Content
2012
Folding of large multidomain proteins by partial encapsulation in the chaperonin TRiC/CCT
26.12.2012
PNAS,
2012,
doi/10.1073/pnas.1218836109,
vol. 109 no. 52, 21208–21215
published on 26.12.2012
PNAS, online article
PNAS, online article
The eukaryotic chaperonin, TRiC/CCT (TRiC, TCP-1 ring complex; CCT, chaperonin containing TCP-1), uses a built-in lid to mediate protein folding in an enclosed central cavity. Recent structural data suggest an effective size limit for the TRiC folding chamber of ∼70 kDa, but numerous chaperonin substrates are substantially larger. Using artificial fusion constructs with actin, an obligate chaperonin substrate, we show that TRiC can mediate folding of large proteins by segmental or domain-wise encapsulation. Single or multiple protein domains up to ∼70 kDa are stably enclosed by stabilizing the ATP-hydrolysis transition state of TRiC. Additional domains, connected by flexible linkers that pass through the central opening of the folding chamber, are excluded and remain accessible to externally added protease. Experiments with the physiological TRiC substrate hSnu114, a 109-kDa multidomain protein, suggest that TRiC has the ability to recognize domain boundaries in partially folded intermediates. In the case of hSnu114, this allows the selective encapsulation of the C-terminal ∼45-kDa domain and segments thereof, presumably reflecting a stepwise folding mechanism. The capacity of the eukaryotic chaperonin to overcome the size limitation of the folding chamber may have facilitated the explosive expansion of the multidomain proteome in eukaryotes.
Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail
26.12.2012
PNAS,
2012,
doi:10.1073/pnas.1214431110,
vol. 109, no. 52, 21348–21353
published on 26.12.2012
PNAS, online article
PNAS, online article
Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis-glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis-glutamine is replaced by a glutamine from RabGAP (trans-glutamine); this differs from the RasGAP mechanism, where the cis-glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans-arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans-glutamine and trans-arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases.
Arabidopsis thaliana Oxa proteins locate to mitochondria and fulfill essential roles during embryo development
21.11.2012
Planta,
2012,
DOI 10.1007/s00425-012-1793-9,
Volume 237, Issue 2, pp 573-588
published on 21.11.2012
Planta, online article
Planta, online article
Members of the Alb3/Oxa1/YidC protein family function as insertases in chloroplasts, mitochondria, and bacteria. Due to independent gene duplications, all organisms possess two isoforms, Oxa1 and Oxa2 except gram-negative bacteria, which encode only for one YidC-like protein. The genome of Arabidopsis thaliana however, encodes for eight different isoforms. The localization of three of these isoforms has been identified earlier: Alb3 and Alb4 located in thylakoid membranes of chloroplasts while AtOxa1 was found in the inner membrane of mitochondria. Here, we show that the second Oxa1 protein, Oxa1b as well as two Oxa2 proteins are also localized in mitochondria. The last two isoforms most likely encode truncated versions of Oxa-like proteins, which might be inoperable pseudogenes. Homozygous mutant lines were only obtained for Oxa1b, which did not reveal any significant phenotypes, while T-DNA insertion lines of Oxa1a, Oxa2a and Oxa2b resulted only in heterozygous plants indicating that these genes are indispensable for plant development. Phenotyping heterozygous lines showed that embryos are either retarded in growth, display an albino phenotype or embryo formation was entirely abolished suggesting that Oxa1a and both Oxa2 proteins function in embryo formation although at different developmental stages as indicated by the various phenotypes observed.
Characterization of Enzymes from Legionella pneumophila Involved in Reversible Adenylylation of Rab1 Protein
12.10.2012
Journal of Biological Chemistry,
2012,
doi: 10.1074/jbc.M112.396861,
287, 42, 35036-35046
published on 12.10.2012
Journal of Biological Chemistry, online article
Journal of Biological Chemistry, online article
After the pathogenic bacterium Legionella pneumophila is phagocytosed, it injects more than 250 different proteins into the cytoplasm of host cells to evade lysosomal digestion and to replicate inside the host cell. Among these secreted proteins is the protein DrrA/SidM, which has been shown to modify Rab1b, a main regulator of vesicular trafficking in eukaryotic cells, by transfer of adenosine monophosphate (AMP) to Tyr77. In addition, Legionella provides the protein SidD that hydrolytically reverses the covalent modification, suggesting a tight spatial and temporal control of Rab1 function by Legionella during infection. Small angle x-ray scattering experiments of DrrA allowed us to validate a tentative complex model built by combining available crystallographic data. We have established the effects of adenylylation on Rab1 interactions and properties in a quantitative way. In addition, we have characterized the kinetics of DrrA-catalyzed adenylylation as well as SidD-catalyzed deadenylylation toward Rab1 and have determined the nucleotide specificities of both enzymes. This study enhances our knowledge of proteins subverting Rab1 function at the Legionella-containing vacuole.
Nucleus-Encoded Light-Harvesting Chlorophyll a/b Proteins are Imported Normally into Chlorophyll b-free Chloroplasts of Arabidopsis
06.10.2012
Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a post-translational manner. Among these nuclear-encoded chloroplast proteins are the light-harvesting chlorophyll a/b-binding proteins (LHCPs). After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes were they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO (chlorophyll a oxigenase) which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators we performed import reactions utilizing a homozygous loss-of-function mutant (cao-1). We imported in vitro translated and 35S-labeled precursor proteins of LHCB1, LHCB4 and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms is not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of LHC proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down regulated. Additionally, reduced steady-state levels of LHCPs do also not occur due to post-translational turn-over of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.
The first alpha-helical domain of the vesicle-inducing protein in plastids 1 promotes oligomerization and lipid binding
02.10.2012
The vesicle-inducing protein in plastids 1 (Vipp1) is an essential component for thylakoid biogenesis in cyanobacteria and chloroplasts. Vipp1 proteins share significant structural similarity with their evolutionary ancestor PspA (bacterial phage shock protein A), namely a predominantly α-helical structure, the formation of oligomeric high molecular weight complexes (HMW-Cs) and a tight association with membranes. Here, we elucidated domains of Vipp1 from Arabidopsis thaliana involved in homo-oligomerization as well as association with chloroplast inner envelope membranes. We could show that the 21 N-terminal amino acids of Vipp1, which form the first α-helix of the protein, are essential for assembly of the 2 MDa HMW-C but are not needed for formation of smaller subcomplexes. Interestingly, removal of this domain also interferes with association of the Vipp1 protein to the inner envelope. Fourier transform infrared spectroscopy of recombinant Vipp1 further indicates that Escherichia coli lipids bind tightly enough that they can be co-purified with the protein. This feature also depends on the presence of the first helix, which strongly supports an interaction of lipids with the Vipp1 HMW-C but not with smaller subcomplexes. Therefore, Vipp1 oligomerization appears to be a prerequisite for its membrane association. Our results further highlight structural differences between Vipp1 and PspA, which might be important in regard to their different function in thylakoid biogenesis and bacterial stress response, respectively.
Essential Role of VIPP1 in Chloroplast Envelope Maintenance in Arabidopsis
03.09.2012
The Plant Cell,
2012,
doi/10.1105/tpc.112.103606,
published on 03.09.2012
The Plant Cell, online article
The Plant Cell, online article
VESICLE-INDUCING PROTEIN IN PLASTIDS1 (VIPP1), proposed to play a role in thylakoid biogenesis, is conserved in photosynthetic organisms and is closely related to Phage Shock Protein A (PspA), which is involved in plasma membrane integrity in Escherichia coli. This study showed that chloroplasts/plastids in Arabidopsis thaliana vipp1 knockdown and knockout mutants exhibit a unique morphology, forming balloon-like structures. This altered morphology, as well as lethality of vipp1, was complemented by expression of VIPP1 fused to green fluorescent protein (VIPP1-GFP). Several lines of evidence show that the balloon chloroplasts result from chloroplast swelling related to osmotic stress, implicating VIPP1 in the maintenance of plastid envelopes. In support of this, Arabidopsis VIPP1 rescued defective proton leakage in an E. coli pspA mutant. Microscopy observation of VIPP1-GFP in transgenic Arabidopsis revealed that VIPP1 forms large macrostructures that are integrated into various morphologies along the envelopes. Furthermore, live imaging revealed that VIPP1-GFP is highly mobile when chloroplasts are subjected to osmotic stress. VIPP1-GFP showed dynamic movement in the transparent area of spherical chloroplasts, as the fluorescent molecules formed filament-like structures likely derived from disassembly of the large VIPP1 complex. Collectively, our data demonstrate that VIPP1 is a multifunctional protein in chloroplasts that is critically important for envelope maintenance.
AtTPR7 is a chaperone-docking protein of the Sec translocon in Arabidopsis
16.08.2012
Journal of Cell Science,
2012,
doi: 10.1242/jcs.111054,
125, 5196–5207
published on 16.08.2012
Journal of Cell Science, online article
Journal of Cell Science, online article
Chaperone-assisted sorting of post-translationally imported proteins is a general mechanism among all eukaryotic organisms. Interaction of some preproteins with the organellar membranes is mediated by chaperones, which are recognised by membrane-bound tetratricopeptide repeat (TPR) domain containing proteins. We have characterised AtTPR7 as an endoplasmic reticulum protein in plants and propose a potential function for AtTPR7 in post-translational protein import. Our data demonstrate that AtTPR7 interacts with the heat shock proteins HSP90 and HSP70 via a cytosol-exposed TPR domain. We further show by in vitro and in vivo experiments that AtTPR7 is associated with the Arabidopsis Sec63 homologue, AtERdj2. Interestingly, AtTPR7 can functionally complement a Δsec71 yeast mutant that is impaired in post-translational protein transport. These data strongly suggest that AtTPR7 not only has a role in chaperone binding but also in post-translational protein import into the endoplasmic reticulum, pointing to a general mechanism of chaperone-mediated post-translational sorting between the endoplasmic reticulum, mitochondria and chloroplasts in plant cells.
Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor
10.04.2012
PNAS,
2012,
doi: 10.1073/pnas.1121161109,
vol. 109 no. 15 5621-5626
published on 10.04.2012
PNAS, online article
PNAS, online article
Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane
extraction/delivery and cytosolic distribution of Rabs is
mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a
bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement
of GDI from Rab:GDI complexes. These activities have
been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of
reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.
Depletion of leaf-type ferredoxin-NADP+ oxidoreductase results in the permanent induction of photoprotective mechanisms in Arabidopsis chloroplasts
16.03.2012
The Plant Journal,
2012,
doi: 10.1111/j.1365-313X.2012.04930.x,
Volume 70, Issue 5, pages 809–817
published on 16.03.2012
The Plant Journal, online article
The Plant Journal, online article
Arabidopsis thaliana contains two photosynthetically competent chloroplast-targeted ferredoxin-NADP+ oxidoreductase (FNR) isoforms that are largely redundant in their function. Nevertheless, the FNR isoforms also display distinct molecular phenotypes, as only the FNR1 is able to directly bind to the thylakoid membrane. We report the consequences of depletion of FNR in the F1 (fnr1 × fnr2) and F2 (fnr1 fnr2) generation plants of the fnr1 and fnr2 single mutant crossings. The fnr1 × fnr2 plants, with a decreased total content of FNR, showed a small and pale green phenotype, accompanied with a marked downregulation of photosynthetic pigment-protein complexes. Specifically, when compared with the wild type (WT), the quantum yield of photosystem II (PSII) electron transport was lower, non-photochemical quenching (NPQ) was higher and the rate of P700+ re-reduction was faster in the mutant plants. The slight over-reduction of the plastoquinone pool detected in the mutants resulted in the adjustment of the reactive oxygen species (ROS) scavenging systems, as both the content and de-epoxidation state of xanthophylls, as well as the content of α-tocopherol, were higher in the leaves of the mutant plants when compared with the WT. The fnr1 fnr2 double mutant plants, which had no detectable FNR and possessed an extremely downregulated photosynthetic machinery, survived only when grown heterotrophically in the presence of sucrose. Intriguingly, the fnr1 fnr2 plants were still capable of sustaining the biogenesis of a few malformed chloroplasts.
Conformational Switching of the Molecular Chaperone Hsp90 via Regulated Phosphorylation
24.02.2012
Molecular Cell,
2012,
doi:10.1016/j.molcel.2011.12.031,
Volume 45, Issue 4, 517-528
published on 24.02.2012
Molecular Cell, online article
Molecular Cell, online article
Hsp90 is an essential molecular chaperone in the eukaryotic cytosol. Its function is modulated by cochaperones and posttranslational modifications. Importantly, the phosphatase Ppt1 is a dedicated regulator of the Hsp90 chaperone system. Little is known about Ppt1-dependent phosphorylation sites and how these affect Hsp90 activity. Here, we identified the major phosphorylation sites of yeast Hsp90 in its middle or the C-terminal domain and determined the subset regulated by Ppt1. In general, phosphorylation decelerates the Hsp90 machinery, reduces chaperone function in vivo, sensitizes yeast cells to Hsp90 inhibition and affects DNA repair processes. Modification of one particular site (S485) is lethal, whereas others modulate Hsp90 activity via distinct mechanisms affecting the ATPase activity, cochaperone binding and manipulating conformational transitions in Hsp90. Our mechanistic analysis reveals that phosphorylation of Hsp90 permits a regulation of the conformational cycle at distinct steps by targeting switch points for the communication of remote regions within Hsp90.
Identification of a Hypochlorite-specific Transcription Factor from Escherichia coli
24.02.2012
The Journal of Biological Chemistry,
2012,
doi: 10.1074/jbc.M111.287219,
287, 6892-6903
published on 24.02.2012
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
Hypochlorite is a powerful oxidant produced by neutrophils to kill invading microorganisms. Despite this important physiological role of HOCl in fighting bacterial infections, no hypochlorite-specific stress response has been identified yet. Here, we identified a hypochlorite-responsive transcription factor, YjiE, which is conserved in proteobacteria and eukaryotes. YjiE forms unusual dodecameric ring-like structures in vitro that undergo large DNA-induced conformational changes to form dimers and tetramers as shown by transmission electron microscopy and analytical ultracentrifugation. Such smaller oligomers are predominant in hypochlorite-stressed cells and are the active species as shown by fluorescence anisotropy and analytical ultracentrifugation. YjiE regulates a large number of genes upon hypochlorite stress. Among them are genes involved in cysteine, methionine biosynthesis, and sulfur metabolism (up-regulated) and genes involved in iron acquisition and homeostasis (down-regulated), thus supposedly replenishing oxidized metabolites and decreasing the hypochlorite-mediated amplification of intracellular reactive oxygen species. As a result, YjiE specifically confers hypochlorite resistance to E. coli cells. Thus, to our knowledge, YjiE is the first described hypochlorite-specific transcription factor.
Programmed cell death in Ricinus and Arabidopsis: the function of KDEL cysteine peptidases in development
17.02.2012
Physiologia Plantarum,
2012,
DOI: 10.1111/j.1399-3054.2012.01580.x,
Volume 145, Issue 1, pages 103–113
published on 17.02.2012
Physiologia Plantarum, online article
Physiologia Plantarum, online article
Programmed cell death (PCD) in plants is a prerequisite for development as well as seed and fruit production. It also plays a significant role in pathogen defense. A unique group of papain-type cysteine endopeptidases, characterized by a C-terminal endoplasmic reticulum (ER) retention signal (KDEL CysEP), is involved in plant PCD. Genes for these endopeptidases have been sequenced and analyzed from 25 angiosperms and gymnosperms. They have no structural relationship to caspases involved in mammalian PCD and homologs to this group of plant cysteine endopeptidases have not been found in mammals or yeast. In castor beans (Ricinus communis), the CysEP is synthesized as pre-pro-enzyme. The pro-enzyme is transported to the cytosol of cells undergoing PCD in ER-derived vesicles called ricinosomes. These vesicles release the mature CysEP in the final stages of organelle disintegration triggered by acidification of the cytoplasm resulting from the disruption of the vacuole. Mature CysEP digests the hydroxyproline (Hyp)-rich proteins (extensins) that form the basic scaffold of the plant cell wall. The KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated Hyp residues of the extensins. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2 and AtCEP3) are expressed in tissues undergoing PCD. In transgenic Arabidopsis plants expressing β-glucuronidase under the control of the promoters for these three genes, cell- and tissue-specific activities were mapped during seedling, flower and seed development. KDEL CysEPs participate in the collapse of tissues in the final stage of PCD and in tissue re-modeling such as lateral root formation.
Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins
03.02.2012
The EMBO Journal,
2012,
doi:10.1038/emboj.2012.16,
31, 1774 - 1784
published on 03.02.2012
The EMBO Journal, online article
The EMBO Journal, online article
The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.
Chloroplast-localized protein kinases: a step forward towards a complete inventory
25.01.2012
Journal of Experimental Botany,
2012,
doi: 10.1093/jxb/err377,
Vol. 63, No. 4, pp. 1713–1723,
published on 25.01.2012
Journal of Experimental Botany, online article
Journal of Experimental Botany, online article
In addition to redox regulation, protein phosphorylation has gained increasing importance as a regulatory principle in chloroplasts in recent years. However, only very few chloroplast-localized protein kinases have been identified to date. Protein phosphorylation regulates important chloroplast processes such as photosynthesis or transcription. In order to better understand chloroplast function, it is therefore crucial to obtain a complete picture of the chloroplast kinome, which is currently constrained by two effects: first, recent observations showed that the bioinformatics-based prediction of chloroplast-localized protein kinases from available sequence data is strongly biased; and, secondly, protein kinases are of very low abundance, which makes their identification by proteomics approaches extremely difficult. Therefore, the aim of this study was to obtain a complete list of chloroplast-localized protein kinases from different species. Evaluation of protein kinases which were either highly predicted to be chloroplast localized or have been identified in different chloroplast proteomic studies resulted in the confirmation of only three new kinases. Considering also all reports of experimentally verified chloroplast protein kinases to date, compelling evidence was found for a total set of 15 chloroplast-localized protein kinases in different species. This is in contrast to a much higher number that would be expected based on targeting prediction or on the general abundance of protein kinases in relation to the entire proteome. Moreover, it is shown that unusual protein kinases with differing ATP-binding sites or catalytic centres seem to occur frequently within the chloroplast kinome, thus making their identification by mass spectrometry-based approaches even more difficult due to a different annotation.
The role of calcium in chloroplasts—an intriguing and unresolved puzzle
08.01.2012
Protoplasma,
2012,
DOI 10.1007/s00709-011-0373-3,
published on 08.01.2012
Protoplasma, online article
Protoplasma, online article
More than 70 years of studies have indicated that chloroplasts contain a significant amount of calcium, are a potential storage compartment for this ion, and might themselves be prone to calcium regulation. Many of these studies have been performed on the photosynthetic light reaction as well as CO2 fixation via the Calvin–Benson–Bassham cycle, and they showed that calcium is required in several steps of these processes. Further studies have indicated that calcium is involved in other chloroplast functions that are not directly related to photosynthesis and that there is a calcium-dependent regulation similar to cytoplasmic calcium signal transduction. Nevertheless, the precise role that calcium has as a functional and regulatory component of chloroplast processes remains enigmatic. Calcium concentrations in different chloroplast subcompartments have been measured, but the extent and direction of intra-plastidal calcium fluxes or calcium transport into and from the cytosol are not yet very well understood. In this review we want to give an overview over the current knowledge on the relationship between chloroplasts and calcium and discuss questions that need to be addressed in future research.
The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop
06.01.2012
The EMBO Journal,
2012,
doi:10.1038/emboj.2011.472,
published on 06.01.2012
The EMBO Journal, online article
The EMBO Journal, online article
Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90- binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous a-helical folds as determined by NMR. These, and especially
DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A– TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal
domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1–DP1 module may serve as an Hsp70–client delivery system for the TPR2A–TPR2B–DP2 segment, which is required for client activation in vivo.
A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana
02.01.2012
Journal of Experimental Botany,
2012,
doi: 10.1093/jxb/err406,
63 (4): 1751-1761
published on 02.01.2012
Journal of Experimental Botany, online article
Journal of Experimental Botany, online article
Calcium has long been acknowledged as one of the most important signalling components in plants. Many abiotic and biotic stimuli are transduced into a cellular response by temporal and spatial changes in cellular calcium concentration and the calcium-sensitive protein aequorin has been exploited as a genetically encoded calcium indicator for the measurement of calcium in planta. The objective of this work was to generate a compatible set of aequorin expression plasmids for the generation of transgenic plant lines to measure changes in calcium levels in different cellular subcompartments. Aequorin was fused to different targeting peptides or organellar proteins as a means to localize it to the cytosol, the nucleus, the plasma membrane, and the mitochondria. Furthermore, constructs were designed to localize aequorin in the stroma as well as the inner and outer surface of the chloroplast envelope membranes. The modular set-up of the plasmids also allows the easy replacement of targeting sequences to include other compartments. An additional YFP-fusion was included to verify the correct subcellular localization of all constructs by laser scanning confocal microscopy. For each construct, pBin19-based binary expression vectors driven by the 35S or UBI10 promoter were made for Agrobacterium-mediated transformation. Stable Arabidopsis lines were generated and initial tests of several lines confirmed their feasibility to measure calcium signals in vivo.
