Copper transport across pea thylakoid membranes

The initial rate of Cu2+ movement across the thylakoid membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membranes loaded with the Cu(2+)-sensitive fluorophore Phen Green SK. Cu2+ transport was rapid, reaching completion within 0.5 s. The initial rate of uptake was dependent upon Cu2+ concentration and saturated at about 0.6 microm total Cu2+. Cu2+ uptake was maximal at a thylakoid lumen pH of 7.0. Cu2+ transport was inhibited by Zn2+ but was largely unaffected by Mn2+ and Cu+. Zn2+ inhibited Cu2+ transport to a maximum of 60%, indicating that there may be more than one transporter for copper in pea thylakoid membranes.     

The action of lipases on chloroplast membranes I. The release of plastocyanin from galactolipase-treated thylakoid membranes

Bean chloroplasts treated with galactolipase (lipolytic acyl hydrolase) isolated from bean leaves showed an inhibition of photosystem I activity as measured by methyl viologen-mediated oxygen uptake and NADP⁺ photoreduction. This inhibition was partially reversed by exogenous plastocyanin added to galactolipase-treated thylakoid membranes. Galactolipase released substantial amounts of endogenous plastocyanin (about 40%) from bean chloroplasts. The results are discussed with regard to the localization of plastocyanin in thylakoid membranes.Abbreviations chlf chlorophyll - DCMU 3-(2,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - MV methyl viologen - NADP⁺ nicotinamide dinucleotide phosphate - PC phosphatidylcholine - PG phosphatidylglycerol - PE phosphatidylethanolamine - PI phosphatidylinositol - SQDG sulphoquinovosyldiacylglycerol - SDS sodium dodecyl sulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethane     

Freezing of isolated thylakoid membranes in complex media

Chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw treatment in a buffered medium containing 70 mM KCl, 30 mM NaNO₃ and 20 mM K₂SO₄ in different combinations. In the presence of the three predominant inorganic electrolytes, inactivation of photophosphorylation was mainly caused by a decrease in the capacity of the photosynthetic electron transport; release of proteins from the membranes was not manifest and light-induced H⁺ gradient and proton permeability were largely unaffected. Omission of nitrate from the medium had little effect. When either sulfate or chloride or both were omitted prior to freezing, inactivation of photophosphorylation was correlated with stimulation of the phosphorylating electron flow, marked increase in H⁺ permeability and loss of the ability of the thylakoids to accumulate protons in the light. In the absence of sulfate, uncoupling was mainly a consequence of the dissociation of chloroplast coupling factor (CF₁). Partial restoration of proton impermeability and pH gradient occurred upon the addition of N,N-dicyclohexylcarbodiimide (DCCD). When sulfate was present but chloride omitted, CF₁ remained attached to the membranes and the addition of DCCD had no effect, indicating that the increase in proton efflux was caused by a different mechanism. It is concluded that sulfate stabilizes the CF₁ and prevents its release from the membranes, but KCl is also necessary for maintaining the low permeability of the membranes to protons. The importance of complex media for investigations on isolated biomembrane systems is stressed.Abbreviations CF₁ chloroplast coupling factor - DCCD N,N-dicyclohexylcarbodiimide - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid I=Santarius 1986 b     

Molecular orientation of plastocyanin on spinach thylakoid membranes as determined by acetylation of lysine residues

To reveal the molecular orientation of plastocyanin (PC) on spinach thylakoid membranes, the position of Lys residues modified by acetic anhydride was compared between thylakoid-bound PC and the isolated one. Digestion of the isolated PC by a trypsin yielded a peptide map with seven spots prior to the acetylation of the protein; none of the spots appeared after the isolated PC was acetylated. On the other hand, there were two spots on the peptide map of the PC acetylated when it was bound to the thylakoids. Those spots were revealed by their amino acid compositions to correspond to the peptide fragments Phe 82-Lys 95 and Val 96-Asn 99. Thus, the Lys residues 81 and 95 of the thylakoid-bound PC were not acetylated. These results suggest that the PC molecule binds to the thylakoids with a specific region including the Lys's 81 and 95 in contact with the membranes. The Lys's 81 and 95 are located near Tyr 83, which has been thought to be the delivery site of electrons from the Cu2+ center.     

Full-length prepro-alpha-factor can be translocated across the mammalian microsomal membrane only if translation has not terminated

We have previously shown that fully synthesized prepro-alpha-factor (pp alpha F), the precursor for the yeast pheromone alpha-factor, can be translocated posttranslationally across yeast rough microsomal (RM) membranes from a soluble, ribosome-free pool. We show here that this is not the case for translocation of pp alpha F across mammalian RM. Rather we found that a small amount of translocation of full-length pp alpha F is observed, but is solely due to polypeptide chains that were still ribosome bound and covalently attached to tRNA, i.e., not terminated. In addition, both signal recognition particle (SRP) and SRP receptor are required, i.e., the same targeting machinery that is normally responsible for the coupling between protein synthesis and translocation. Thus, the molecular requirements for targeting are distinct from posttranslational translocation across yeast RM. As termination is generally regarded as part of translation, the translocation of full-length pp alpha F across mammalian RM does not occur "posttranslationally," albeit independent of elongation. Most other proteins for which posttranslational translocation across mammalian RM was previously claimed fall into the same category in that ribosome attachment as peptidyl-tRNA is required. To clearly separate these two distinct processes, we suggest that the term posttranslational be reserved for those processes that occur in the complete absence of the translational machinery. We propose the term "ribosome-coupled translocation" for the events described here.     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

Integration of early light-inducible proteins into isolated thylakoid membranes.

An in-vitro system has been established to study the integration of early light-inducible proteins (ELIP) into isolated thylakoid membranes. The in-vitro-expressed ELIP precursor proteins exist in two forms, a high-molecular-mass aggregate which is accessible to trypsin but no longer to the stromal processing protease and a soluble form which is readily cleaved to the mature form by the stromal protease. The mature form of ELIP is integrated into thylakoid membranes; its correct integration can be deduced from the observation that the posttranslationally transported products and the in-vitro integrated ELIP species are cleaved by trypsin to products of the same apparent molecular mass. Trypsin-resistant fragments of high-molecular-mass and low-molecular-mass ELIP appear to have the same size. The processed ELIP species, as well as an engineered mature form of ELIP, are integrated into isolated thylakoid membranes. Integration of the mature protein occurs in the absence of stroma, into sodium-chloride-washed, and trypsin-treated thylakoid membranes. The process of integration is almost temperature independent over 0-30 degrees C. Analysis of the time course of integration leads to the conclusion that, under in-vitro conditions, processing but not integration into membranes is the rate-limiting step. In the absence of stroma, the ELIP precursor is bound to the thylakoid membranes, however, it is no longer accessible to the stromal maturating protease when added after binding has occurred. In conclusion, integration of ELIP differs in many essential details from that of its relatives, the light-harvesting chlorophyll a/b protein family.     

The precursor of mitochondrial aspartate aminotransferase is translocated into mitochondria as apoprotein

Mitochondrial aspartate aminotransferase is synthesized on free polysomes as a higher molecular weight precursor (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). The present study examines whether the coenzyme pyridoxal phosphate or pyridoxamine phosphate is required for the uptake of the precursor into mitochondria. Chicken embryo fibroblasts were cultured in medium prepared with and without pyridoxal. In cells grown in the presence of pyridoxal only holoform of aspartate aminotransferase and no apoenzyme was detected. Cells cultured under pyridoxal deficiency contained about 30% of apoenzyme in secondary cultures. All of this apoform was identified as mitochondrial isoenzyme. In order to differentiate whether this apoenzyme corresponded to newly synthesized protein or originated from pre-existing holoenzyme, double isotope-labeling experiments were performed. Secondary cultures of chicken embryo fibroblasts grown under pyridoxal depletion were labeled with [3H]methionine, and then pulsed with [35S]methionine. In another series of experiments, the 3H-labeled cells were pulsed with [35S]methionine in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone in order to accumulate the precursor. Subsequently, the accumulated precursor was chased into the mitochondria by addition of the carbonyl cyanide m-chlorophenylhydrazone antagonist cysteamine. The holo- and apoenzyme from the ultrasonic extract of the double-labeled cells were separated by affinity chromatography on a phosphopyridoxyl-AH-Sepharose column, immunoprecipitated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Under both experimental conditions, the 3H/35S ratio of the apoenzyme was less than half of that of the holoenzyme. Therefore, the apoenzyme and not the holoenzyme is the first product of the precursor in the mitochondria. Apparently, the precursor of mitochondrial aspartate aminotransferase is transported into mitochondria as apoprotein and is processed there independently of the coenzyme.     

Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes

The energy requirements for the import of nuclear-encoded proteins into isolated chloroplasts have been reinvestigated. We have shown that, in contrast to protein import into mitochondria, the translocation of the precursors to ferredoxin, plastocyanin (prPC) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (prSS) across all chloroplastic membranes is independent of a protonmotive force and requires only ATP. This extends previous works in which investigations were limited to prSS and demonstrates that our results are probably general to all chloroplastic protein precursors. Our results are particularly interesting for the import of prPC, since in addition to the two envelope membranes, this protein must traverse the energy-transducing thylakoid membranes en route to its proper location in the thylakoid lumen. This lack of involvement of a protonmotive force, specifically of a transmembrane electric potential, demonstrates that separate mechanisms operate during the import of proteins into chloroplasts and mitochondria. We also examined the question of whether ATP is utilized inside or outside of chloroplasts during protein import. Previous attempts to resolve this question have resulted in conflicting answers. We found, by two independent approaches, that ATP for protein import is utilized inside chloroplasts. The implications of these results on the possible mechanisms of protein import into chloroplasts are discussed.     

Cytochrome distribution across chloroplast thylakoid membranes. Controlled proteolysis of inside-out and right-side-out vesicles

The transverse distribution of chloroplast cytochromes b-559 (high and low potentials), b-563 and f in pea thylakoid membranes was studied by the effects of trypsin and pronase on inside-out and right-side-out thylakoid vesicles. The high potential (HP) form of cytochrome b-559 was degraded to a low potential (LP) form most rapidly in right-side-out vesicles. In either type of vesicle there was no overall loss of the cytochrome from the membrane. This suggests that the haem group is buried in the membrane but that the cytochrome environment is most labile at the outer surface. Cytochrome b-563 was unaffected by trypsin and only slightly degraded by pronase in inverted vesicles. However, pronase caused the loss of an M_r 1000, non-haem fraction from the cytochrome f polypeptide in inside-out vesices only. The total cytochrome f content (measured spectrophotometrically and by staining polyacrylamide gels for haem associated peroxidase activity) decayed only slightly in either type of vesicle. These observations suggest that cytochrome f is, in part, exposed to the intrathylakoid lumen, whilst its haem group is retained in a more hydrophobic region.     

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.     

Response of isolated thylakoid membranes with altered fluidity to short term heat stress

The effect of alterations of lipid phase order of thylakoid membranes on the thermosensitivity of photosystem I (PS I) and photosystem II (PS II) was studied. Plant sterols stigmasterol and cholesterol were applied to decrease the fluidity in isolated membranes. After sterol treatment, a decrease of the temperature of 50 % inhibition of PSII activity was observed. Heat stress-induced stimulation of PSI-mediated electron transport rate was registered for control, but not for sterol-treated membranes. Effect of altered lipid order on oxygen evolving complex was evaluated by means of flash oxygen yields revealing changes in the stoichiometry of PSII_α and PSII_β centers. The effect of sterol incorporation on the changes in the thermotropic behavior of the main pigment-protein complexes was studied by differential scanning calorimetry (DSC). DSC traces of control thylakoids in the temperature range 20–98 °C exhibited several irreversible endothermic transitions. Incorporation of cholesterol and stigmasterol results in superimposition of the transitions and only two main bands could be resolved. While high temperature band peaks at the same temperature after treatment with both sterols, the band that combines low temperature transitions shows different melting temperature (T_m): 70 °C for stigmasterol- and 65 °C for cholesterol-treated membranes. The data presented here emphasise the crucial role of lipid order for the response of thylakoids to high temperatures, mediated not only by changes in the fluidity of bulk lipid phase as result of sterol incorporation but also by changes in the thermotropic properties of pigment-protein complexes.Key words: Cholesterol, Fluidity, Heat stress, Oxygen flash yields, Thylakoid membrane, Stigmasterol     

Positive charges of polyamines protect PSII in isolated thylakoid membranes during photoinhibitory conditions

The effects of the positive charges of amines such as spermine (SPM), putrescine (PUT) and methylamine (MET) on the protection of PSII against excessive illumination were investigated in isolated thylakoid membranes. Under photoinhibition conditions, water oxidation, the kinetics of the Chl fluorescence rise and charge recombination in PSII were affected. A low concentration of SPM (1 mM) added before photoinhibition produced a significant improvement of F(v)/F(0), the oxygen yield and the amplitude of the B-band of thermoluminescence compared with the other amines. Amongst the amines studied, only SPM could protect the photosynthetic apparatus under photoinhibition conditions. This protection was probably provided by the polycationic nature of SPM (four positive charges at physiological pH), which can stabilize surface-exposed proteins of PSII through electrostatic interaction.     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.