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 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 [(3)H]-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.

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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-adipalmitoylglycerophosphoethanolamine (Pam2GroPEtn) 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 "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 (ql). Extrapolation to q 1 = 0 Pa . s yielded a correlation time of $ = 20 5 ns, t = 30T, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be D, = 2.0 x lo9 rad' . s-' and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was q2 z 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 q 1 = 1 mPa . s yielded a value of $r z 450 ns corresponding to D, = 8.8 x lo7 rad' . s -' 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 z 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 2 1.5 x lo4: 1 aggregation occurred in the model membranes below 30°C. However, above 30°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.The triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) from the inner membrane of the chloroplast envelope catalyzes the export of fixed carbon (triose phosphates) from the chloroplast into the cytosol. This process plays a key role in the regulation of plant carbon metabolism (for review see [I, 21).The apparent molecular mass of the protein is 29 kDa and hydrodynamic studies in detergent solution indicate that the active unit is a dimer [3]. The phosphate translocator seems to function as a strict antiporter in a gated-pore fashion as proposed for the ADP/ATP carrier from the inner membrane of mitochondria [4].When reconstituted into liposomes the activity of the phosphate translocator was shown to be regulated by ...

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