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K Cline

Publications and source records attributed to K Cline.

At least 19 recordsLinked to original sources
PubMed2001

Post-translational protein translocation into thylakoids by the Sec and DeltapH-dependent pathways.

Two distinct protein translocation pathways that employ hydrophobic signal peptides function in the plant thylakoid membrane. These two systems are precursor specific and distinguished by their energy and component requirements. Recent studies have shown that one pathway is homologous to the bacterial general export system called Sec. The other one, called the DeltapH-dependent pathway, was originally considered to be unique to plant thylakoids. However, it is now known that homologous transport systems are widely present in prokaryotes and even present in archaea. Here we review these protein transport pathways and discuss their capabilities and mechanisms of operation.

Amino Acid Sequence↗
PubMed2001

Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.

The thylakoid DeltapH-dependent pathway transports folded proteins with twin arginine-containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC-Hcf106 complex serves as receptor for specific binding of twin arginine-containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an approximately 700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC-Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of DeltapH. These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

Membrane Proteins↗
PubMed2001

Chloroplast TatC plays a direct role in thylakoid (Delta)pH-dependent protein transport.

The thylakoid (Delta)pH-dependent pathway transports folded proteins. Identified components include Hcf106 and Tha4. Orthologs of these proteins plus a membrane protein called TatC are essential for the homologous bacterial Tat system. Here we report identification of a chloroplast TatC (cpTatC). cpTatC is an integral thylakoid membrane protein as determined by in vitro chloroplast import and immunoblotting. Antibody to cpTatC specifically inhibited the thylakoid (Delta)pH-dependent pathway in vitro. cpTatC is present in about the same quantity as estimated translocation sites, whereas Hcf106 and Tha4 are present in 5-8-fold excess. These results are relevant to mechanistic models for this system.

Amino Acid Sequence↗
PubMed2001

Localization and targeting of the VP14 epoxy-carotenoid dioxygenase to chloroplast membranes.

Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges. ABA is synthesized via an oxidative cleavage of 9-cis epoxy-carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway. Vp14 of maize encodes an epoxy-carotenoid dioxygenase that is soluble when expressed in E. coli. An important goal has been to determine how the soluble VP14 protein is targeted to epoxy-carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids. Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma-targeting domain. The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes. Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N-terminal sequence. A putative amphipathic alpha-helix within this region is essential, but not sufficient, for the membrane targeting. Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S-transferase failed to associate with the thylakoid membrane. The membrane-bound VP14 was partially resistant to chaotropic washes such as 0.1 M Na2CO3 (pH 11.5) and 6 M urea. Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane.

Amino Acid Sequence↗
PubMed2000

The thylakoid delta pH-dependent pathway machinery facilitates RR-independent N-tail protein integration.

The thylakoidal DeltapH-dependent and bacterial twin arginine transport systems are structurally and functionally related protein export machineries. These recently discovered systems have been shown to transport folded proteins but are not known to assemble integral membrane proteins. We determined the translocation pathway of a thylakoidal FtsH homologue, plastid fusion/protein translocation factor, which is synthesized with a chloroplast-targeting peptide, a hydrophobic signal peptide, and a hydrophobic membrane anchor. The twin arginine motif in its signal peptide and its sole integration requirement of a DeltapH suggested that plastid fusion/protein translocation factor employs the DeltapH pathway. Surprisingly, changing the twin arginine to twin lysine or deleting the signal peptide did not abrogate integration capability or characteristics. Nevertheless, three criteria argue that all three forms require the DeltapH pathway for integration. First, integration was competed by an authentic DeltapH pathway precursor. Second, antibodies to DeltapH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the hcf106 null mutant were unable to integrate Pftf into their thylakoids. Thus, DeltapH pathway machinery facilitates both signal peptide-directed and N-tail-mediated membrane integration and does not strictly require the twin arginine motif.

Arginine↗
PubMed2000

Precursors bind to specific sites on thylakoid membranes prior to transport on the delta pH protein translocation system.

The Delta pH pathway is one of two systems for protein transport to the thylakoid lumen. It is a novel transport system that requires only the thylakoidal DeltapH to power translocation. Several substrates of the Delta pH pathway, including the intermediate precursor form of OE17 (iOE17) and the truncated precursor form of OE17 (tOE17), were shown to bind to the membrane in the absence of the DeltapH and be transported into the lumen when the DeltapH was restored. Binding occurred without energy or soluble factors, and efficient transport from the bound state ( approximately 80-90%) required only the DeltapH. Binding is due to protein-protein interactions because protease pretreatment of thylakoids destroyed their binding capability. Precursors are bound to a specific site on the Delta pH pathway because binding was competed by saturating amounts of Delta pH pathway precursor proteins, but not by a Sec pathway precursor protein. These results suggested that precursor tOE17 binds to components of the Delta pathway translocation machinery. Hcf106 and Tha4 are two components of the Delta pH pathway machinery. Antibodies to Hcf106 or Tha4, when prebound to thylakoids, specifically inhibited precursor transport on the Delta pH pathway. However, only Hcf106 antibodies reduced the level of precursor binding. These results suggest that Hcf106 functions in early steps of the transport process.

Antibodies↗
PubMed2000

Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma.

The 14-3-3 proteins are acidic, dimeric proteins that have been implicated in many eukaryotic cellular processes because of direct protein association with enzymes and other metabolic and regulatory proteins. 14-3-3 proteins are largely considered to be cytoplasmic, but a search for proteins that specifically interact with a plant 14-3-3 resulted in the isolation of a nuclear-encoded, thylakoid-targeted chloroplast precursor, the full-length Arabidopsis photosystem I N-subunit At pPSI-N (P.C. Sehnke, R.J. Ferl ¿1995 Plant Physiol 109: 1126). Using precursor truncations in the two-hybrid system, it was determined that the leader sequence is the site of PSI-N that associates with 14-3-3. This suggested the novel possibility that 14-3-3 would be found within chloroplasts. Immuno-electron microscopy of leaf tissue and western analysis of chloroplast fractions with monoclonal anti-14-3-3 antibodies localized 14-3-3 proteins to the chloroplast stroma and the stromal side of thylakoid membranes. Using peptide-generated, isoform-specific antibodies, GF14nu, GF14epsilon, GF14mu, and GF14upsilon were shown to be present in the chloroplast stromal extract. These isoforms represent two distinct phylogenetic 14-3-3 groupings. These data suggest a novel interorganellar role for these phylogenetically distinct 14-3-3 proteins.

14-3-3 Proteins↗
PubMed1999

Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways.

Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in approximately 10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

Amino Acid Sequence↗
PubMed1999

Red bell pepper chromoplasts exhibit in vitro import competency and membrane targeting of passenger proteins from the thylakoidal sec and DeltapH pathways but not the chloroplast signal recognition particle pathway.

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and DeltapH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane DeltapH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and DeltapH pathways.

Biological Transport, Active↗
PubMed1998

A signal peptide that directs non-Sec transport in bacteria also directs efficient and exclusive transport on the thylakoid Delta pH pathway.

Signal peptides that specifically direct precursor proteins to the thylakoid Delta pH pathway possess an N domain RR motif. Signal peptides that direct transport of bacterial proteins across a non-Sec export pathway possess an N domain RRXFLK consensus motif. Recent genetic studies suggest an evolutionary link between these two protein translocation pathways. To further explore this relationship, we examined the thylakoid targeting capability of the signal peptide for Escherichia coli hydrogenase 1 small subunit (HyaA) by linking it to plastocyanin and assaying the chimeric protein in an in vitro thylakoid transport assay. The chimeric precursor was transported across thylakoids with high efficiency. Transport was characteristic of the Delta pH but not the Sec pathway, i.e. it was eliminated by ionophores that dissipate the DeltapH but occurred in the absence of stromal extract or ATP. This result was confirmed by competition with chemical quantities of a Delta pH pathway precursor. This indicates that the HyaA signal peptide has the necessary elements for efficient and exclusive targeting to the Delta pH pathway and further supports the notion that the alternate targeting pathways in prokaryotes and plant thylakoids are analogous.

Adenosine Triphosphatases↗
PubMed1998

Evidence for a loop mechanism of protein transport by the thylakoid Delta pH pathway.

The thylakoid Delta pH pathway is a protein transport system with unprecedented characteristics. To investigate its mechanism, the topology of precursor insertion was determined. A fusion protein comprising a large polypeptide domain fused to the amino terminus of pOE17 (a Delta pH pathway precursor) was efficiently processed by thylakoid membranes. The amino terminus, including the targeting peptide, remained on the cis side of the membrane. Mature OE17 was transported to the lumen. These experiments demonstrate that Delta pH directed precursors enter the thylakoid membrane in a loop, implying that the Delta pH pathway has evolved from an export-type protein translocation system.

Animals↗
PubMed1997

Sec-independent protein translocation by the maize Hcf106 protein.

The bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts. A third translocation mechanism in chloroplasts [the proton concentration difference (DeltapH) pathway] was previously thought to be unique. The hcf106 mutation of maize disrupts the localization of proteins transported through this DeltapH pathway in isolated chloroplasts. The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the DeltapH pathway has been conserved since the endosymbiotic origin of chloroplasts. Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.

Amino Acid Sequence↗
PubMed1997

Chloroplast SRP54 interacts with a specific subset of thylakoid precursor proteins.

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.

Amino Acid Sequence↗
PubMed1997

Targeting determinants and proposed evolutionary basis for the Sec and the Delta pH protein transport systems in chloroplast thylakoid membranes.

Transport of proteins to the thylakoid lumen is accomplished by two precursor-specific pathways, the Sec and the unique Delta pH transport systems. Pathway selection is specified by transient lumen-targeting domains (LTDs) on precursor proteins. Here, chimeric and mutant LTDs were used to identify elements responsible for targeting specificity. The results showed that: (a) minimal signal peptide motifs consisting of charged N, hydrophobic H, and cleavage C domains were both necessary and sufficient for pathway-specific targeting; (b) exclusive targeting to the Delta pH pathway requires a twin arginine in the N domain and an H domain that is incompatible with the Sec pathway; (c) exclusive targeting to the Sec pathway is achieved by an N domain that lacks the twin arginine, although the twin arginine was completely compatible with the Sec system. A dual-targeting signal peptide, constructed by combining Delta pH and Sec domains, was used to simultaneously compare the transport capability of both pathways when confronted with different passenger proteins. Whereas Sec passengers were efficiently transported by both pathways, Delta pH passengers were arrested in translocation on the Sec pathway. This finding suggests that the Delta pH mechanism evolved to accommodate transport of proteins incompatible with the thylakoid Sec machinery.

Amino Acid Sequence↗
PubMed1997

Chromoplast development in ripening tomato fruit: identification of cDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein.

During tomato fruit ripening, photosynthetically competent thylakoid membranes are broken down and replaced by membranous deposits of carotenoids. Few of the proteins involved in this transition have been identified. We have used chloroplast protein import assays as a means to identify two cDNAs that encode proteins destined for the developing chromoplast. One of the cDNAs had unexpected properties and its biological function has not been determined. However, the other cDNA encodes a plastid-localized low-MW heat shock protein (hsp). The steady-state level of RNA corresponding to this cDNA increased several-fold during tomato ripening, and the amount of RNA induced by heat stress increased dramatically during this process. These observations suggest a new role for this stress protein in protecting the plastid during the dismantling of the thylakoid membranes or during the buildup of carotenoids.

Amino Acid Sequence↗