AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.     

Tid1, the Mammalian Homologue of Drosophila Tumor Suppressor Tid56, Mediates Macroautophagy by Interacting with Beclin1-containing Autophagy Protein Complex

One of the fundamental functions of molecular chaperone proteins is to selectively conjugate cellular proteins, targeting them directly to lysosome. Some of chaperones, such as the stress-induced Hsp70, also play important roles in autophagosome-forming macroautophagy under various stress conditions. However, the role of their co-chaperones in autophagy regulation has not been well defined. We here show that Tid1, a DnaJ co-chaperone for Hsp70 and the mammalian homologue of the Drosophila tumor suppressor Tid56, is a key mediator of macroautophagy pathway. Ectopic expression of Tid1 induces autophagy by forming LC3+ autophagosome foci, whereas silencing Tid1 leads to drastic impairment of autophagy as induced by nutrient deprivation or rapamycin. In contrast, Hsp70 is dispensable for a role in nutrient deprivation-induced autophagy. The murine Tid1 can be replaced with human Tid1 in murine fibroblast cells for induction of autophagy. We further show that Tid1 increases autophagy flux by interacting with the Beclin1-PI3 kinase class III protein complex in response to autophagy inducing signal and that Tid1 is an essential mediator that connects IκB kinases to the Beclin1-containing autophagy protein complex. Together, these results reveal a crucial role of Tid1 as an evolutionarily conserved and essential mediator of canonical macroautophagy.     

Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.     

Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions

Heat shock protein 40s (Hsp40s) and heat shock protein 70s (Hsp70s) form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of a broad range of client proteins. While the Hsp40 family of proteins is highly diverse with multiple forms occurring in any particular cell or compartment, all its members are characterized by a J domain that directs their interaction with a partner Hsp70. Specific Hsp40-Hsp70 chaperone partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. The elucidation of the mechanism by which these specific Hsp40-Hsp70 partnerships are formed will greatly enhance our understanding of the way in which chaperone pathways are integrated into finely regulated protein folding networks. From in silico analyses, domain swapping and rational protein engineering experiments, evidence has accumulated that indicates that J domains contain key specificity determinants. This review will critically discuss the current understanding of the structural features of J domains that determine the specificity of interaction between Hsp40 proteins and their partner Hsp70s. We also propose a model in which the J domain is able to integrate specificity and chaperone activity.     

Multiple Molecules of Hsc70 and a Dimer of DjA1 Independently Bind to an Unfolded Protein

Protein folding is a prominent chaperone function of the Hsp70 system. Refolding of an unfolded protein is efficiently mediated by the Hsc70 system with either type 1 DnaJ protein, DjA1 or DjA2, and a nucleotide exchange factor. A surface plasmon resonance technique was applied to investigate substrate recognition by the Hsc70 system and demonstrated that multiple Hsc70 proteins and a dimer of DjA1 initially bind independently to an unfolded protein. The association rate of the Hsc70 was faster than that of DjA1 under folding-compatible conditions. The Hsc70 binding involved a conformational change, whereas the DjA1 binding was bivalent and substoichiometric. Consistently, we found that the bound ¹⁴C-labeled Hsc70 to the unfolded protein became more resistant to tryptic digestion. The gel filtration and cross-linking experiments revealed the predominant presence of the DjA1 dimer. Furthermore, the Hsc70 and DjA1 bound to distinct sets of peptide array sequences. All of these findings argue against the generality of the widely proposed hypothesis that the DnaJ-bound substrate is targeted and transferred to Hsp70. Instead, these results suggest the importance of the bivalent binding of DjA1 dimer that limits unfavorable transitions of substrate conformations in protein folding.     

Zuotin, a DnaJ molecular chaperone, stimulates cap-independent translation in yeast

A small inhibitor RNA (IRNA) isolated from yeast has previously been shown to efficiently block poliovirus and hepatitis C virus IRES-mediated translation by sequestering mammalian RNA-binding (transacting) factors that play important roles in cap-independent translation. Here we have investigated the IRNA-binding proteins that might be involved in cap-independent translation in the yeast Saccharomyces cerevisiae. We have identified Zuotin, a DnaJ chaperone protein similar to mammalian HSP-40 chaperone, which interacts strongly with IRNA. Using ZUO1-deleted S. cerevisiae, we demonstrate a preferential requirement of Zuo1p for cap-independent translation mediated by the 5' untranslated region of the yeast TFIID mRNA. Further studies using zuo1delta S. cerevisiae complemented with various Zuo1p mutants indicate that the DnaJ domain of Zuo1p, known to influence its interaction with HSP-70, significantly affects cap-independent translation. These results demonstrate for the first time a role for an established chaperone protein in cap-independent translation of a cellular mRNA.