurch auf beiden Seiten der Lipidschicht gebildete, dünne Protein-film erscheint nach der Osmium-oder Permanganatfixierung als kontrastreiche 20 Å dicke Schicht. Dieses neue Modell der Thylakoidmembran wurde mit einigen neuen Strukturschemata verglichen.