Sex-biased dispersal of great white sharks.
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Animals↗
Biomedical subjects
B D Bruce
Publications and source records attributed to B D Bruce.
Cytometric analysis of an epitope-tagged transit peptide bound to the chloroplast translocation apparatus.
Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope-tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His-S-SStp, contains a removable dual-epitope tag, His-S, at its N-terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser-scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C-terminal to the transit peptide, His-S-SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non-radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre- and post-import thermolysin treatment, photochemical cross-linking and organelle fractionation. The interaction was specific for the transit peptide since His-S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry, showing saturation of binding yet only slight ATP-dependence. The addition of GTP showed inhibition of the binding of His-S-SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocation intermediate. In addition, direct visualization of His-S-SStp and Toc75 by confocal microscopy revealed a patch-like labeling, suggesting a co-ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast-binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.
Amino Acid Sequence↗
The cellular level of PR500, a protein complex related to the 19S regulatory particle of the proteasome, is regulated in response to stresses in plants.
In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of approximately 800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the "lid" subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.
Arabidopsis↗
Nanoscale photosynthesis: photocatalytic production of hydrogen by platinized photosystem I reaction centers.
A study of the photocatalytic production of molecular hydrogen from platinized photosystem I (PSI) reaction centers is reported. At pH 7 and room temperature metallic platinum was photoprecipitated at the reducing end of PSI according to the reaction, [PtCl6]2- + 4e- + hv-->Pt decreases + 6Cl-, where it interacted with photogenerated PSI electrons and catalyzed the evolution of molecular hydrogen. The reaction mixture included purified spinach PSI reaction centers, sodium ascorbate and spinach plastocyanin. Experimental data on real-time catalytic platinum formation as measured by the onset and rates of hydrogen photoevolution as a function of time are presented. The key objective of the experiments was demonstration of functional nanoscale surface metalization at the reducing end of isolated PSI by substituting negatively charged [PtCl6]2- for negatively charged ferredoxin, the naturally occurring water-soluble electron carrier in photosynthesis. The data are interpreted in terms of electrostatic interactions between [PtCl6]2- and the positively charged surface of psaD, the ferredoxin docking site situated at the stromal interface of the photosynthetic membrane and which is presumably retained in our PSI preparation. A discussion of the rates of hydrogen evolution in terms of the structural components of the various PSI preparations as well as of those of the intact thylakoid membranes is presented.
Catalysis↗
Involvement of an ATP-dependent peptide chaperone in cross-presentation after DNA immunization.
Immunization with plasmid DNA holds promise as a vaccination strategy perhaps useful in situations that currently lack vaccines, since the major means of immune induction may differ from more conventional approach. In the present study, we demonstrate that exposure of macrophages to plasmid DNA encoding viral proteins or OVA generates Ag-specific material that, when presented in vitro by dendritic cells to naive T cells, induces primary CTL response or elicits IL-2 production from an OVA peptide-specific T-T hybridoma. The immunogenic material released was proteinaceous in nature, free of apoptotic bodies, and had an apparent m.w. much larger than a 9-11-aa CTL-recognizable peptide. The macrophage-released factor(s) specifically required a hydrolyzable ATP substrate and was inhibited by procedures that removed or hydrolyzed ATP; in addition, anti-heat-shock protein 70 antiserum abrogated the activity to a large extent. These results indicate the possible involvement of a heat-shock protein 70-linked peptide chaperone in a cross-priming method of immune induction by DNA vaccination. Such a cross-priming process may represent a principal mechanism by which plasmid DNA delivered to cells such as myocytes effectively shuttle Ag to DC or other APC to achieve CTL induction in vivo.
Adenosine Triphosphate↗
Chloroplast transit peptides: structure, function and evolution.
It is thought that two to three thousand different proteins are targeted to the chloroplast, and the 'transit peptides' that act as chloroplast targeting sequences are probably the largest class of targeting sequences in plants. At a primary structural level, transit peptide sequences are highly divergent in length, composition and organization. An emerging concept suggests that transit peptides contain multiple domains that provide either distinct or overlapping functions. These functions include direct interaction with envelope lipids, chloroplast receptors and the stromal processing peptidase. The genomic organization of transit peptides suggests that these domains might have originated from distinct exons, which were shuffled and streamlined throughout evolution to yield a modern, multifunctional transit peptide. Although still poorly characterized, this evolutionary process could yield transit peptides with different domain organizations. The plasticity of transit peptide design is consistent with the diverse biological functions of chloroplast proteins.
Biological Transport↗
Identification of a Hsp70 recognition domain within the rubisco small subunit transit peptide.
The interaction between SStp, the transit peptide of the precursor protein to the small subunit of Rubisco (prSSU) and two Hsp70 molecular chaperones, Escherichia coli DnaK and pea (Pisum sativum) CSS1, was investigated in detail. Two statistical analyses were developed and used to investigate and predict regions of SStp recognized by DnaK. Both algorithms suggested that DnaK would have high affinity for the N terminus of SStp, moderate affinity for the central region, and low affinity for the C terminus. Furthermore, both algorithms predicted this affinity pattern for >75% of the transit peptides analyzed in the chloroplast transit peptide (CHLPEP) database. In vitro association between SStp and these Hsp70s was confirmed by three independent assays: limited trypsin resistance, ATPase stimulation, and native gel shift. Finally, synthetic peptides scanning the length of SStp and C-terminal deletion mutants of SStp were used to experimentally map the region of greatest DnaK affinity to the N terminus. CSS1 displayed a similar affinity for the N terminus of SStp. The major stromal Hsp70s affinity for the N terminus of SStp and other transit peptides supports a molecular motor model in which the chaperone functions as an ATP-dependent translocase, committing chloroplast precursor proteins to unidirectional movement across the envelope.
Adenosine Triphosphatases↗
In vivo and in vitro interaction of DnaK and a chloroplast transit peptide.
Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones. Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone. In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His6-S-peptide tag (His-S) via an ATP-dependent mechanism. Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro. The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex. Therefore, both GST-SStp and His-S-SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo. The combination of these results provides the first experimental support for a transit peptide-dependent interaction between a chloroplast precursor and Hsp70. These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria.
Adenosine Triphosphate↗
The C terminus of a chloroplast precursor modulates its interaction with the translocation apparatus and PIRAC.
The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (Delta52, Delta67, and Delta74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of (35)S-prSSU. Both IC(50) and K(i) values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and Delta52 were properly processed and accumulated within the chloroplast, whereas Delta67 and Delta74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.
Binding, Competitive↗
An agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice.
Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiDeltabasic) was expressed in transgenic mice under the control of the beta-actin promoter (BAPaDeltabasic). Three independent lines of BAPaDeltabasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiDeltabasic protein was functional in vivo. However, none of the BAPaDeltabasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiDeltabasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiDeltabasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaDeltabasic transgenic mice. These findings implicate a functional role for the agouti basic domain in vivo, possibly influencing the biogenesis of secreted agouti protein or modulating protein-protein interactions that contribute to effective antagonism of melanocortin receptors.
Agouti Signaling Protein↗
The mechanism of inactivation of a 50-pS envelope anion channel during chloroplast protein import.
The mechanism of import-competent precursor protein-induced inactivation of a 50-pS anion channel of the chloroplast envelope is investigated using single-channel recordings. The inactivation by precursor protein is the result of the induction of a long-lived closed state of the channel. The mean duration of this state does not depend on precursor concentration. From this it can be concluded that the protein import related anion channel enters the inactive state less frequently when the precursor concentration is lowered, but that the time spent in this state remains the same. Furthermore, it was found that the presence of precursor protein also decreases the mean durations of preexisting open and closed states of the channel. This decrease is found to be dependent on the precursor concentration. From this it is concluded that there is a direct interaction between the precursor protein and a protein complex of which the channel is a constituent. The mean duration of the precursor-induced long-lived closed state does not depend on the length of the translocation-competent precursor. This suggests that the duration of import is independent of precursor length.
Biological Transport, Active↗
Organelle isolation by magnetic immunoabsorption.
Currently, most organelle isolation procedures rely on physical parameters and centrifugation for separation. Here, we report the rapid and gentle isolation of a variety of organelles by immunolabeling whole cell lysates with organelle-specific antibodies and streptavidin magnetic particles followed by separation in a magnetic field. Using magnetic immunoabsorption, we have been able to specifically label mouse metaphase chromosomes and a variety of plant organelles, including: amyloplasts, choroplasts and nuclei from whole cell lysates of various plant tissues. We find that the distinct magnetic properties, surface characteristics and mean diameter-size ranges of different particle preparations significantly influence their specific utility for organelle isolations. By using an internal-field magnetic separation device, we have developed a method for quantitative recovery of labeled organelles in microarrays and tested a variety of antibodies to chloroplast outer envelope proteins for their ability to immune-isolate chloroplasts.
Animals↗
The role of lipids in plastid protein transport.
The elaborate compartmentalization of plant cells requires multiple mechanisms of protein targeting and trafficking. In addition to the organelles found in all eukaryotes, the plant cell contains a semi-autonomous organelle, the plastid. The plastid is not only the most active site of protein transport in the cell, but with its three membranes and three aqueous compartments, it also represents the most topologically complex organelle in the cell. The chloroplast contains both a protein import system in the envelope and multiple protein export systems in the thylakoid. Although significant advances have identified several proteinaceous components of the protein import and export apparatuses, the lipids found within plastid membranes are also emerging as important players in the targeting, insertion, and assembly of proteins in plastid membranes. The apparent affinity of chloroplast transit peptides for chloroplast lipids and the tendency for unsaturated MGDG to adopt a hexagonal II phase organization are discussed as possible mechanisms for initiating the binding and/or translocation of precursors to plastid membranes. Other important roles for lipids in plastid biogenesis are addressed, including the spontaneous insertion of proteins into the outer envelope and thylakoid, the role of cubic lipid structures in targeting and assembly of proteins to the prolamellar body, and the repair process of D1 after photoinhibition. The current progress in the identification of the genes and their associated mutations in galactolipid biosynthesis is discussed. Finally, the potential role of plastid-derived tubules in facilitating macromolecular transport between plastids and other cellular organelles is discussed.
Biological Transport↗
Characterization of the molecular-chaperone function of the heat-shock-cognate-70-interacting protein.
A histidine-tagged form of the recently discovered molecular chaperone, 70-kDa heat-shock cognate (Hsc70)-interacting protein (Hip), has been expressed in Escherichia coli and purified to near homogeneity. This protein remains soluble when expressed in E. coli. Several important properties of this chaperone have been investigated. HPLC size-exclusion chromatography indicates that the chaperone forms a tetramer similar to what has been reported for the native protein from rat liver cytosol. The recombinant form of Hip did not catalyze the hydrolysis of ATP and ATP analogs, although fluorescence measurements indicated that the chaperone recognizes anthraniloyl-dATP, anthraniloyl-ADP, and 2'-O-trinitrophenyl-ATP. The role of Hip as a molecular chaperone has been confirmed by its ability to strongly bind to the reduced, carboxymethylated form of alpha-lactalbumin. This interaction is specific for non-native domains since native alpha-lactalbumin fails to interact with Hip. Fluorescence-anisotropy measurements indicate that reduced, carboxymethylated lactalbumin binds Hip with a Kd of 5 microM. Although Hip appears to be able to bind nucleotides and non-native proteins, it is unable to facilitate the refolding of two denatured proteins, E. coli alkaline phosphatase and mitochondrial malate dehydrogenase. Hip inhibited the refolding of alkaline phosphatase and malic dehydrogenase. Inhibition occurred at near stoichiometric levels of Hip and could not be reversed by the addition of ATP. These results suggest that Hip may regulate the function of the Hsp70 molecular chaperone complex in vivo and play a critical role in protein folding in the eukaryotic cytoplasm.
Adenosine Triphosphate↗
In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent.
Interaction of artificial lipid bilayers (liposomes) with the purified transit peptide (SS-tp) of the precursor form of the small subunit for ribulose-2,5-bisphosphate carboxylase/oxygenase (prSSU) has been studied using a vesicle-disruption assay (calcein dye release) and electron microscopy. Employing purified forms of Escherichia coli-expressed prSSU, mature small subunit, glutathione S-transferase-transit peptide fusion protein, and SS-tp in dye release studies demonstrated that lipid interaction is mediated primarily through the transit peptide. Using chemically synthesized peptides (20-mers), the lipid-interacting domain of the transit peptide was partially mapped to the C-terminal 20 amino acids of the transit peptide. Peptides corresponding to other regions of the transit peptide and control peptides promoted significantly less calcein release. Interaction between the transit peptide and the bilayer was very rapid and could not be resolved by stopped-flow fluorometry with a mixing time of <50 ms. Interaction between the peptides and bilayer was also lipid class-dependent. Disruption occurred only when the bilayer contained the galactolipid monogalactosyldiacylglycerol (MGDG). The extent of bilayer disruption directly correlated with the relative concentration of MGDG in the liposome, with maximum calcein release occurring in 20 mol % MGDG liposomes. Lipid bilayers with greater than 20 mol % MGDG could not be achieved as determined by calcein entrapment. Electron microscopy of the liposomes before and after addition of the transit peptide suggested that the transit peptide induced a dramatic reorganization of lipids. These results are discussed in light of a possible mechanism for the early steps in protein transport that may involve polymorphic changes in the envelope membrane organization to include localized non-bilayer HII structures.
Amino Acid Sequence↗
Isolation of a carotenoid-containing sub-membrane particle from the chloroplastic envelope outer membrane of pea (Pisum sativum).
Chloroplastic envelope membranes isolated from pea (Pisum sativum) leaves are rich in carotenoids, containing approximately 2 micrograms of carotenoid mg-1 protein. We report here that envelopes can be surfactant-solubilized while maintaining association of carotenoids with protein components of the membrane. Treatment of isolated chloroplastic envelope membranes with 0.5% Deriphat 160 (N-lauryl-beta-imminodipropionate) causes general solubilization but preserves an envelope sub-membrane fragment which is fractionated by centrifugation in a sucrose gradient and by chromatography on a column of DEAE-Sephacel. The isolated submembrane complex contained five major proteins with M(r) values equivalent to 75,000, 36,000, 34,000 17,500, and 14,500. Spectroscopic and chromatographic analyses revealed that the complex contains violaxanthin and at least one other carotenoid. Carotenoid content of the fractionated complex was estimated as 4.8 micrograms mg-1 protein. Immunoblot analysis reveals that the constituent proteins of this complex are derived from the chloroplastic outer envelope membrane. These data suggest that at least some of the carotenoids of the chloroplastic envelope may be organized by apoproteins.
Carotenoids↗
Biosynthesis of the chloroplast cytochrome b6f complex: studies in a photosynthetic mutant of Lemna.
The biosynthesis of the cytochrome b6f complex has been studied in a mutant, no. 1073, of Lemna perpusilla that contained less than 1% of the four protein subunits when compared with a wild-type strain. RNA gel blot analyses of the mutant indicated that the chloroplast genes for cytochrome f, cytochrome b6, and subunit IV (petA, petB, and petD, respectively) are transcribed and that the petB and petD transcripts undergo their normal processing. Analysis of polysomal polyA+ RNA indicated that the level of translationally active mRNA for the nuclear-encoded Rieske Fe-S protein (petC) was reduced by greater than 100-fold in the mutant. Immunoprecipitation of in vivo labeled proteins indicated that both cytochrome f and subunit IV are synthesized and that subunit IV has a 10-fold higher rate of protein turnover in the mutant. These results are discussed in terms of the assembly of the cytochrome complex and the key role of the Rieske Fe-S protein in this process.
Chloroplasts↗
Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus.
Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.
Chlamydomonas↗