Content
2011
The phosphorylation state of chloroplast transit peptides regulates preprotein import
15.12.2011
Plant Signaling & Behavior,
2011,
http://dx.doi.org/10.4161/psb.6.12.18127,
Volume 6, Issue 12, Pages 1918 - 1920
published on 15.12.2011
Plant Signaling & Behavior, online article
Plant Signaling & Behavior, online article
Import of nuclear encoded proteins into chloroplast is an essential and well-regulated mechanism. The cytosolic kinases STY8, STY17 and STY46 have been shown to phosphorylate chloroplast preprotein transit peptides advantaging the binding of a 14-3-3 dimer. Analyses of sty8 sty17 sty46 mutant plants revealed a role for the kinases in chloroplast differentiation, possibly due to lack of transit peptide phosphorylation. Moreover we could show that not only phosphorylation but also transit peptide dephosphorylation appears to be required for the fine regulation of the back-transport of nuclear encoded proteins to the chloroplast.
The plastid outer envelope protein OEP16 affects metabolic fluxes during ABA-controlled seed development and germination
09.12.2011
Journal of Experimental Botany,
2011,
doi: 10.1093/jxb/err375,
Vol. 63, No. 5, pp. 1919–1936
published on 09.12.2011
Journal of Experimental Botany, online article
Journal of Experimental Botany, online article
Previously, the OEP16.1 channel pore in the outer envelope membrane of mature pea (Pisum sativum) chloroplasts in vitro has been characterized to be selective for amino acids. Isolation of OEP16.2, a second OEP16 isoform from pea, in the current study allowed membrane localization and gene expression of OEP16 to be followed throughout seed development and germination of Arabidopsis thaliana and P. sativum. Thereby it can be shown on the transcript and protein level that the isoforms OEP16.1 and OEP16.2 in both plant species are alternating: whereas OEP16.1 is prominent in early embryo development and first leaves of the growing plantlet, OEP16.2 dominates in late seed development stages, which are associated with dormancy and desiccation, as well as early germination events. Further, OEP16.2 expression in seeds is under control of the phytohormone abscisic acid (ABA), leading to an ABA-hypersensitive phenotype of germinating oep16 knockout mutants. In consequence, the loss of OEP16 causes metabolic imbalance, in particular that of amino acids during seed development and early germination. It is thus concluded that in vivo OEP16 most probably functions in shuttling amino acids across the outer envelope of seed plastids.
The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively
25.11.2011
Plant Molecular Biology,
2011,
DOI: 10.1007/s11103-011-9856-z,
published on 25.11.2011
Plant Molecular Biology, online article
Plant Molecular Biology, online article
Calmodulin (CaM) is a ubiquitous sensor/ transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized
in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of
peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.
How kinesin-2 forms a stalk
15.11.2011
Mol. Biol. Cell,
2011,
doi: 10.1091/mbc.E11-02-0112,
vol. 22 no. 22 4279-4287
published on 15.11.2011
Mol. Biol. Cell, online article
Mol. Biol. Cell, online article
The heterotrimeric structure of kinesin-2 makes it a unique member of the kinesin superfamily; however, molecular details of the oligomer formation are largely unknown. Here we demonstrate that heterodimerization of the two distinct motor domains KLP11 and
KLP20 of Caenorhabditis elegans kinesin-2 requires a dimerization seed of merely two heptads at the C terminus of the stalk. This heterodimeric seed is sufficient to promote dimerization along the entire length of the stalk, as shown by circular dichroism spectroscopy, Förster resonance energy transfer analysis, and electron microscopy. In addition to explaining the formation of the kinesin-2 stalk, the seed sequence identified here bears great potential for generating specific heterodimerization in other protein biochemical applications.
Vibralactone as a Tool to Study the Activity and Structure of the ClpP1P2 Complex from Listeria monocytogenes
11.11.2011
Angewandte Chemie,
2011,
DOI: 10.1002/ange.201104391,
Volume 50, Issue 46, pages 11001–11004
published on 11.11.2011
Nature provides a rich source of bioactive compounds comprising a diverse set of electrophilic core structures that are poised to react with corresponding nucleophilic residues such as serine and cysteine in enzyme active sites.These residues are usually relevant for catalysis and therefore display fine-tuned reactivity towards their dedicated substrates.We and others previously investigated the dedicated targets of monocyclic b-lactones which turned out to be
potent and selective inhibitors of diverse disease-associated enzyme classes. Covalent inhibition of the caseinolytic peptidase ClpP, for instance, resulted in a dramatic attenuation of bacterial virulence. ClpP is an important, highly conserved heat shock protein with additional regulatory functions in many pathogens. Some organisms such as Listeria monocytogenes genetically encode for two functionally and structurally uncharacterized ClpP isoforms (ClpP1 and ClpP2). So far, all b-lactones were reported to target
solely ClpP2 and not ClpP1, raising the question whether monocyclic lactones lack suitable reactivity to interact with the ClpP1 active-site nucleophile.
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Arabidopsis calcium-binding mitochondrial carrier proteins as potential facilitators of mitochondrial ATP-import and plastid SAM-import
02.11.2011
FEBS Letters,
2011,
doi:10.1016/j.febslet.2011.10.039,
Volume 585, Issue 24 , Pages 3935-3940,
published on 02.11.2011
FEBS Letters, online article
FEBS Letters, online article
Chloroplasts and mitochondria are central to crucial cellular processes in plants and contribute to a whole range of metabolic pathways. The use of calcium ions as a secondary messenger in and around organelles is increasingly appreciated as an important mediator of plant cell signaling, enabling plants to develop or to acclimatize to changing environmental conditions. Here, we have studied the four calcium-dependent mitochondrial carriers that are encoded in the Arabidopsis genome. An unknown substrate carrier, which was previously found to localize to chloroplasts, is proposed to present a calcium-dependent S-adenosyl methionine carrier. For three predicted ATP/phosphate carriers, we present experimental evidence that they can function as mitochondrial ATP-importers.
Firefly luciferase mutants as sensors of proteome stress
04.09.2011
Nature Methods,
2011,
doi:10.1038/nmeth.1697,
8, 879–884,
published on 04.09.2011
Nature Methods, online article
Nature Methods, online article
Maintenance of cellular protein homeostasis (proteostasis) depends on a complex network of molecular chaperones, proteases and other regulatory factors. Proteostasis deficiency develops during normal aging and predisposes individuals for many diseases, including neurodegenerative disorders. Here we describe sensor proteins for the comparative measurement of proteostasis capacity in different cell types and model organisms. These sensors are increasingly structurally destabilized versions of firefly luciferase. Imbalances in proteostasis manifest as changes in sensor solubility and luminescence activity. We used EGFP-tagged constructs to monitor the aggregation state of the sensors and the ability of cells to solubilize or degrade the aggregated proteins.
A set of three sensor proteins serves as a convenient toolkit to assess the proteostasis status in a wide range of experimental systems, including cell and organism models of stress, neurodegenerative disease and aging.
Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53
04.09.2011
Nature Structural & Molecular Biology,
2011,
doi:10.1038/nsmb.2114,
published on 04.09.2011
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
In eukaryotes, the essential dimeric molecular chaperone Hsp90 is required for the activation and maturation of specific substrates such as steroid hormone receptors, tyrosine kinases and transcription factors. Hsp90 is involved in the establishment of cancer and has become an attractive target for drug design. Here we present a structural characterization of the complex between Hsp90 and the tumor suppressor p53, a key mediator of apoptosis whose structural integrity is crucial for cell-cycle control. Using biophysical methods, we show that the human p53 DNA-binding domain interacts with multiple domains of yeast Hsp90. p53 binds to the Hsp90 C-terminal domain in its native-like state in a charge-dependent manner, but it also associates weakly with binding sites in the middle and the N-terminal domains. The fine-tuned interplay between several Hsp90 domains provides the interactions required for efficient chaperoning of p53.
The Cytosolic Kinases STY8, STY17, and STY46 Are Involved in Chloroplast Differentiation in Arabidopsis
28.07.2011
Plant Physiology,
2011,
doi: http://dx.doi.org/10.1104/pp.111.182774,
Vol. 157, pp. 70–85,
published on 28.07.2011
Plant Physiology, online article
Plant Physiology, online article
In Arabidopsis (Arabidopsis thaliana), transit peptides for chloroplast-destined preproteins can be phosphorylated by the protein kinases STY8, STY17, and STY46. In this study, we have investigated the in vitro properties of these plant-specific kinases. Characterization of the mechanistic functioning of STY8 led to the identification of an essential threonine in the activation segment, which is phosphorylated by an intramolecular mechanism. STY8 is inhibited by specific tyrosine kinase inhibitors, although it lacked the ability to phosphorylate tyrosine residues in vitro. In vivo analysis of sty8, sty17, and sty46 Arabidopsis knockout/knockdown mutants revealed a distinct function of the three kinases in the greening process and in the efficient differentiation of chloroplasts. Mutant plants displayed not only a delayed accumulation of chlorophyll but also a reduction of nucleus-encoded chloroplast proteins and a retarded establishment of photosynthetic capacity during the first 6 h of deetiolation, supporting a role of cytosolic STY kinases in chloroplast differentiation.
Protein Import into Chloroplasts: Dealing with the (Membrane) Integration Problem
21.06.2011
ChemBioChem,
2011,
DOI: 10.1002/cbic.201100118,
Volume 12, Issue 11, pages 1655–1661,
published on 21.06.2011
ChemBioChem, online article
ChemBioChem, online article
Due to their endosymbiotic origin, chloroplasts are completely reliant on post-translational import of their resident proteins.1 Integral membrane proteins comprise approximately 20 % of the total estimated chloroplast proteome.2, 3 Based on several proteomic analyses, 20 % of all membrane proteins are located in the inner envelope (IE).4–6 Judging from the protein pattern on an SDS-PAGE gel, it seems there are fewer proteins in the outer envelope (OE) than in the IE.7 In light of the great importance of membrane proteins in many essential chloroplast processes, it appears puzzling that not much is known about the mechanisms of their targeting and import. Considering that in chloroplasts an additional membrane system, the thylakoids, is present and makes coordinated targeting and insertion even more complicated, reliable sorting becomes of vital importance.
Cytosolic HSP90 Cochaperones HOP and FKBP Interact with Freshly Synthesized Chloroplast Preproteins of Arabidopsis
18.05.2011
Molecular Plant,
2011,
doi: 10.1093/mp/ssr037,
Volume 4, Number 6, Pages 1133–1145,
published on 18.05.2011
Molecular Plant, online article
Molecular Plant, online article
Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing protein aggregation of freshly translated preproteins and assist in maintaining the preproteins in an import competent state. Preproteins can associate with HSP70, HSP90, and 14–3–3 proteins in the cytosol. In this study, we analyzed a large set of wheat germ-translated chloroplast preproteins with respect to their chaperone binding. Our results demonstrate that the formation of distinct 14–3–3 or HSP90 containing preprotein complexes is a common feature in post-translational protein transport in addition to preproteins that seem to interact solely with HSP70. We were able to identify a diverse and extensive class of preproteins as HSP90 substrates, thus providing a tool for the investigation of HSP90 client protein association. The analyses of chimeric HSP90 and 14–3–3 binding preproteins with exchanged transit peptides indicate an involvement of both the transit peptide and the mature part of the proteins, in HSP90 binding. We identified two partner components of the HSP90 cycle, which were present in the preprotein containing high-molecular-weight complexes, the HSP70/HSP90 organizing protein HOP, as well as the immunophilin FKBP73. The results establish chloroplast preproteins as a general class of HSP90 client proteins in plants using HOP and FKBP as novel cochaperones.
Technical note: Brassica rapa: a new model plant for the isolation of chloro-plasts and biochemical studies
01.05.2011
Endocytobiosis and Cell Research,
2011,
21,
52 - 58
published on 01.05.2011
Journal of Endocytobiosis and Cell Research, online article
Journal of Endocytobiosis and Cell Research, online article
A major problem for chloroplast research remains that plant species which are utilized in order to obtain a high amount of isolated intact chloroplasts are not sequenced on a genomic base. Thus it is difficult to combine the data from biochemical experiments with genomic data and employ techniques such as MS to identify low abundant proteins. There are plant species like Arabidopsis thaliana with a completely available genome but the yield of chloroplasts compared to plants like Pisum sativum is comparatively low. Brassica rapa subsp. pekinensis, commonly known as chinese or napa cabbage, is a fast growing plant with a high yield of biomass. In addition, it has a small genome which will soon be completed and available for the plant science community (http://www.brassicarapa.org). The availability of a complete genome sequence and its close kinship to Arabidopsis thaliana makes this plant an interesting new model organism. In this paper we show that a high yield of pure intact chloroplasts can be obtained from 4 weeks old leave material making the plant suitable for biochemical studies. In addition we demonstrate that protoplasts can be obtained and used for PEG mediated transient expression of GFPtagged proteins allowing for experiments such as the investigation of protein localisation.
Total Chemical Synthesis of an Integral Membrane Enzyme: Diacylglycerol Kinase from Escherichia coli
18.04.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201006686,
Volume 50, Issue 17, pages 3988–3992,
published on 18.04.2011
Angew. Chemie, online article
Angew. Chemie, online article
Recent progress in chemical protein synthesis has provided access to many small to medium-sized proteins. However, the highly important class of membrane proteins comprising multimembrane-spanning receptors and ion channels as well as integral membrane enzymes remains elusive with regard to chemical synthesis. Only certain moderately sized membrane proteins have been generated by chemical protein synthesis or semisynthesis. Synthesis of the constituent hydrophobic membrane-spanning peptide segments remains challenging owing to incomplete amino acid coupling steps and subsequent purification problems. Herein we report the chemical synthesis of Escherichia coli diacylglycerol kinase (DAGK),
an integral membrane enzyme consisting of monomers with 121 amino acids each that form a homotrimer in a membrane environment as well as in detergent micelles. These homotrimers catalyze the conversion of diacylglycerol into phosphatidic acid and play a vital role in the lipid metabolism of Gram-negative bacteria, especially under conditions of environmental stress.
Closing In on the Hsp90 Chaperone-Client Relationship
13.04.2011
The molecular chaperone Hsp90 regulates the activity and stability of a set of client proteins. Despite prog- ress in understanding its mechanism, the interaction of Hsp90 with clients has remained enigmatic. Now, in a recent issue of Molecular Cell, Street and coworkers present results that integrate the client in the Hsp90 chaperone cycle.
Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction
16.02.2011
Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its Alpha-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chainmodifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that Alpha-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association.
Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptidebinding activity
24.01.2011
Protein Science,
2011,
DOI: 10.1002/pro.592,
VOL 20:588—596
published on 24.01.2011
Protein Science, online article
Protein Science, online article
Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI‐aba′c) where the b′ domain was partially deleted, thus abolishing peptide binding but maintaining a PDI‐like redox potential. PDI‐aba′c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI‐aba′c isomerizes non‐native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.
Reduction of disulphide bonds unmasks potent antimicrobial activity of human Beta-defensin 1
19.01.2011
Nature,
2011,
doi:10.1038/nature09674,
Nature Volume: 469, Pages: 419–423
published on 19.01.2011
Nature, online article
Nature, online article
Human epithelia are permanently challenged by bacteria and
fungi, including commensal and pathogenic microbiota. In the
gut, the fraction of strict anaerobes increases from proximal to
distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of 2200mV to –300mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at
mucosal surfaces. Human Beta-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous
expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Grampositive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.
Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions
09.01.2011
Nature Strucrural & Molecular Biology,,
2011,
doi:10.1038/nsmb.1970,
Pages:150–158 (2011)
published on 09.01.2011
Nature Strucrural & Molecular Biology, online article
Nature Strucrural & Molecular Biology, online article
The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain–binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered CH1 1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.
