The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin.     

Disruption of the gene for Hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in import of proteins into mitochondria

The gene for Hsp30, the only known alpha-crystallin-related heat shock protein of Neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. Hsp30, which is not synthesized at 30 degrees C, associates reversibly with the mitochondria at high temperature (45 degrees C). In this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired at high temperature in a strain mutant in Hsp30. After 70 min of cell incubation at 45 degrees C, most matrix, inner membrane, and intermembrane-space proteins tested were reduced in import by about 50-70% in the mutant, as compared to wild-type cells. In contrast, assembly of selected proteins into the outer mitochondrial membrane was not reduced, except for one component of the preprotein translocase complex of the mitochondrial outer membrane. Three proteins of this complex co-immunoprecipitated with Hsp30 of wild-type cells incubated at 45 degrees C. We propose that Hsp30 interacts with the preprotein translocase of the mitochondrial outer membrane and that it chaperones the activity of one or more components of this translocase complex at high temperature.     

The Hsp40 molecular chaperone Ydj1p, along with the protein kinase C pathway, affects cell-wall integrity in the yeast Saccharomyces cerevisiae

Molecular chaperones, such as Hsp40, regulate cellular processes by aiding in the folding, localization, and activation of multi-protein machines. To identify new targets of chaperone action, we performed a multi-copy suppressor screen for genes that improved the slow-growth defect of yeast lacking the YDJ1 chromosomal locus and expressing a defective Hsp40 chimera. Among the genes identified were MID2, which regulates cell-wall integrity, and PKC1, which encodes protein kinase C and is linked to cell-wall biogenesis. We found that ydj1delta yeast exhibit phenotypes consistent with cell-wall defects and that these phenotypes were improved by Mid2p or Pkc1p overexpression or by overexpression of activated downstream components in the PKC pathway. Yeast containing a thermosensitive allele in the gene encoding Hsp90 also exhibited cell-wall defects, and Mid2p or Pkc1p overexpression improved the growth of these cells at elevated temperatures. To determine the physiological basis for suppression of the ydj1delta growth defect, wild-type and ydj1delta yeast were examined by electron microscopy and we found that Mid2p overexpression thickened the mutant's cell wall. Together, these data provide the first direct link between cytoplasmic chaperone function and cell-wall integrity and suggest that chaperones orchestrate the complex biogenesis of this structure.     

Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria

Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E. coli GroEL. Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin. To define the in vivo role of the co- chaperonin, we have used the genetic and biochemical potential of the yeast S. cerevisiae. The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated. Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function. Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space. The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro. The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature.     

The protein chaperone Ssa1 affects mRNA localization to the mitochondria

Many nuclear-transcribed mRNAs encoding mitochondrial proteins are localized near the mitochondrial outer membrane. A yet unresolved question is whether protein synthesis is important for transport of these mRNAs to their destination. Herein we present a connection between mRNA localization in yeast and the protein chaperone Ssa1. Ssa1 depletion lowered mRNA association with mitochondria while its overexpression increased it. A genome-wide analysis revealed that Ssa proteins preferentially affect mRNAs encoding hydrophobic proteins, which are expected targets for these protein chaperones. Importantly, deletion of the mitochondrial receptor Tom70 abolished the impact of Ssa1 overexpression on mRNAs encoding Tom70 targets. Taken together, our results suggest a role for Ssa1 in mediating localization of nascent peptide-ribosome-mRNA complexes to the mitochondria, consistent with a co-translational transport process.     

SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.     

The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica

Ultrastructural analysis of Entamoeba histolytica reveals that this intestinal human pathogen lacks recognizable mitochondria, but the presence in its genome of genes encoding proteins of mitochondrial origin suggests the existence of a mitochondrially derived compartment. We have cloned the full-length E. histolytica gene encoding one such protein, chaperonin CPN60, and have characterized its structure and expression. Using an affinity-purified antibody raised against recombinant protein, we have localized native E. histolytica CPN60 to a previously undescribed organelle of putative mitochondrial origin, the mitosome. Most cells contain only one mitosome, as determined by immunofluorescence studies. Entamoeba histolytica CPN60 has an amino-terminal extension reminiscent of known mitochondrial and hydrogenosomal targeting signals. Deletion of the first 15 amino acids of CPN60 leads to an accumulation of the truncated protein in the cytoplasm. However, this mutant phenotype can be reversed by replacement of the deleted amino acids with a mitochondrial targeting signal from Trypanosoma cruzi HSP70. The observed functional conservation between mitochondrial import in trypanosomes and mitosome import in Entamoeba is strong evidence that the E. histolytica organelle housing chaperonin CPN60 represents a mitochondrial remnant.     

A cell model to study different degrees of Hsp60 deficiency in HEK293 cells

Mitochondrial dysfunction is associated with neurodegenerative diseases and mutations in the HSPD1 gene, encoding the mitochondrial Hsp60 chaperone, are the causative factors of two neurodegenerative diseases, hereditary spastic paraplegia and MitChap60 disease. In cooperation with Hsp10, Hsp60 forms a barrel-shaped complex, which encloses unfolded polypeptides and provides an environment facilitating folding. We have generated an Hsp60 variant with a mutation (Asp423Ala) in the ATPase domain and established a stable human embryonic kidney (HEK293) cell line allowing tetracycline-controlled expression of this mutant variant. We monitored expression of the Hsp60–Asp423Ala variant protein following induction and examined its effects on cellular properties. We showed that the folding of mitochondrial-targeted green fluorescent protein, a well-known substrate protein of Hsp60, was consistently impaired in cells expressing Hsp60–Asp423Ala. The level of the Hsp60–Asp423Ala variant protein increased over time upon induction, cell proliferation stopped after 48-h induction and mitochondrial membrane potential decreased in a time-dependent manner. In summary, we have established a stable cell line with controllable expression of an Hsp60 variant, which allows detailed studies of different degrees of Hsp60 deficiency.     

The molecular anatomy of human Hsp60 and its similarity with that of bacterial orthologs and acetylcholine receptor reveal a potential pathogenetic role of anti-chaperonin immunity in myasthenia gravis

Heat-shock protein 60 (Hsp60) is ubiquitous and highly conserved being present in eukaryotes and prokaryotes, including pathogens. This chaperonin, although typically a mitochondrial protein, can also be found in other intracellular sites, extracellularly, and in circulation. Thus, it can signal the immune system and participate in the development of inflammation and immune reactions. Both phenomena can be elicited by human and foreign Hsp60 (e.g., bacterial GroEL), when released into the blood by infectious agents. Consequently, all these Hsp60 proteins become part of a complex autoimmune response characterized by multiple cross reactions because of their structural similarities. In this study, we demonstrate that Hsp60 proteins from humans and two common pathogens, Chlamydia trachomatis and Chlamydia pneumoniae, share various sequence segments of potentially highly immunogenic epitopes with acetylcholine receptor α1 subunit (AChRα1). The structural data indicate that AChRα1 antibodies, implicated in the pathogenesis of myasthenia gravis, could very well be elicited and/or maintained by self- and/or bacterial Hsp60.     

Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica

Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC. 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic.     

The Hsp60-(p.V98I) mutation associated with hereditary spastic paraplegia SPG13 compromises chaperonin function both in vitro and in vivo

We have previously reported the association of a mutation (c.292G > A/p.V98I) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin with a dominantly inherited form of hereditary spastic paraplegia. Here, we show that the purified Hsp60-(p.V98I) chaperonin displays decreased ATPase activity and exhibits a strongly reduced capacity to promote folding of denatured malate dehydrogenase in vitro. To test its in vivo functions, we engineered a bacterial model system that lacks the endogenous chaperonin genes and harbors two plasmids carrying differentially inducible operons with human Hsp10 and wild-type Hsp60 or Hsp10 and Hsp60-(p.V98I), respectively. Ten hours after shutdown of the wild-type chaperonin operon and induction of the Hsp60-(p.V98I)/Hsp10 mutant operon, bacterial cell growth was strongly inhibited. No globally increased protein aggregation was observed, and microarray analyses showed that a number of genes involved in metabolic pathways, some of which are essential for robust aerobic growth, were strongly up-regulated in Hsp60-(p.V98I)-expressing bacteria, suggesting that the growth arrest was caused by defective folding of some essential proteins. Co-expression of Hsp60-(p.V98I) and wild-type Hsp60 exerted a dominant negative effect only when the chaperonin genes were expressed at relatively low levels. Based on our in vivo and in vitro data, we propose that the major effect of heterozygosity for the Hsp60-(p.V98I) mutation is a moderately decreased activity of chaperonin complexes composed of mixed wild-type and Hsp60-(p.V98I) mutant subunits.     

STU1, a suppressor of a beta-tubulin mutation, encodes a novel and essential component of the yeast mitotic spindle

We have isolated a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in S. cerevisiae, that confers a specific defect in spindle microtubule function. At 14 degrees C, tub2-406 cells lack a normal bipolar spindle but do assemble functional cytoplasmic microtubules. In an attempt to identify proteins that are important for spindle assembly, we screened for suppressors of the cold-sensitivity of tub2-406 and obtained four alleles of a novel gene, STU1. Genetic interactions between stu1 alleles and alleles of TUB1 and TUB2 suggest that Stu1p specifically interacts with microtubules. STU1 is essential for growth and disruption of STU1 causes defects in spindle assembly that are similar to those produced by the tub2-406 mutation. The nucleotide sequence of the STU1 gene predicts a protein product of 174 kD with no significant similarity to known proteins. An epitope-tagged Stulp colocalizes with microtubules in the mitotic spindle of yeast. These results demonstrate that Stulp is an essential component of the yeast mitotic spindle.     

Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60

SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.     

The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction: humans versus Escherichia coli

Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a beta-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the beta-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.     

Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48 degrees C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50 degrees C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30 degrees C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, Delta clpB or Delta ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.