Identification of a 66 KDa protein associated with yeast mitochondrial ATP synthase as heat shock protein hsp60

A 66 kDa protein, denoted P66, not hitherto classified as an integral component of yeast mitochondrial ATPase, is often observed in preparations of this enzyme complex. A physical association exists between P66 and the assembled ATPase complex since both components are coimmunoprecipitated by anti-F1 beta monoclonal antibody. Two recombinant clones expressing proteins immunologically similar to P66 were isolated from a yeast genomic library in lambda gt11 by screening with a polyclonal anti-holo-ATPase antibody. Based on restriction site mapping and partial nucleotide sequence analysis, both clones encompass the gene encoding the yeast heat shock protein hsp60. The identification of P66 with hsp60, taken together with its demonstrated association with the mitochondrial ATPase complex, is consistent with recent suggestions that hsp60 is involved in assembly of the ATP synthase complex.     

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10.

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.     

cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock

Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric proteins encoded by both nuclear and mitochondrial genes. We have isolated and sequenced full-length complementary DNAs coding for this mitochondrial chaperonin in Arabidopsis thaliana and Zea mays. Southern-blot analysis indicates the presence of a single hsp60 gene in the genome of A. thaliana. There is a high degree of homology at the predicted amino acid levels (43 to 60%) between plant HSP60s and their homologues in prokaryotes and other eukaryotes which indicates that these proteins must have similar evolutionarily conserved functions in all organisms. Northern- and western-blot analyses indicate that the expression of the hsp60 gene is developmentally regulated during seed germination. It is also heat-inducible. Developmental regulation of the (-subunit) of F₁-ATPase, an enzyme complex that is involved in the cyanide-sensitive mitochondrial electron transport system, indicates that imbibed embryos undergo rapid mitochondrial biogenesis through the early stages of germination. Based on the functional role of HSP60 in macromolecular assembly, these data collectively suggest that the presence of higher levels of HSP60 is necessary during active mitochondrial biogenesis, when the need for this protein is greatest in assisting the rapid assembly of the oligomeric protein structures.     

Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins.

We have created yeast strains in which the mitochondrial chaperonin, hsp60, can be either physically depleted or functionally inactivated. Cells completely depleted of hsp60 stop growing but retain for awhile the capacity to reaccumulate hsp60. While this newly made hsp60 is targeted to and processed correctly within the mitochondrion, assembly of a functional hsp60 complex does not occur. Rather, the hsp60 monomers are localized in different-size soluble complexes containing another mitochondrial chaperone, the mitochondrial form of hsp70. A number of other mitochondrial matrix-targeted proteins synthesized in the absence of functional hsp60 are imported into mitochondria but often show some buildup of precursor forms and, unlike hsp60, accumulate as insoluble aggregates. By contrast, several mitochondrial proteins normally targeted to the intermembrane space show normal processing in the complete absence of a functional hsp60 complex. Similar and complementary results were obtained when we examined the metabolism of matrix- and intermembrane space-localized proteins in cells expressing three different temperature-sensitive alleles of HSP60. In all cases, matrix-targeted proteins synthesized at nonpermissive (i.e., hsp60-inactivating) temperatures were correctly targeted to and processed within mitochondria but accumulated predominantly or totally as insoluble aggregates. The metabolism of two intermembrane space proteins, cytochrome b2 and cytochrome c1, was unaffected at the nonpermissive temperature, as judged by the correct processing and complete solubility of newly synthesized forms of both proteins. These findings are discussed with regard to current models of intermembrane targeting.     

Synaptophysin-containing microvesicles transport heat-shock protein hsp60 in insulin-secreting beta cells

58–62 kDa heat-shock proteins (hsp60) are molecular chaperonins involved in the process of protein folding, transmembrane translocation and assembly of oligomeric protein complexes. In eukaryotic cells hsp60 proteins have been found in mitochondria and chloroplasts. However, we have recently documented that, in addition to mitochondria, a hsp60-like protein is present in secretory granules of insulin-secreting beta cells. The pathway by which hsp60 is targeted to secretory granules was unknown. Here we report the existence of microvesicles involved in the transport of hsp60 protein. Immunoelectron microscopy of serial thin-sections of beta cells directly visualized stages associated with hsp60 delivery: attachment of microvesicles to a secretory granule, fusion with the secretory granule membrane and release of hsp60 molecules. Further biochemical and immunological analysis of microvesicles revealed the presence in their membrane of synaptophysin, a major component of synaptic-like microvesicles (SLMV) of neuroendocrine cells. Double immunogold labelling with antibodies to synaptophysin and hsp60 demonstrated co-localization of both proteins in the same microvesicles. Moreover, fusion of synaptophysin-positive microvesicles leaves synaptophysin incorporated, at least transiently, to secretory granule membranes. These findings suggest that, in beta cells, synaptic-like vesicles are involved in the transport and delivery of hsp60 and represent a novel pathway for protein transport and secretion.     

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

The hsp60B gene of Drosophila melanogaster is essential for the spermatid individualization process

The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts. The Hsp60 proteins promote proper protein folding by preventing aggregation. In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis. In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility. In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation. At these stages, mitochondria also displayed a differentiation process similar to wild-types. However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process. In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst. Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails. The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria. Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.     

The heat shock response in hydra: immunological relationship of hsp60, the major heat shock protein of Hydra vulgaris,to the ubiquitous hsp70 family

The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins.     

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.     

The Drosophila melanogaster homologue of the hsp60 gene is encoded by the essential locus l(1)10Ac and is differentially expressed during fly development

 The hsp60 (heat-shock protein 60) gene family of molecular chaperones has been a subject of study in numerous systems due to its important role in the correct folding of non-native proteins in development as well as after heat-shock treatment. Here we present the characterization of the first Drosophila hsp60 homologue. Drosophila HSP60 is most closely related (72% identity across the entire protein sequence) to the mouse mitochondrial HSP60. Western blot experiments indicate that Drosophila HSP60 is enriched in the mitochondrial fraction. The distribution of HSP60 protein is dynamic during fly embryogenesis, suggesting that various cell types might have different HSP60 requirements. The molecular analysis of a P-element-induced mutation that affects the l(1)10Ac locus shows that the transposon is inserted in a 3-kb intron present in the hsp60 gene. By genetic rescue experiments we prove that Drosophila HSP60 is encoded by the essential locus l(1)10Ac opening the possibility for detailed genetic analysis of HSP60 functions in the fly. Received: 24 March 1997 / Accepted: 16 June 1997     

Co-operative binding of hsp60 may promote transfer from hsp70 and correct folding of imported proteins in mitochondria [corrected]

I propose that a molecular chaperone hsp60 binds to and dissociates from the unfolded polypeptide or folding intermediate in a positively co-operative manner, but another chaperone hsp70 shows no such co-operativity. This could simply explain the fact that the protein newly imported in the mitochondrial matrix is transferred from hsp70 to hsp60 promotes correct folding of the substrate protein while hsp70 does not.     

The involvement of p23, hsp90, and immunophilins in the assembly of progesterone receptor complexes

To better understand the assembly mechanism for the progesterone receptor (PR), we have developed cell-free systems for studying interactions of PR, hsp90, and other associated proteins. When PR is incubated in rabbit reticulocyte lysate, its association with hsp90, hsp70, the three immunophilins FKBP54, FKBP52 and CyP-40, and with p23 is observed. These interactions require ATP/Mg²⁺ and when ATP is limiting the PR complex is altered to one containing the proteins p60 and p48, but lacking immunophilins and p23. We have studied two pre-formed hsp90 complexes that may participate in the assembly of PR complexes. One contains hsp90 bound to hsp70 and p60 and this complex forms spontaneously in the absence of ATP. A second complex contains hsp90 bound to p23 plus the three immunophilins and some hsp70. The formation of this complex requires ATP. In further studies we have shown that purified hsp90 can bind to purified p23 and this interaction requires both ATP and molybdate. This explains, in part, the known effects of ATP and molybdate on assembly of PR complexes.     

Molecular Comparison of the Mitochondrial and Cytoplasmic hsp 70 of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major

ABSTRACT. We compared the expression and localization of the mitochondrial and cytoplasmic hsp70 of the protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. The mitochondrial protein is encoded by multiple mRNA in all species, while the cytoplasmic protein is encoded by a single mRNA. In all three species, the mitochondrial hsp70 is concentrated in the kinetoplast, a submitochondrial structure that houses the unusual DNA (kDNA) that characterizes this group of organisms, while the cytoplasmic protein is distributed throughout the cell. These results suggest that, in all kinetoplastid species, mt-hsp70 has a specific function in kDNA biology, possibly in the processes of kDNA replication, RNA editing or kinetoplast structure.     

A mitochondrial chaperonin: genetic, biochemical, and molecular characteristics

Mitochondria contain a matrix-localized protein complex composed of subunits homologous to the E.coli protein groEL. As with groEL in E.coli, the nuclear gene coding for the mitochondrial protein is essential for cell survival and the accumulation of the protein is elevated at heat shock-inducing temperatures. Biochemical analyses of wild type and mutant yeast strains have shown that this protein, hsp60, is required for the correct folding and assembly of newly imported, mitochondrially-targeted proteins. There is evidence suggesting a mandatory interaction between hsp60 and many imported, as well as mitochondrially-synthesized, proteins. The implications of these and other data are discussed.     

Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix

We previously reported that hsp70 in the mitochondrial matrix (mt-hsp 70 = Ssclp) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70. We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.     

Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels.

Martinez, J., Perez-Serrano, J., Bernadina, W. E., Rodriguez-Caabeiro, F. 1999 Echinococcus granulosus: In vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. Experimental Parasitology93, 171-180. Organisms or cells exposed to injurious stresses such as heat shock or chemicals respond by increased (or altered) expression of heat-shock proteins (HSPs). Conversely, an earlier exposure to stress can prepare cells to cope with a subsequent more severe stress. In the present study, protoscolices of Echinococcus granulosus were subjected to several anthelmintic treatments, involving storage of the protoscolices for 18, 30, and 50 h with 0.1 mg/ml of ivermectin (IV), praziquantel (PZ), and a combination of each with albendazole (ALB). The organisms were analyzed for the effects of drug treatment on cell integrity and on levels of hsp60 and hsp70 production. Drug efficacy was evaluated by microscopy and by protein content measurement. Hsp60 and hsp70 were detected by Western blotting and incubation with anti-hsp60 and anti-hsp70 antibody, respectively, and quantitation of these proteins was obtained using image analysis. Incubation with IV alone produced the most damage to the protoscolices as indicated by viability loss, decreased protein content, and altered hsp60 and hsp70 levels; incubation with IV + ALB produced less damage as manifested by fewer changes in the aforementioned damage parameters but PZ and PZ + ALB, in this context, were poor anthelmintics. Exposure of protoscolices to thermal stress prior to anthelmintic treatment, in most cases, increased drug efficacy. It is concluded that in the E. granulosus model system drug efficacy is associated with decreased levels of hsp70 expression and increased levels of hsp60 expression.