Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana

The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.     

Novel proteins interacting with peroxisomal protein receptor PEX7 in Arabidopsis thaliana

Peroxisomal matrix protein transport relies on 2 cytosolic receptors, PEX5 and PEX7, which import peroxisomal targeting signal type 1 (PTS1) and PTS2-containing proteins, respectively. To better understand the transport mechanism of PEX7, we isolated PEX7 complexes using proteomics. We identified PEX5 as well as PTS1- and PTS2-containing proteins within the complex, thereby confirming the interaction between PEX5 and PEX7 during cargo transport that had been previously characterized by biochemical approaches. In addition, a chaperone T-complex and 2 small Rab GTPases were identified. We recently reported that the RabE1c is involved in the degradation of the PEX7 when abnormal PEX7 is accumulated on the peroxisomal membrane. This study expands our knowledge on the transport machinery via PEX7 by identifying both known and novel PEX7-interacting proteins and thus is helpful for further investigation of the regulation of the peroxisomal protein receptor during its translocation.     

The Hsp90 family of proteins in Arabidopsis thaliana

The 90-kDa heat shock protein (Hsp90) is an essential molecular chaperone in eukaryotic cells, with key roles in the folding and activation of proteins involved in signal transduction and control of the cell cycle. A search for Hsp90 sequences in the Arabidopsis thaliana genome revealed that this family includes 7 members. The AtHsp90-1 through AtHsp90-4 proteins constitute the cytoplasmic subfamily, whereas the AtHsp90-5, AtHsp90-6, and AtHsp90-7 proteins are predicted to be within the plastidial, mitochondrial, and endoplasmic reticulum compartments, respectively. The deduced amino acid sequences of each of the cytoplasmic proteins contains the highly conserved C-terminal pentapeptide MEEVD. All of the AtHsp90 sequences include a conserved adenosine triphosphate-binding domain, whereas only the cytoplasmic and endoplasmic reticulum-resident sequences include an adjacent charged linker domain that is common in mammalian and yeast sequences. The occurrence of multiple AtHsp90 proteins in the cytoplasm and of family members in other subcellular compartments suggests a range of specific functions and target polypeptides.     

Alternative processing of Arabidopsis Hsp70 precursors during protein import into chloroplasts

During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.     

Stress and development phenotyping of Hsp101 and diverse other Hsp mutants of Arabidopsis thaliana

Heat shock proteins or Hsps are critical in mounting plant resistance against heat stress. The complex Hsp spectrum of Arabidopsis thaliana plant contains over two hundred proteins belonging to six different families namely Hsp20, Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100. Importantly, the cellular function(s) of most Hsps remains to be established. We aimed at phenotyping of stress and development response of the selected, homozygous hsp mutant lines produced by T-DNA insertional mutagenesis method. The heat stress phenotype was assessed for basal and acquired heat stress response at seed and seedling stages. Distinct phenotype was noted for the hot1-3 mutant (knockout mutant of Hsp101 gene) showing higher heat sensitivity and for the salk_087844 mutant (knockout mutant of Hsc70-2 gene) showing higher heat tolerance than the wild type seedlings. The homozygous cs808162 mutant (mutant of ClpB-p gene encoding for the chloroplast-localized form of Hsp101) did not survive even under unstressed, control condition. salk_064887C mutant (mutant of cpn60β4 gene) showed accelerated development cycling. The hot1-3 mutant apart from showing different heat response, exhibited development lesions like bigger size of seeds, buds, siliques, and pollen compared to the wild type plants. In response to controlled deterioration treatment of seeds, hot1-3 seeds showed higher accumulation of reactive oxygen species molecules, higher rates of protein and lipid oxidation and a faster decline in germination rate as compared to wild type seeds. Our findings show that Hsps perform diverse metabolic functions in plant response to stress, growth, and development.

     

Modeling Hsp70-mediated protein folding

The Hsp70 chaperone system is the major molecular chaperone system that assists protein-folding processes in all cells. To understand these processes, we analyzed the kinetic characteristics of the Escherichia coli homologs of this chaperone system during folding of a denatured protein using computer simulations and compared the results with in vitro refolding experiments. Rate constants used for the model were derived from recent literature or were determined and scrutinized for their applicability to the refolding reaction. Our simulation results are consistent with reported laboratory experiments, not only simulating the refolding reaction of wild-type proteins but also the behavior of mutant variants. Variation of kinetic parameters and concentrations of components of the Hsp70 system demonstrate the robustness of the chaperone system in assisting protein folding. Furthermore, the importance of the synergistic stimulation of the ATPase activity of Hsp70 is demonstrated. The limitations of our kinetic model indicate sore spots in our understanding of this chaperone system. Our model provides a platform for further research on chaperone action and the mechanism of chaperone-assisted refolding of denatured proteins.     

Redox control of Hsp70-Co-chaperone interaction revealed by expression of a thioredoxin-like Arabidopsis protein

By using a yeast functional complementation assay, we have identified AtTDX, a new Arabidopsis thaliana gene, encoding a two-domain 42-kDa protein. The amino-terminal domain of AtTDX is closely related to the co-chaperone Hsp70-interacting protein HIP, whereas its carboxyl-terminal part contains a thioredoxin domain. Both in vivo and in vitro assays showed that AtTDX is a protein-disulfide reductase. We next found that the HIP domain of AtTDX is capable of interacting with the ATPase domain of Ssb2, a yeast heat-shock protein 70 chaperone. Strikingly, the AtTDX-Ssb2 interaction can be released under oxidative stress, a redox-dependent regulation involving the thioredoxin activity of AtTDX. A mutation inactivating the cysteine 20 of the ATPase domain of Ssb2 was found to stabilize the AtTDX-Ssb2 interaction that becomes redox-insensitive. As cysteine 20 is conserved in virtually all the Hsp70 chaperones, our results suggest that this residue might be more generally the target of redox regulations of chaperone binding activity.     

N-Ethylmaleimide-modified Hsp70 inhibits protein folding.

Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.     

Structural studies of the Hsp70/Hsp90 organizing protein of Plasmodium falciparum and its modulation of Hsp70 and Hsp90 ATPase activities

HOP is a cochaperone belonging to the foldosome, a system formed by the cytoplasmic Hsp70 and Hsp90 chaperones. HOP acts as an adapter protein capable of transferring client proteins from the first to the second molecular chaperone. HOP is a modular protein that regulates the ATPase activity of Hsp70 and Hsp90 to perform its function. To obtain more detailed information on the structure and function of this protein, we produced the recombinant HOP of Plasmodium falciparum (PfHOP). The protein was obtained in a folded form, with a high content of α-helix secondary structure. Unfolding experiments showed that PfHOP unfolds through two transitions, suggesting the presence of at least two domains with different stabilities. In addition, PfHOP primarily behaved as an elongated dimer in equilibrium with the monomer. Small-angle X-ray scattering data corroborated this interpretation and led to the reconstruction of a PfHOP ab initio model as a dimer. Finally, the PfHOP protein was able to inhibit and to stimulate the ATPase activity of the recombinant Hsp90 and Hsp70–1, respectively, of P. falciparum. Our results deepened the knowledge of the structure and function of PfHOP and further clarified its participation in the P. falciparum foldosome.     

Metazoan Hsp70 machines use Hsp110 to power protein disaggregation

Accumulation of aggregation‐prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP‐dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70‐Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70‐Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP‐driven chaperoning is not required. Knock‐down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat‐induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70‐Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress.     

Lipid interacting regions in phosphate stress glycosyltransferase atDGD2 from Arabidopsis thaliana

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227-245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130-148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227-245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.     

Hsp70 chaperone machine remodels protein aggregates at the initial step of Hsp70-Hsp100-dependent disaggregation

Exposure to temperatures over a certain limit leads to massive protein aggregation in the cell. Disaggregation of such aggregates is largely dependent on the Hsp100 and Hsp70 chaperones. The exact role of the Hsp70 chaperone machine (composed of DnaK, DnaJ, and GrpE) in the Hsp100-dependent process remains unknown. In this study we focused on the Hsp70 role at the initial step of the disaggregation process. Two different aggregated model substrates, green fluorescent protein (GFP) and firefly luciferase, were incubated with the Hsp70 machine resulting in efficient fragmentation of large aggregates into smaller ones. Our data suggest that the observed fragmentation is achieved first by extraction of polypeptides from aggregates in Hsp70 chaperone machine-dependent manner and not by direct fragmentation of large aggregates. In the absence of Hsp100 (ClpB) these "extracted" polypeptides were not able to fold properly and promptly reassociated into new aggregates. The extracted GFP molecules were efficiently recognized and sequestered by a molecular trap, the mutant GroEL D87K, which binds stably to unfolded but not to native polypeptides. The binding of extracted GFP molecules to the GroEL trap prevented their reaggregation. We propose that the Hsp70 machine disentangles polypeptides from protein aggregates prior to Hsp100 action.     

Successive and synergistic action of the Hsp70 and Hsp100 chaperones in protein disaggregation

Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.