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S M Theg

Publications and source records attributed to S M Theg.

At least 19 recordsLinked to original sources
PubMed2000

Proton transfer limits protein translocation rate by the thylakoid DeltapH/Tat machinery.

The thylakoid transmembrane DeltapH is the sole energy source driving translocation of precursor proteins by the DeltapH/Tat machinery. Consequently, proton translocation must be coupled to precursor translocation. For the precursor of the 17 kDa protein of the oxygen-evolving complex (pOE17), the protein translocation process is characterized by a steep drop in efficiency at an external pH below 7.0 and above 8.7. As the membrane DeltapH is virtually unaffected from pH 6.5 to 9.2, the loss in import efficiency is a consequence of the titration of multiple residues within the translocation machinery. Transport is retarded by a factor of 2-3 in deuterium oxide (D(2)O) relative to water, strongly suggesting that proton-transfer reactions limit translocation rate. The solvent isotope effect manifests itself after the precursor binds to the membrane, indicating that the rate-limiting step is a later event in the transport process.

Biological Transport↗
PubMed2000

Characterization of the early steps of OE17 precursor transport by the thylakoid DeltapH/Tat machinery.

In order to probe the structure and protein translocation function of the thylakoid Tat machinery, a 25-residue C-terminal extension containing a 13-residue in vivo biotinylation tag and a 6x His tag was added to a mutant precursor of the 17-kDa subunit of the oxygen-evolving complex to form pOE17(C)-BioHis. When avidin was attached to biotinylated precursor in situ, the precursor-avidin complex was neither imported nor did it form a membrane-spanning translocation intermediate. It did, however, competitively inhibit the translocation of unbiotinylated precursor with an apparent KI unaffected by avidin. It is shown that the precursor protein achieves a stable folded structure upon dilution from urea, suggesting that the avidin-induced inhibition of transport results from a folding-induced proximity of N-terminal and C-terminal domains. It is further demonstrated that the majority of precursor rapidly binds to the thylakoid membrane, remaining import competent and yet undissociable by high salt or high pH treatment at ice temperature. The membrane binding event is unaffected by avidin. Import kinetics reveal that nonproton motive force-driven transport steps make up a major fraction of the transport time. These observations suggest that the N-terminal presequence on the avidin-bound precursor is available for membrane binding and initial recognition by the transport machinery, but the attached avidin signals the machinery that the precursor is an incorrectly configured substrate and thus import is aborted. Consequently, the DeltapH/Tat machinery's proofreading mechanism must operate after precursor recognition but before the committed step in transport.

Amino Acid Sequence↗
PubMed1998

Energy-transducing thylakoid membranes remain highly impermeable to ions during protein translocation.

We investigated the operation of a posttranslational protein translocation pathway to determine whether ions are excluded from the translocase during protein transport. The membrane capacitance during protein translocation across chloroplast thylakoid membranes was monitored via electric-field-indicating carotenoid electrochromic bandshift measurements. Evidence is presented that shows that the membrane ion conductance is not increased during the complete cycle of binding, transport, and substrate release by the DeltapH-dependent translocase; i.e., the membrane remains ion-tight during protein translocation. We further demonstrate that a synthetic targeting peptide that directs proteins across this membrane does not gate translocation pores. We conclude that protein transport across the thylakoid membrane does not compromise its ability to maintain ion gradients and is, thus, unlikely to affect its functions in energy transduction.

Biological Transport, Active↗
PubMed1997

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.

Animals↗
PubMed1996

Unassembled subunits of the photosynthetic oxygen-evolving complex present in the thylakoid lumen are long-lived and assembly-competent.

Physiologically healthy pea chloroplasts contain unassembled, soluble subunits of the oxygen-evolving complex in the thylakoid lumen. We report that the lifetimes of two of these subunits, both on and off the membrane, exceed 8 h in vitro. We also demonstrate that each of the subunits present in the thylakoid lumen is competent for assembly into the membrane-bound complex. These data are consistent with the postulate that the soluble lumen-resident subunits play a role in photosystem II homeostasis. We also demonstrate that the reconstitution of the 33 kDa subunit is inhibited by extremely low concentrations of Triton X-100, suggesting that hydrophobic interactions are involved in the binding of this subunit to the photosystem II reaction center.

Cell Fractionation↗
PubMed1996

A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: energetics and mechanistic implications.

Chloroplast protein import presents a complex membrane traversal problem: precursor proteins must cross two envelope membranes to reach the stromal compartment. This work characterizes a new chloroplast protein import intermediate which has completely traversed the outer envelope membrane but has not yet reached the stroma. The existence of this intermediate demonstrates that distinct protein transport machineries are present in both envelope membranes, and that they are able to operate independently of one another under certain conditions. Energetic characterization of this pathway led to the identification of three independent energy-requiring steps: binding of the precursor to the outer envelope membrane, outer membrane transport, and inner membrane transport. Localization of the sites of energy utilization for each of these steps, as well as their respective nucleotide specificities, suggest that three different ATPases mediate chloroplast envelope transport.

Adenosine Triphosphatases↗
PubMed1994

Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana.

Cyclophilin (CyP), a protein with peptidyl-prolyl cis-trans isomerase (rotamase) activity, is the specific cellular target of cyclosporin A. We have isolated cDNA clones of two genes (designated ROC1 and ROC4) encoding CyP homologs from Arabidopsis thaliana (L.). The protein products of these genes are distinct from a previously identified Arabidopsis CyP. ROC1 is expressed in all tested plant organs and encodes a protein which is highly similar to previously described cytosolic CyP isoforms of other plants. In contrast, ROC4 is expressed only in photosynthetic organs and encodes a protein which includes an amino-terminal extension with properties of known chloroplast transit peptides. In vitro import experiments using the putative precursor protein to ROC4 showed that the protein is imported into chloroplasts where it is processed to the predicted mature size. Rotamase assays and immunoblot analysis of subcellular fractions indicate the presence of a CyP isoform in the stroma of chloroplasts but not in the thylakoid membranes or thylakoid lumen. Together, these data show that ROC4 is a novel CyP isoform which is located in the stroma of chloroplasts. In vitro chloroplast import of precursors of other chloroplast proteins was unaffected by concentrations of cyclosporin A which completely inhibit rotamase activity of chloroplast stromal CyP. Thus, this activity is not essential for protein import into chloroplasts.

Amino Acid Isomerases↗
PubMed1993

Protein import into chloroplasts.

The structural complexity of chloroplasts is reflected in their intriguing protein-targeting system. Not only must nucleus-encoded proteins be targeted to the chloroplast, but also, once inside the chloroplast, these polypeptides must be directed to their final destination in one of six intrachloroplastic compartments. Although the details of this process remain elusive, many recent advances have improved our vantage point for examining this system.

Journal Article↗
PubMed1992

Biophysical characterization of a transit peptide directing chloroplast protein import.

We have investigated the biophysical properties of a 35 amino acid peptide representing the entire length of a chloroplastic targeting sequence. The peptide, termed gamma-tp, corresponds in sequence to the transit peptide of the gamma subunit of the chloroplast ATP synthase from Chlamydomonas reinhardtii. We found that gamma-tp blocks the import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase into isolated pea chloroplasts (KI approximately 5 microM), suggesting that it interacts with higher plant plastids in a physiological manner. We also found the gamma-tp to have a high affinity for nonpolar environments, but not to cause a general disruption of membrane integrity. Hydrophobic moment analysis suggests that the gamma-tp can adopt an amphipathic beta structure. However, circular dichroism measurements indicate that the peptide is largely a random coil, in both the presence and absence of sodium laurylsulfate micelles. In the absence of a recognizable secondary structural targeting motif, we asked whether the presence of a transit peptide on a chloroplast protein increases the protein's overall affinity for nonpolar environments. Phase-partition experiments with Triton X-114 suggest that this is not the case. These results are discussed in relation to the mechanism of protein targeting to chloroplasts.

Algal Proteins↗
PubMed1992

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.

Adenosine Triphosphate↗
PubMed1991

Evidence that localized energy coupling in thylakoids can continue beyond the energetic threshold onset into steady illumination.

Energy transduction from proton gradients into ATP formation in chloroplast thylakoids has been hypothesized to be driven equally efficiently by localized domain delta mu H+ or by a delocalized delta mu H+ (Beard, W. A. and Dilley, R. A. (1988) J. Bioenerg. Biomembr. 20, 129-154). An important question is whether the apparent localized protonmotive force energy coupling mode can be observed only in the dark-to-light transient in the flash excitation protocol commonly used, or whether the localized energy coupling gradient can be maintained under conditions of continuous illumination ATP formation. The assay in the previous work was to use permeable amines, added to thylakoids in the dark, and observe the effect of the amine on the length of the energization lag (number of single-turnover flashes) required to initiate ATP formation in the dark-to-light transition. Amine buffers delayed the ATP onset in high-salt-stored membranes but did not delay the onset with low salt-stored membranes. This work tested whether permeable amines show the different effects in low- or high-salt-stored thylakoids which had attained a steady-state ATP formation rate (in continuous light) for 20-40 s prior to adding the amine. Hydroxyethylmorpholine was the preferred amine for such experiments, a suitable choice inasmuch as it behaves similarly to pyridine in the flash-induced ATP formation onset experiments, but it permeates more rapidly than pyridine and it has a higher pKa, which enhances its buffering effects. With high-salt-stored thylakoids, 0.5 or 1.0 mM hydroxyethylmorpholine added after 40 s of continuous illumination caused a marked, but transient, slowing of the ATP formation rate, but little or no slowing of the rate was observed with low-salt-stored thylakoids (at similar phosphorylation rates for the two thylakoid samples). Those data indicate that in continuous illumination conditions the proton gradient driving ATP formation in thylakoids from the low-salt-stored treatment did not equilibrate with the lumen, but in thylakoids stored in high-salt the delta mu H+ freely equilibrated with the lumen. That suggestion was supported by measurement of the luminal pH under coupling conditions by the [14C]methylamine distribution method using low- or high-salt-stored thylakoids. Further supportive evidence was obtained from measuring the effect of permeable amine buffers on H+ uptake under coupled and basal conditions with both types of thylakoid.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen.

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.

Calcium↗
PubMed1991

Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration.

A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.

Journal Article↗
PubMed1990

Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.

Most chloroplastic proteins are cytosolically synthesized and posttranslationally transported to their proper locations. Two examples of this group of proteins are ferredoxin and plastocyanin, both of which are metal-containing components of the photosynthetic electron-transport chain. The import process for these two proteins includes the insertion of the metal ions to produce the holo forms of the proteins. We show here that in vitro translated precursor proteins of ferredoxin and plastocyanin are synthesized as apo forms and are assembled into their respective holo forms after being imported into isolated chloroplasts. We also provide evidence that only mature-sized proteins are competent to be assembled into holo forms.

Journal Article↗
PubMed1989

ATP is required for the binding of precursor proteins to chloroplasts.

One of the first steps in the transport of nuclear-encoded, cytoplasmically synthesized precursor proteins into chloroplasts is a specific binding interaction between precursor proteins and the surface of the organelle. Although protein translocation into chloroplasts requires ATP hydrolysis, binding is generally thought to be energy independent. A more detailed investigation of precursor binding to the surface of chloroplasts showed that ATP was required for efficient binding. Protein translocation is known to require relatively high levels (1 mM or more) of ATP. As little as 50-100 microM ATP caused significant stimulation of precursor binding over controls with no ATP. Several different precursors were tested and all showed increased binding upon addition of low levels of ATP. Nonhydrolyzable analogs of ATP did not substitute for ATP, indicating that ATP hydrolysis was required for binding. A protonmotive force was not involved in the energy requirement for binding. Other (hydrolyzable) nucleotides could substitute for ATP but were less effective at stimulating binding. Binding was stimulated by ATP generated inside chloroplasts even when an ATP trap was present to destroy external ATP. We conclude that internal ATP is required for stimulation of precursor binding to chloroplasts.

Adenosine Triphosphate↗
PubMed1989

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.

Adenosine Triphosphate↗
PubMed1988

Protons in the thylakoid membrane-sequestered domains can directly pass through the coupling factor during ATP synthesis in flashing light.

Thylakoid membranes contain sequestered domains in which protons are held in a metastable state out of equilibrium with those in the inner (lumen) or the outer aqueous bulk phases unless the membranes are made leaky by, for instance, the addition of uncouplers. Previously, it has not been clear whether such sequestered domain protons are: 1) directly on a localized pathway into the CF0-CF1; 2) on an ultimately delocalized pathway comprising domains----lumen----CF0-CF1; or 3) perhaps not importantly involved in any way with proton gradient-linked ATP formation. Recent developments now permit a test of the above possibilities. The test for the possible mode of involvement of domain protons in energy coupling utilized single turnover flashes to energize electric field-driven ATP formation, as influenced by both the proton depletion level of the sequestered domains and whether the thylakoids were prepared in a way to show either a localized or delocalized proton gradient response in ATP formation (this response is reversibly controlled by incubation in low (localized coupling mode) or high KCl (delocalized mode) incubations (Beard, W. A., and Dilley, R. A. (1986) FEBS Lett. 201, 57-62; Chiang, G. and Dilley, R. A. (1987) Biochemistry 26, 4911-1916). Using thylakoids showing delocalized coupling responses, there was no influence of the reversible depletion of the sequestered domain buffering pool on the delta psi-driven ATP formation onset lag. Lumenal protons rather than domain protons in that case appear to be the first to be driven through the CF0-CF1 complex by the delta psi field. However, using thylakoids prepared to be in the localized gradient coupling mode, protons in the domains, rather than those in the lumen, are the first to pass through the CF0-CF1 complex in the onset of energization. We conclude that the sequestered domain protons, in the latter case only, are obligatorily in the main proton diffusion pathway for energization of ATP formation and are the first protons (rather than lumen protons) driven through the CF1-CF0 complex by a delta psi field.

Adenosine Triphosphate↗