Partner proteins determine multiple functions of Hsp70.

The 70 kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that are best known for their participation in protein folding. However, evidence is accumulating that Hsp70s perform several other cellular functions in cooperation with specific soluble or membrane-bound partner proteins. While the basic function of Hsp70s is explained by their ability to bind unfolded polypeptide segments, the partner proteins appear to customize them for specific roles such as involvement in protein traffic and folding, translocation of preproteins across membranes, and gene regulation.

Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common membrane insertion site.

The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1-complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44.

The import of preproteins into mitochondria involves translocation of the polypeptide chains through putative channels in the outer and inner membranes. Preprotein-binding proteins are needed to drive the unidirectional translocation of the precursor polypeptides. Two of these preprotein-binding proteins are the peripheral inner membrane protein MIM44 and the matrix heat shock protein hsp70. We report here that MIM44 is mainly exposed on the matrix side, and a fraction of mt-hsp70 is reversibly bound to the inner membrane. Mt-hsp70 binds to MIM44 in a 1:1 ratio, suggesting that mt-hsp70 is localizing to the membrane via its interaction with MIM44. Formation of the complex requires a functional ATPase domain of mt-hsp70. Addition of Mg-ATP leads to dissociation of the complex. Overexpression of mt-hsp70 rescues the protein import defect of mutants in MIM44; conversely, overexpression of MIM44 rescues protein import defects of mt-hsp70 mutants. In addition, yeast strains with conditional mutations in both MIM44 and mt-hsp70 are barely viable, showing a synthetic growth defect compared to strains carrying single mutations. We propose that MIM44 and mt-hsp70 cooperate in translocation of preproteins. By binding to MIM44, mt-hsp70 is recruited at the protein import sites of the inner membrane, and preproteins arriving at MIM44 may be directly handed over to mt-hsp70.

Protein biogenesis: chaperones for nascent polypeptides.

A number of molecular chaperones have been found to interact with nascent polypeptides attached to ribosomes, allowing these protein-synthesis machines to play a key part in protein folding and targeting.

Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates translocation and reduces the membrane potential dependence of loosely folded preproteins.

The mitochondrial heat shock protein Hsp70 (mtHsp70) is essential for driving translocation of preproteins into the matrix. Two models, trapping and pulling by mtHsp70, are discussed, but positive evidence for either model has not been found so far. We have analyzed a mutant mtHsp70, Ssc1-2, that shows a reduced interaction with the membrane anchor Tim44, but an enhanced trapping of preproteins. Unexpectedly, at a low inner membrane potential, ssc1-2 mitochondria imported loosely folded preproteins more efficiently than wild-type mitochondria. The import of a tightly folded preprotein, however, was not increased in ssc1-2 mitochondria. Thus, enhanced trapping by mtHsp70 stimulates the import of loosely folded preproteins and reduces the dependence on the import-driving activity of the membrane potential, directly demonstrating that trapping is one of the molecular mechanisms of mtHsp70 action.

Mitochondrial protein import motor: the ATPase domain of matrix Hsp70 is crucial for binding to Tim44, while the peptide binding domain and the carboxy-terminal segment play a stimulatory role.

The import motor for preproteins that are targeted into the mitochondrial matrix consists of the matrix heat shock protein Hsp70 (mtHsp70) and the translocase subunit Tim44 of the inner membrane. mtHsp70 interacts with Tim44 in an ATP-dependent reaction cycle, binds to preproteins in transit, and drives their translocation into the matrix. While different functional mechanisms are discussed for the mtHsp70-Tim44 machinery, little is known about the actual mode of interaction of both proteins. Here, we have addressed which of the three Hsp70 regions, the ATPase domain, the peptide binding domain, or the carboxy-terminal segment, are required for the interaction with Tim44. By two independent means, a two-hybrid system and coprecipitation of mtHsp70 constructs imported into mitochondria, we show that the ATPase domain interacts with Tim44, although with a reduced efficiency compared to the full-length mtHsp70. The interaction of the ATPase domain with Tim44 is ATP sensitive. The peptide binding domain and carboxy-terminal segment are unable to bind to Tim44 in the absence of the ATPase domain, but both regions enhance the interaction with Tim44 in the presence of the ATPase domain. We conclude that the ATPase domain of mtHsp70 is essential for and directly interacts with Tim44, clearly separating the mtHsp70-Tim44 interaction from the mtHsp70-substrate interaction.

Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane.

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.

Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential.

Two different functions have been proposed for the phosphate carrier protein/p32 of Saccharomyces cerevisiae mitochondria: transport of phosphate and requirement for import of precursor proteins into mitochondria. We characterized a yeast mutant lacking the gene for the phosphate carrier/p32 and found both a block in the import of phosphate and a strong reduction in the import of preproteins transported to the mitochondrial inner membrane and matrix. Binding of preproteins to the surface of mutant mitochondria and import of outer membrane proteins were not inhibited, indicating that the inhibition of protein import occurred after the recognition step at the outer membrane. The membrane potential across the inner membrane of the mutant mitochondria was strongly reduced. Restoration of the membrane potential restored preprotein import but did not affect the block of phosphate transport of the mutant mitochondria. We conclude that the inhibition of protein import into mitochondria lacking the phosphate carrier/p32 is indirectly caused by a reduction of the mitochondrial membrane potential (delta(gamma)), and we propose a model that the reduction of delta(psi) is due to the defective phosphate import, suggesting that phosphate transport is the primary function of the phosphate carrier/p32.

Membrane potential-driven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence.

The transport of preproteins into or across the mitochondrial inner membrane requires the membrane potential Deltapsi across this membrane. Two roles of Deltapsi in the import of cleavable preproteins have been described: an electrophoretic effect on the positively charged matrix-targeting sequences and the activation of the translocase subunit Tim23. We report the unexpected finding that deletion of a segment within the sorting sequence of cytochrome b(2), which is located behind the matrix-targeting sequence, strongly influenced the Deltapsi-dependence of import. The differential Deltapsi-dependence was independent of the submitochondrial destination of the preprotein and was not attributable to the requirement for mitochondrial Hsp70 or Tim23. With a series of preprotein constructs, the net charge of the sorting sequence was altered, but the Deltapsi-dependence of import was not affected. These results suggested that the sorting sequence contributed to the import driving mechanism in a manner distinct from the two known roles of Deltapsi. Indeed, a charge-neutral amino acid exchange in the hydrophobic segment of the sorting sequence generated a preprotein with an even better import, i.e. one with lower Deltapsi-dependence than the wild-type preprotein. The sorting sequence functioned early in the import pathway since it strongly influenced the efficiency of translocation of the matrix-targeting sequence across the inner membrane. These results suggest a model whereby an electrophoretic effect of Deltapsi on the matrix-targeting sequence is complemented by an import-stimulating activity of the sorting sequence.

Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway.

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

Contact sites between inner and outer membranes: structure and role in protein translocation into the mitochondria.

Contact sites between both mitochondrial membranes play a predominant role in the transport of nuclear-coded precursor proteins into mitochondria. The characterization of contact sites was greatly advanced by the reversible accumulation of precursor proteins in transit (translocation intermediates). It was found that the sites are saturable, apparently contain proteinaceous components and mediate extensive unfolding of the polypeptide chain in translocation. Some components of mitochondrial contact sites are currently being identified.

Biogenesis of the dicarboxylate carrier (DIC): translocation across the mitochondrial outer membrane and subsequent release from the TOM channel are membrane potential-independent.

The mitochondrial inner membrane of Saccharomyces cerevisiae contains a group of homologous carrier proteins that mediate the exchange of several metabolites. The members of this protein family are synthesized in the cytosol and reach their final topology after translocation across the mitochondrial outer membrane. Using the ADP/ATP carrier (AAC) as a model protein, previous studies have established four distinct steps of the import pathway (stages I-IV). In the absence of the mitochondrial membrane potential (deltapsi), the AAC accumulates at the inner surface of the outer membrane (stage IIIa) and remains bound to the outer membrane import channel. Only in the presence of the membrane potential, can a complex of small Tim proteins mediate transfer of the AAC to the inner membrane. In this study, we characterized the import pathway of the dicarboxylate carrier (DIC). Different from the AAC, the DIC showed complete deltapsi-independent translocation across the outer membrane, release from the import pore, and mainly accumulated in a soluble state in the intermembrane space, thus defining a new translocation intermediate (stage III*). The DIC should be a suitable model protein for the characterization of deltapsi-independent functions of the intermembrane space Tim proteins.

The mitochondrial ClpB homolog Hsp78 cooperates with matrix Hsp70 in maintenance of mitochondrial function.

The mitochondrial heat shock protein Hsp78 is a member of the Hsp104/Clp family with unknown function. Saccharomyces cerevisiae deletion mutants of HSP78 show wild-type like growth. We report that deletion of the HSP78 gene in yeast strains with point mutations in the SSC1 gene (encoding matrix Hsp70) led to loss of mitochondrial DNA, indicating that at least one of the heat shock proteins Hsp78 and mt-Hsp70 is needed to maintain a rho+ state of the mitochondrial genome. Mitochondria isolated from these double mutants had a strongly reduced membrane potential, explaining defects in the rate of preprotein import. The lack of Hsp78 led to aggregation of the mutant mt-Hsp70 while other matrix chaperones stayed soluble. We conclude that Hsp78 is required to keep mutant forms of mt-Hsp70 soluble and suggest a cooperation of Hsp78 and mt-Hsp70 in maintenance of essential mitochondrial functions.

The preprotein translocase of the inner mitochondrial membrane: evolutionary conservation of targeting and assembly of Tim17.

The preprotein translocase of the inner mitochondrial membrane has only been described in Saccharomyces cerevisiae to date. We report that the essential subunit Tim17 is highly conserved in evolution. The targeting and assembly of yeast Tim17 as well as that of human and Drosophila melanogaster Tim17 were characterized with isolated yeast mitochondria. Targeting signals in the mature protein direct the Tim17 precursors to the receptor Tom70 on the mitochondrial surface. In a membrane potential-dependent step the precursors insert into the inner membrane, adopt a characteristic topology and assemble with Tim23. The mechanisms of targeting and assembly were indistinguishable between the Tim17s from distinct organisms, indicating a high evolutionary conservation.

Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme.

Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step.

The preprotein translocase of the mitochondrial inner membrane: function and evolution.

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.

Mitochondrial preproteins en route from the outer membrane to the inner membrane are exposed to the intermembrane space.

Mitochondrial precursor proteins are known to be imported at sites of close contact between mitochondrial outer and inner membranes. We have identified translocation intermediates exposed to the intermembrane space, including the precursor of the ADP/ATP carrier accumulated at the general insertion site GIP, and the precursor of F1-ATPase subunit beta accumulated on its import pathway at low levels of ATP. These results suggest that mitochondrial contact sites are not sealed structures, but that polypeptides pass (at least partly) through the intermembrane space on their route from the outer membrane to the inner membrane.