The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology. Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, proteins to proteolytic enzymes in the mitochondrial matrix.

Animals↗

PubMed1994

The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria.

The entry of segments of preproteins of defined lengths into the matrix space of mitochondria was studied. The mitochondrial chaperone Hsp70 (mtHsp70) interacted with proteins emerging from the protein import channel and stabilized translocation intermediates across the membranes in an adenosine triphosphate-dependent fashion. The chaperone bound to the presequence and mature parts of preproteins. In the absence of mtHsp70 binding, preproteins with less than 30 to 40 residues in the matrix diffused out of mitochondria. Thus, protein translocation was reversible up to a late stage. The import channels in both mitochondrial membranes constitute a passive pore that interacts weakly with polypeptide chains entering the matrix.

Adenosine Triphosphate↗

PubMed1994

Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.

ATP-Dependent Proteases↗

PubMed1994

Mitochondrial Hsp70/MIM44 complex facilitates protein import.

Protein translocation into mitochondria requires the mitochondrial protein Hsp70. This molecular chaperone of the mitochondrial matrix is recruited to the protein import machinery by MIM44, a component associated with the inner membrane of the mitochondria. Formation of the mt-Hsp70/MIM44 complex is regulated by ATP. MIM44 and mt-Hsp 70 interact in a sequential manner with incoming segments of unfolded preproteins and thereby facilitate stepwise vectorial translocation of proteins across the mitochondrial membranes. The complex appears to act as a molecular ratchet which is energetically driven by the hydrolysis of ATP.

Adenosine Triphosphate↗

PubMed1994

Yta10p is required for the ATP-dependent degradation of polypeptides in the inner membrane of mitochondria.

Incompletely synthesized polypeptides in the mitochondrial inner membrane are subject to rapid proteolysis. We demonstrate that Yta10p, a mitochondrial homologue of a conserved family of putative ATPases in Saccharomyces cerevisiae, is essential for this proteolytic process. Yta10p-dependent degradation requires divalent metal ions and the hydrolysis of ATP. Yta10p is an integral protein of the inner mitochondrial membrane exposing the carboxy terminus to the mitochondrial matrix space. Based on the presence of consensus binding sites for ATP, and for divalent metal ions found in a number of metal dependent endopeptidases, a direct role of Yta10p in the proteolytic breakdown of membrane-associated polypeptides in mitochondria is suggested.

Adenosine Triphosphatases↗

PubMed1994

Mitochondrial protein import: mechanisms, components and energetics.

The transport of nuclear-encoded proteins from the cytosol into mitochondria is mediated by targeting (signal) sequences present on precursor forms. Most precursors of the mitochondrial matrix possess amino-terminal signals which characteristically contain hydroxylated and basic amino acids and lack acidic residues. With a minority of precursor proteins, internal sequence motifs can direct proteins to the mitochondria (Pfanner, N., Hoeben, P., Tropschug, M. and Neupert, W. (1987) J. Biol. Chem. 262, 14851-14854). The presence of a mitochondrial targeting sequence alone, however, is not sufficient for specific targeting to the organelle and further to the various subcompartments. There is the need for components which recognise the targeting sequences and others which keep the precursor protein in a translocation-competent form. Beyond the recognition step, components are required which mediate translocation across the mitochondrial membranes. Mitochondria posses two translocation machineries, one in the outer membrane and one in the inner membrane. The matrix space harbors a number of factors which participate in the import of proteins, in their unfolding and folding. Energy is required at several steps of these processes.

Cytosol↗

PubMed1994

Protein synthesis. Cradle at the ribosome.

Explore the source record for details and available documents.

Peptides↗

PubMed1994

XDJ1, a gene encoding a novel non-essential DnaJ homologue from Saccharomyces cerevisiae.

The gene encoding a novel DnaJ-like protein, termed Xdj1, has been identified by amplification of Saccharomyces cerevisiae genomic DNA. An open reading frame of 1380 bp was detected. Disruption of XDJ1 did not yield any detectable new phenotype. A double-deletion strain containing a disruption of both XDJ1 and YDJ1, another gene coding for a DnaJ-like protein, was still viable. Under a variety of growth conditions, no XDJ1 transcripts could be detected by Northern blot analysis and no translation product was found by immunoblotting with antibody against Xdj1 produced in Escherichia coli. Thus, XDJ1 is either expressed only under very specific conditions or represents a silent gene.

Amino Acid Sequence↗

PubMed1994

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding.

Mdj1p, a novel member of the DnaJ family, is a heat shock protein that is associated with the inner membrane of mitochondria of Saccharomyces cerevisiae. Disruption of the MDJ1 gene resulted in a petite phenotype, loss of mitochondrial DNA, and inviability at 37 degrees C. Import of precursor proteins was not affected by a lack of Mdj1p, but folding of newly imported proteins was markedly impaired. The efficiency of refolding of a tester protein, dihydrofolate reductase, was significantly reduced in mitochondria lacking Mdj1p after incubation at elevated temperature. We conclude that Mdj1p is an important mitochondrial chaperone that participates in the folding of newly imported proteins and in the protection of proteins against heat denaturation and aggregation.

Amino Acid Sequence↗

PubMed1994

Specific recognition of mitochondrial preproteins by the cytosolic domain of the import receptor MOM72.

The import receptor MOM72 constitutes part of the protein translocation machinery of the outer mitochondrial membrane, the receptor-general insertion pore complex. The protein contains a membrane anchor at the NH2 terminus and a large cytosolic domain. In yeast and Neurospora crassa the cytosolic domain comprises about 570-580 amino acid residues. The cytosolic domain of yeast MOM72 was purified after expression in Escherichia coli as a homogeneous monomeric protein. It can recognize precursor proteins as demonstrated by its ability to compete for binding and import into the mitochondria and to physically interact with preproteins. A subset of preproteins including the ADP/ATP carrier and the phosphate carrier interact with very high affinity, precursors that are known to be targeted via MOM72. Thus, the cytosolic domain of MOM72 plays a critical function in the recognition of preproteins by directly binding to precursor proteins and thereby facilitating their targeting to mitochondria.

Base Sequence↗

PubMed1994

A morphological view on mitochondrial protein targeting.

Mitochondrial protein targeting includes both intramitochondrial sorting of proteins encoded by the organellar genome and import and subsequent sorting of nuclear encoded precursor proteins. Only a few proteins are encoded by the mitochondrial genome and synthesized in the organellar matrix. These include predominantly inner membrane proteins that are perhaps co-translationally inserted into this membrane. Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane. Ultrastructurally, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established. The majority of the mitochondrial proteins are nuclear encoded and synthesized as precursors in the cytosol. Electron microscopic studies revealed that import of precursor proteins is generally confined to sites where both mitochondrial envelope membranes are closely apposed. In line with these observations, biochemical studies indicated that precursor proteins destined for the inner membrane or matrix have to interact with the energized inner membrane to allow complete passage of the precursor through the outer membrane. As a consequence, the mitochondrial envelope membranes have to be in close proximity at protein import sites. In isolated mitochondria distinct sites (designated as contact sites) exist where both envelope membranes are closely apposed and presumably stably associated. In situ, however, mitochondrial boundary membranes are in close proximity over large areas that cover almost the entire mitochondrial periphery. Consequently, the relative area of the mitochondrial surface, where both boundary membranes are in sufficient proximity for allowing protein translocation, is generally larger in situ compared to that in isolated organelles. Immunocytochemical localization studies showed a rather random distribution of components of the mitochondrial protein translocation machinery over the entire mitochondrial surface and not confined to contact sites. Based on these ultrastructural data and recent biochemical findings we propose that mitochondrial protein import sites are dynamic in nature and include relatively labile regions of close association of the boundary membranes. In vitro, however, mitochondrial protein import may preferentially take place at or near the presumably stable contact sites.

Biological Transport↗

PubMed1994

Characterization of the mitochondrial processing peptidase of Neurospora crassa.

The mitochondrial processing peptidase (MPP) of Neurospora crassa is constituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome b2 by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a approximately 30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH-terminal portion of the presequence of cytochrome b2 rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal to the cleavage site. Non-cleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping.

Base Sequence↗

PubMed1994

The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space.

The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b2 (pb2), a precursor carrying a bipartite presequence, into the intermembrane space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb2 fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb2) was largely independent of matrix ATP. For those intermembrane-space-targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b2 domain. These results suggest the following: (a) The import of matrix-targeted preproteins, in addition to a membrane potential delta psi, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat-shock protein 70 (mt-Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of intermembrane-space-targeted precursors with bipartite signals, the function of ATP/mt-Hsp70 is not obligatory, as components of the intermembrane-space-sorting pathway may substitute for ATP/mt-Hsp70 function (however, if a tightly folded domain is present in the precursor, ATP/mt-Hsp70 is indispensable); (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix-ATP/mt-Hsp70.

Adenosine Triphosphate↗

PubMed1994

Heinrich Wieland--prize lecture. Transport of proteins across mitochondrial membranes.

The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of pre-proteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments--the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix--are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c1 heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c1 preproteins, and the mitochondrial processing peptidase which cleaves signal sequences after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle.

Animals↗