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Steven M Theg

Publications and source records attributed to Steven M Theg.

15 recordsLinked to original sources
PubMed2006

Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts.

Lipopolysaccharide (LPS) (a.k.a., endotoxin) is an essential component of the outer leaflet of the outer membrane of gram-negative bacteria and is a potent activator of the innate immune system of animals. Lipid A, the glycolipid core of LPS, is the agent responsible for disease and death from gram-negative sepsis, an important cause of human mortality and morbidity. Although it is generally accepted that lipid A is restricted to the prokaryotes, recent efforts to purify molecules from green algae with structural features unique to lipid A have met with success. Furthermore, the vascular plant Arabidopsis thaliana has been found to contain genes that encode all of the enzymes of the biosynthetic pathway for lipid A. It is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. For the present study, we used affinity reagents for lipid A to probe green alga and tissues of the garden pea for a light microscopic localization of lipid A in these eukaryote cells. We find staining for lipid A in free-living and endosymbiotic green algae and in the chloroplasts of vascular plants, indicating that this molecule is not restricted to prokaryotes, but is found also in select eukaryotes.

Chlorophyta↗
PubMed2006

Evidence for an ER to Golgi to chloroplast protein transport pathway.

Chloroplast protein import is generally believed to occur posttranslationally through the interaction of a precursor protein with the Toc and Tic transport apparatus in the plastid envelope membranes. The cleavable N-terminal transit peptide present on translocated proteins has been considered to be essential and sufficient for targeting. This idea was recently challenged when an analysis of the chloroplast proteome revealed many proteins without a predicted transit peptide. A recent study demonstrates the existence of a novel chloroplast targeting pathway, starting with protein entry into the endoplasmic reticulum and involving the Golgi apparatus.

Arabidopsis↗
PubMed2005

Complete maturation of the plastid protein translocation channel requires a type I signal peptidase.

The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

Arabidopsis↗
PubMed2005

Carbonic anhydrase activity of the photosystem II OEC33 protein from pea.

The purpose of this study was to identify the location of one of the two sources of carbonic anhydrase (CA) activity associated with the PSII complex in chloroplast membranes. We tested the hypothesis that the extrinsic 33 kDa protein, OEC33, associated with the oxygen-evolving complex (OEC), is one source of CA activity. We found that precursor OEC33 expressed in Escherichia coli exhibits CA activity, but the expressed precursors of OEC24 or OEC17 do not. The CA activity of OEC33 remained after treatment at 90 degrees C for 15 min. Additional biochemical evidence supports the hypothesis. Only those wash treatments that remove the OEC33 from PSII also remove CA activity. Both immunoblot and CA activity show that the CA tracks the OEC33, in parallel, when PSII undergoes washing at different CaCl2 concentrations. The OEC33 protein purified by HiTrap Q anion exchange chromatography has CA activity that is inhibited by an antibody against OEC33. PSII membranes washed with 1 M CaCl2 to remove OEC33 can be reconstituted either with extracted, purified, OEC33 or with the E. coli-expressed precursor OEC33. Reconstitution partially restores both oxygen evolution and CA activity. For maximal CA activity, OEC33 requires manganese as a cofactor.

Antibodies↗
PubMed2005

Chloroplast outer membrane protein targeting and insertion.

Proteins in the chloroplast outer envelope membrane are nuclear encoded and post-translationally targeted to the chloroplast. The targeting and membrane insertion of these proteins is not well understood. Although early work suggested otherwise, the best-studied outer membrane proteins (OMPs) use both proteins within the chloroplast and NTPs for insertion. There have been conflicting reports in the field regarding protein targeting and insertion, which have probably arisen because of differences in experimental methodology and different interpretations of reduction (versus abolition) of integration. This review summarizes what is known to date about the mechanism of chloroplast OMP targeting.

Amino Acid Sequence↗
PubMed2005

Toc64 is not required for import of proteins into chloroplasts in the moss Physcomitrella patens.

Toc64 has been suggested to be part of the chloroplast import machinery in Pisum sativum. A role for Toc64 in protein transport has not been established, however. To address this, we generated knockout mutants in the moss Physcomitrella patens using the moss's ability to perform homologous recombination with nuclear DNA. Physcomitrella patens contains two genes that encode Toc64-like proteins. Both of those proteins appear to be localized in the chloroplast. The double-mutant plants were lacking Toc64 protein in the chloroplasts but showed no growth phenotype. In addition, these plants accumulated other plastid proteins at wild-type levels and showed no difference from wild type in in vitro protein import assays. These plants did have a slightly altered chloroplast shape in some tissues, however. The evidence therefore indicates that Toc64 proteins are not required for import of proteins in Physcomitrella, but may point to involvement in the determination of plastid shape.

Amino Acid Sequence↗
PubMed2005

Protein- and energy-mediated targeting of chloroplast outer envelope membrane proteins.

While the import of nuclear-encoded chloroplast proteins is relatively well studied, the targeting of proteins to the outer membrane of the chloroplast envelope is not. The insertion of most outer membrane proteins (OMP) is generally considered to occur without the utilization of energy or proteinaceous components. Recently, however, proteins have been shown to be involved in the integration of outer envelope protein 14 (OEP14), whose outer membrane insertion was previously thought to be spontaneous. Here we investigate the insertion of two proteins from Physcomitrella patens, PpOEP64-1 and PpOEP64-2 (formerly known as PpToc64-1 and PpToc64-2), into the outer membrane of chloroplasts. The association of PpOEP64-1 with chloroplasts was not affected by chloroplast pre-treatments. Its insertion into the membrane was affected, however, demonstrating the importance of measuring insertion specifically in these types of assays. We found that the insertion of PpOEP64-1, PpOEP64-2 and two other OMPs, OEP14 and digalactosyldiacylglycerol synthase 1 (DGD1), was reduced by either nucleotide depletion or proteolysis of the chloroplasts. Integration was also inhibited in the presence of an excess of an imported precursor protein. In addition, OEP14 competed with the insertion of the OEP64s and DGD1. These data demonstrate that the targeting of several OMPs involves proteins present in chloroplasts and requires nucleotides. Together with previous reports, our data suggest that OMPs in general do not insert spontaneously.

Adenosine Triphosphate↗
PubMed2004

A homolog of prokaryotic thiol disulfide transporter CcdA is required for the assembly of the cytochrome b6f complex in Arabidopsis chloroplasts.

The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b(6)f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.

Alkaline Phosphatase↗
PubMed2003

Protein transport via the cpTat pathway displays cooperativity and is stimulated by transport-incompetent substrate.

Kinetic analyses of cpTat-mediated protein transport across the thylakoid membrane were conducted, revealing three important characteristics of this translocation pathway. First, transport via the cpTAT system displays a non-Michaelis-Menten, sigmoidal rate-substrate relationship with an apparent Hill coefficient of 1.80, indicative of positive homotropic cooperativity. Second, the presence of transport-incompetent substrates was found not to competitively inhibit the translocation of transport-competent substrates. However, the presence of low concentrations of transport-incompetent protein enhances the transport of wild type substrate. Together, these findings suggest that interaction between Tat machinery components and both transport-competent and transport-incompetent protein may elicit a cooperative effect on the translocation rate.

Gene Products, tat↗
PubMed2003

Energetics of protein transport across biological membranes. a study of the thylakoid DeltapH-dependent/cpTat pathway.

Among the pathways for protein translocation across biological membranes, the DeltapH-dependent/Tat system is unusual in its sole reliance upon the transmembrane pH gradient to drive protein transport. The free energy cost of protein translocation via the chloro-plast DeltapH-dependent/Tat pathway was measured by conducting in vitro transport assays with isolated thylakoids while concurrently monitoring energetic parameters. These experiments revealed a substrate-specific energetic barrier to cpTat-mediated transport as well as direct utilization of protons from the gradient, consistent with a H+/protein antiporter mechanism. The magnitude of proton flux was assayed by four independent approaches and averaged 7.9 x 10(4) protons released from the gradient per transported protein. This corresponds to a DeltaG transport of 6.9 x 10(5) kJ.mol protein translocated(-1), representing the utilization of an energetic equivalent of 10(4) molecules of ATP. At this cost, we estimate that the DeltapH-dependent/cpTat pathway utilizes approximately 3% of the total energy output of the chloroplast.

Adenosine Triphosphate↗
PubMed2003

Energy use by biological protein transport pathways.

The targeting of proteins into and across biological membranes to their correct cellular locations is mediated by a variety of transport pathways. These systems must couple the thermodynamically unfavorable processes of substrate translocation and integration with the expenditure of metabolic energy, using the free energy of ATP and GTP hydrolysis and/or a transmembrane protonmotive force. Several recent advances in our knowledge of the structure and function of these transport systems have provided insights into the mechanisms of energy transduction, force generation and energy use by different protein transport pathways.

Adenosine Triphosphatases↗
PubMed2003

Physcomitrella patens as a model for the study of chloroplast protein transport: conserved machineries between vascular and non-vascular plants.

A single general import pathway in vascular plants mediates the transport of precursor proteins across the two membranes of the chloroplast envelope, and at least four pathways are responsible for thylakoid protein targeting. While the transport systems in the thylakoid are related to bacterial secretion systems, the envelope machinery is thought to have arisen with the endosymbiotic event and to be derived, at least in part, from proteins present in the original endosymbiont. Recently the moss Physcomitrella patens has gained worldwide attention for its ability to undergo homologous recombination in the nuclear genome at rates unseen in any other land plants. Because of this, we were interested to know whether it would be a useful model system for studying chloroplast protein transport. We searched the large database of P. patens expressed sequence tags for chloroplast transport components and found many putative homologues. We obtained full-length sequences for homologues of three Toc components from moss. To our knowledge, this is the first sequence information for these proteins from non-vascular plants. In addition to identifying components of the transport machinery from moss, we isolated plastids and tested their activity in protein import assays. Our data indicate that moss and pea (Pisum sativum) plastid transport systems are functionally similar. These findings identify P. patens as a potentially useful tool for combining genetic and biochemical approaches for the study of chloroplast protein targeting.

Bryopsida↗
PubMed2002

Chemical modification of amine groups on PS II protein(s) retards photoassembly of the photosynthetic water-oxidizing complex.

Four Mn atoms function as catalysts in the water-oxidizing complex located on the oxidizing side of PS II. We have studied the involvement of amine groups of the PS II proteins in photoligation of Mn2+ to the apo water-oxidizing complex, using the combined techniques of photoactivation and chemical modification with the modifiers methyl acetimidate (MAI), acetic acid N-hydroxysuccinimide ester (NHS), and 2,4,6-trinitrobenzenesulfonic acid (TNBS). Chemical modification of hydroxylamine-treated PS II core complexes decreased their capacity for restoration of oxygen evolution and photoligation of Mn2+ to the apo water-oxidizing complex (WOC), but did not affect their electron transfer activity in the vicinity of PS II. The number of functional high-affinity Mn-binding sites, but not of low-affinity sites, was significantly modulated by chemical modification. Kinetic analysis of photoactivation with the repetitive flashes revealed that the intermediate generated during a photoactivation process was destabilized by the chemical modification. To identify which proteins possess the amine groups involved in ligation of functional Mn, we examined the difference in NHS biotinylation between PS II core complexes with and without the Mn cluster. NHS biotinylation resulting in altered ligation of functional Mn apparently occurred on three proteins: an antenna chlorophyll binding protein (CP47), a light-harvesting chlorophyll protein (CP29), and another chlorophyll binding protein (PS II-S). Of these proteins, only the Mn-dependent biotinylation of CP47 was found to occur independently of the application of an NHS-masking concentration before removal of the functional Mn. These results suggest that lysyl residues of CP47, and perhaps also CP29 and PS II-S, function in direct photoligation of Mn2+ to the apo WOC.

Amines↗
PubMed2002

Reciprocal expression of two candidate di-iron enzymes affecting photosystem I and light-harvesting complex accumulation.

Crd1 (Copper response defect 1), which is required for the maintenance of photosystem I and its associated light-harvesting complexes in copper-deficient (-Cu) and oxygen-deficient (-O(2)) Chlamydomonas reinhardtii cells, is localized to the thylakoid membrane. A related protein, Cth1 (Copper target homolog 1), is shown to have a similar but not identical function by genetic suppressor analysis of gain-of-function sct1 (suppressor of copper target 1) strains that are transposon-containing alleles at CTH1. The pattern of Crd1 versus Cth1 accumulation is reciprocal; Crd1 abundance is increased in -Cu or -O(2) cells, whereas Cth1 accumulates in copper-sufficient (+Cu), oxygenated cells. This expression pattern is determined by a single trans-acting regulatory locus, CRR1 (COPPER RESPONSE REGULATOR 1), which activates transcription in -Cu cells. In +Cu cells, a 2.1-kb Cth1 mRNA is produced and translated, whereas Crd1 is transcribed only at basal levels, leading to Cth1 accumulation in +Cu cells. In -Cu cells, CRR1 function determines the activation of Crd1 expression and the production of an alternative 3.1-kb Cth1 mRNA that is extended at the 5' end relative to the 2.1-kb mRNA. Synthesis of the 3.1-kb mRNA, which encodes six small upstream open reading frames that possibly result in poor translation, blocks the downstream promoter through transcriptional occlusion. Fluorescence analysis of wild-type, crd1, and sct1 strains indicates that copper-responsive adjustment of the Cth1:Crd1 ratio results in modification of the interactions between photosystem I and associated light-harvesting complexes. The tightly coordinated CRR1-dependent regulation of isoenzymes Cth1 and Crd1 reinforces the notion that copper plays a specific role in the maintenance of chlorophyll proteins.

Algal Proteins↗