Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones.

We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import-stimulating factor (MSF). The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor-MSF-Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor-Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl. In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor-MSF-Tom70 complex to Tom20. The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex. Formation of the Tom20-precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl. Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal. Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence.     

Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt- hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.     

Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein.

In vitro mitochondrial import of the purified precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) is stimulated by a cytosolic factor(s) contained in rabbit reticulocyte lysate. A protein factor that binds to pOTC but not to mature OTC and was named presequence binding factor or PBF, was purified 91,000-fold from the lysate by affinity chromatography using pOTC-bound Sepharose, DEAE-5PW HPLC and sucrose gradient centrifugation. The purified PBF migrated as a single polypeptide of 50,000 daltons on SDS-PAGE. On sucrose gradients, urea-denatured pOTC sedimented to the bottom, whereas PBF sedimented with an S20,w value of 5.5S. When pOTC and PBF were centrifuged together, both polypeptides sedimented as a complex of 7.1S. Formation of the pOTC-PBF complex was inhibited by micromolar concentrations of the synthetic presequence of pOTC and those of other mitochondrial precursor proteins. The purified PBF markedly stimulated the import of purified or in vitro synthesized pOTC into the mitochondria. PBF-stimulated pOTC import was further enhanced by a 70 kd heat shock protein (hsp 70) purified from yeast; the hsp70 alone had little effect. Thus, PBF binds to the presequence portion of the precursors and may hold them in a transport-competent form in cooperation with hsp70.     

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.     

Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal.

Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a second cleavable sorting signal that functions in directing the precursor into the mitochondrial intermembrane space. The role of the second sorting sequence was analyzed by replacing one, two or all of the three positively charged amino acid residues which are present at the amino-terminal side of the hydrophobic core by uncharged residues or an acidic residue. With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the same time targeting to the matrix space occurred. The accumulation in the matrix depended on a high level of intramitochondrial ATP. At low levels of matrix ATP, the mutant proteins were sorted into the intermembrane space like the wild-type precursors. The results: (i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have reduced ability to interact with the recognition component(s) and then embark on the default pathway into the matrix by interacting with mitochondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue against a mechanism by which the hydrophobic segment of the sorting sequence stops translocation in the hydrophobic phase of the inner membrane.     

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.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

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.     

The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.

The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.     

Role of heat shock cognate 70 protein in import of ornithine transcarbamylase precursor into mammalian mitochondria.

The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfolding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein)-specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major form of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However, when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreased when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent form.     

Structure and expression of a human gene coding for a 71 kd heat shock ''cognate'' protein.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.     

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.     

A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites

Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.     

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.     

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 cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix

The cleavable prepiece of the precursor to yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) was attached to the amino-terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase.     

The cleavable pre-sequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria

The cleavable pre-sequences of imported chloroplast and mitochondrial proteins have several features in common. This structural similarity prompted us to test whether a chloroplast pre-sequence (`transit peptide') can also be decoded by the mitochondrial import machinery. In the green alga, Chlamydomonas reinhardtii, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (a chloroplast protein) is nuclear-encoded and synthesized in the cytosol with a transient pre-sequence of 45 residues. The 31 amino-terminal residues of this chloroplast pre-sequence were fused to mouse dihydrofolate reductase (a cytosolic protein) and to yeast cytochrome oxidase subunit IV (an imported mitochondrial protein) from which the authentic pre-sequence had been removed. The chloroplast pre-sequence transported both attached proteins into the yeast mitochondrial matrix or inner membrane, although it functioned less efficiently than an authentic mitochondrial pre-sequence. We conclude that mitochondrial and chloroplast pre-sequences perform their function by a similar mechanism.     

Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria.

Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP. The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins.