The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties

The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens. Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila. Immunofluorescence data show that human TRAP1 is localized to mitochondria. This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins. Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone. TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60). Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state. However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin. In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol. Thus, TRAP1 has functions that are distinct from those of hsp90.     

Function of the maize mitochondrial chaperonin hsp60: specific association between hsp60 and newly synthesized F1-ATPase alpha subunits.

Mitochondria contain a protein, hsp60, that is induced by heat shock and has been shown to function as a chaperonin in the assembly of mitochondrial enzyme complexes composed of proteins encoded by nuclear genes and imported from the cytosol. To determine whether products of mitochondrial genes are also assembled through an interaction with hsp60, we looked for association between hsp60 and proteins synthesized by isolated mitochondria. We have determined by electrophoretic, centrifugal, and immunological assays that at least two of those proteins become physically associated with hsp60. In mitochondrial matrix extracts, this association could be disrupted by the addition of Mg-ATP. One of the proteins that formed a stable association with hsp60 was the alpha subunit of the multicomponent complex F1-ATPase. We have not identified the other protein. These results indicate that hsp60 can function in the folding and assembly of mitochondrial proteins encoded by both mitochondrial and nuclear genes.     

Molecular cloning of a Chinese hamster mitochondrial protein related to the "chaperonin" family of bacterial and plant proteins

The complete cDNA sequence of a mitochondrial protein from Chinese hamster ovary cells, designated P1, which was originally identified as a microtubule-related protein (Gupta, R.S., Ho, T.K.W., Moffat, M.R.K., and Gupta, R. (1982) J. Biol. Chem. 257, 1071-1078), has been determined. The P1 cDNA encodes a protein of 60,983 Da including a 26-amino acid putative mitochondrial targeting sequence at its N-terminal end. The deduced amino acid sequence of Chinese hamster P1 shows 97% identity to the human P1 protein. Most interestingly, the amino acid sequences of mammalian P1 proteins show extensive sequence homology (42-60% identical residues and an additional 15-25% conservative replacements) to the "chaperonin" family of bacterial, yeast, and plant proteins (viz. groEL protein of Escherichia coli, hsp 60 protein of yeast, and ribulose-1,5-bisphosphate carboxylase subunit binding protein of plant chloroplasts) and to the 60-65-kDa major antigenic protein of mycobacteria and Coxiella burnetii. The homology between mammalian P1 and other proteins begins after the putative mitochondrial presequence and extends up to the C-terminal end. Furthermore, similar to the chaperonin family of proteins, P1 appears to exist in cells as a homooligomeric complex of seven subunits and shows ATPase activity. These observations strongly indicate that P1 protein is a member of the chaperonin family and that it may be involved in a similar function in mammalian cells.     

The mitochondrial F1ATPase alpha-subunit is necessary for efficient import of mitochondrial precursors.

The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.     

Intramitochondrial sorting of the precursor to yeast cytochrome c oxidase subunit Va

We have continued our studies on the import pathway of the precursor to yeast cytochrome c oxidase subunit Va (pVa), a mitochondrial inner membrane protein. Previous work on this precursor demonstrated that import of pVa is unusually efficient, and that inner membrane localization is directed by a membrane-spanning domain in the COOH- terminal third of the protein. Here we report the results of studies aimed at analyzing the intramitochondrial sorting of pVa, as well as the role played by ancillary factors in import and localization of the precursor. We found that pVa was efficiently imported and correctly sorted in mitochondria prepared from yeast strains defective in the function of either mitochondrial heat shock protein (hsp)60 or hsp70. Under identical conditions the import and sorting of another mitochondrial protein, the precursor to the beta subunit of the F1 ATPase, was completely defective. Consistent with previous results demonstrating that the subunit Va precursor is loosely folded, we found that pVa could be efficiently imported into mitochondria after translation in wheat germ extracts. This results suggests that normal levels of extramitochondrial hsp70 are also not required for import of the protein. The results of this study enhance our understanding of the mechanism by which pVa is routed to the mitochondrial inner membrane. They suggest that while the NH2 terminus of pVa is exposed to the matrix and processed by the matrix metalloprotease, the protein remains anchored to the inner membrane before being assembled into a functional holoenzyme complex.     

A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins

The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix- targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt- hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt- hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.     

Hsp60-independent protein folding in the matrix of yeast mitochondria.

Proteins that are imported from the cytosol into mitochondria cross the mitochondrial membranes in an unfolded conformation and then fold in the matrix. Some of these proteins require the chaperonin hsp60 for folding. To test whether hsp60 is required for the folding of all imported matrix proteins, we monitored the folding of four monomeric proteins after import into mitochondria from wild-type yeast or from a mutant strain in which hsp60 had been inactivated. The four precursors included two authentic matrix proteins (rhodanese and the mitochondrial cyclophilin Cpr3p) and two artificial precursors (matrix-targeted variants of dihydrofolate reductase and barnase). Only rhodanese formed a tight complex with hsp60 and required hsp60 for folding. The three other proteins folded efficiently without, and showed no detectable binding to, hsp60. Thus, the mitochondrial chaperonin system is not essential for the folding of all matrix proteins. These data agree well with earlier in vitro studies, which had demonstrated that only a subset of proteins require chaperones for efficient folding.     

A heat shock protein complex isolated from rabbit reticulocyte lysate can reconstitute a functional glucocorticoid receptor-Hsp90 complex.

When unliganded glucocorticoid receptor that has been stripped free of associated proteins is incubated with rabbit reticulocyte lysate, the receptor becomes associated with the 70- and 90-kDa heat shock proteins (hsp70 and hsp90), and the untransformed state of the receptor is functionally reconstituted [Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., & Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400]. Recently, an hsp70-containing protein complex (200-250 kDa) purified from rabbit reticulocyte lysate was shown to maintain a fusion protein bearing the mitochondrial matrix-targeting signal in a state that is competent for mitochondrial import [Sheffield, W. P., Shore, G. C., & Randall, S. K. (1990) J. Biol. Chem. 265, 11069-11076]. In this work, we show that this partially purified mitochondrial import-competent fraction contains both hsp90 and hsp70. When the purified fraction is immunoadsorbed with a monoclonal antibody specific for hsp90, a significant portion of the hsp70 is co-immunoadsorbed, suggesting that hsp90 and hsp70 are present together as a complex. The partially purified fraction maintains a hybrid precursor protein containing the mitochondrial matrix-targeting signal of rat pre-ornithine carbamyl transferase in an import-competent state. Incubation of immunopurified glucocorticoid receptor with this fraction of reticulocyte lysate results in ATP-dependent association of the receptor with both hsp70 and hsp90, and the resulting complexes are functional as assessed by return of the receptor to the high-affinity steroid binding conformation. The glucocorticoid receptor hetero-complex reconstituting activity of the lysate fraction is low relative to its mitochondrial import activity. Importantly, however, this is the first demonstration of the functional and structural reconstitution of the untransformed state of any steroid receptor utilizing a partially purified system.     

Regulation of heat shock protein 90 ATPase activity by sequences in the carboxyl terminus.

Hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with a k(cat) of 0.02 min(-1) and a K(m) greater than 300 microm. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10-100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1-573, was found to be monomeric, and yet the catalytic efficiency (k(cat)/K(m)) was greater than 100 times that of the full-length protein (k(cat) of 0.24 min(-1) and K(m) of 60 microm). In contrast, an internal deletion mutant, Delta661-677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to full-length hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis.     

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.     

Yeast genomic clones encoding polypeptides immunologically related to an 18 kDa subunit of mitochondrial ATP synthase

A yeast genomic library in the bacteriophage expression vector lambda gt11 was screened with a polyclonal anti-holo-ATPase antiserum resulting in the isolation of 54 immunoreactive clones. Four of these phage clones express in bacteria a polypeptide antigenically related to an 18 kDa subunit (P18) of the yeast mitochondrial ATPase complex. Molecular analysis of the yeast DNA inserts in these phage clones revealed two classes of yeast DNA that share little homology at the nucleotide sequence level and therefore may represent distinct separate genes. The polypeptides potentially encoded by these yeast DNA segments do show scattered short blocks of strong amino acid sequence homology, which may underlie the observed immunochemical relatedness between the proteins expressed in bacteria.     

A protein inhibitor of mitochondrial adenosine triphosphatase (F1) from Saccharomyces cerevisiae.

A heat-stable protein has been detected in Saccharomyces cerevisiae which inhibits mitochondrial ATPase activity. The protein inhibitor has been isolated from extracts prepared by brief heat treatment of unbroken cell suspensions. The isolated inhibitor is a small basic protein (molecular weight close to 7000, isoelectric proint 9.05) devoid of tryptophan, tyrosine, and cysteine as well as proline. The NHP2-terminal amino acid is serine. The ultraviolet absorption spectrum shows the vibrational fine structure of the phenyl-alanine band. Like the ATPase inhibitor from bovine heart mitochondria the yeast inhibitor is rapidly destroyed by trypsin. It is also inactivated by the yeast proteinases A and B. Radioimmunological analysis indicates that the inhibitor is synthesized on cytoplasmic ribosomes. Its accumulation seems to be connected to the formation of the mitochondrial ATPase complex, since its specific activity is greatly reduced both in extracts obtained from the F1-ATPase-deficient nuclear mutant pet 936 and from the cytoplasmic petite mutant D 273-10B-1.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.     

A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein.

We have probed the environment of a precursor protein stuck in mitochondrial import sites using cleavable bifunctional crosslinking reagents. The stuck precursor was crosslinked to a 70 kd protein which, by immunological techniques, was shown to be a matrix protein. The protein was purified to homogeneity by ATP-Sepharose chromatography and partially sequenced. Fourteen of its 15 N-terminal amino acids were identical to residues 24-38 of the protein encoded by the nuclear gene SSC1, which had been proposed to encode a dnaK-like 70 kd mitochondrial stress protein. Our data imply that this mitochondrial hsp70 is made with a cleavable matrix-targeting sequence composed of 23 residues. The complex containing stuck precursor, mitochondrial hsp70, and ISP42 could be solubilized from mitochondria by the non-ionic detergent Triton X-100 even without crosslinking, suggesting tight association of these three components. As the stuck precursor is arrested at an early stage of translocation, mitochondrial hsp70 may initiate the events that lead to refolding of imported precursors in the matrix space.     

ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae

The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon. Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis. Recently, putative roles for hsp60 and hsp70-like proteins have been proposed in Saccharomyces cerevisiae. hsp70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix. In this paper, we report that a brief temperature shock (44 degrees C) impairs coupling of oxidative phosphorylation in S. cerevisiae as measured indirectly by the Cl-CCP/oligomycin assay. Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions. Pretreatment of cells for a short period of time at 37 degrees C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport. Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity. We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.     

The plasma membrane of yeast acquires a novel heat-shock protein (hsp30) and displays a decline in proton-pumping ATPase levels in response to both heat shock and the entry to stationary phase.

Recent studies have revealed that the action of the proton-translocating ATPase of the plasma membrane of yeast is an important determinant of several stress tolerances and affects the capacity of cells to synthesise heat shock proteins in response to heat shock [Panaretou, B. & Piper, P. W. (1990) J. Gen. Microbiol. 136, 1763-1770; Coote, P. J., Cole, M. B. & Jones, M. V. (1991) J. Gen. Microbiol. 137, 1701-1708]. This study investigated the changes to the protein composition of the Saccharomyces cerevisiae plasma membrane that result from a heat shock to dividing cultures and the entry to stationary growth caused by carbon source limitation. Plasma membranes were prepared from exponential, heat-shocked and stationary yeast cultures. The proteins of these membrane preparations were then analysed by polyacrylamide gel electrophoresis and immunoblot measurement of ATPase levels. The protein composition of plasma membranes displayed two prominent changes in response to both heat shock and the entry to stationary phase: (a) a reduction in the level of the plasma membrane ATPase; and (b) the acquisition of a previously uncharacterised 30 kDa heat-shock protein (hsp30). The ATPase decline with heat shock probably exerts an important influence over the ability of the cell to maintain ATPase activity, and therefore intracellular pH, during extended periods of stress. Through in vivo pulse-labelling of plasma membrane proteins synthesised before and during heat shock, followed by subcellular fractionation, it was shown that hsp30 is the only protein induced by the yeast heat-shock response that substantially copurifies with plasma membranes. It might therefore exert a stress-protective function specifically at this membrane.     

SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein.

SSC1 is an essential member of the yeast HSP70 multigene family (E. Craig, J. Kramer, and J. Kosic-Smithers, Proc. Natl. Acad. Sci. USA 84:4156-4160, 1987). Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton protein that is more similar to DnaK, an Escherichia coli hsp70 protein, than other yeast hsp70s whose sequences have been determined. Ssc1p was found to have an amino-terminal extension of 28 amino acids, in comparison with either Ssa1p, another hsp70 yeast protein, or Dnak. This putative leader is rich in basic and hydroxyl amino acids, characteristic of many mitochondrial leader sequences. Ssc1p that was synthesized in vitro could be imported into mitochondria and was cleaved in the process. The imported protein comigrated with an abundant mitochondrial protein that reacted with hsp70-specific antibodies. We conclude that Ssc1p is a mitochondrial protein and that hsp70 proteins perform functions in many compartments of the cell.