Showing papers on "Mitochondrial carrier published in 1994"
TL;DR: The MIM44/Hsp70 complex appears to act as a molecular ratchet which is energetically driven by the hydrolysis of ATP and facilitates stepwise vectorial translocation of proteins across the mitochondrial membranes.
Abstract: Protein translocation into mitochondria requires the mitochondria! 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-Hsp70 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.
384 citations
TL;DR: All mitochondrial carriers investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport, the only exception is the carnitine carrier that proceeds via a ping‐pong mechanism.
328 citations
TL;DR: Results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.
Abstract: Yeast cells with the mdm10 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance. The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division. Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDM10 gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells. MDM10 encodes a protein of 56.2 kD located in the mitochondrial outer membrane. Depletion of Mdm10p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology. These results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.
275 citations
TL;DR: It is proposed that Mmm1p maintains mitochondria in an elongated shape by attaching the mitochondrion to an external framework, such as the cytoskeleton, which is located in the mitochondrial outer membrane, with a large carboxyl-terminal domain facing the cytOSol.
Abstract: In the yeast Saccharomyces cerevisiae, mitochondria are elongated organelles which form a reticulum around the cell periphery. To determine the mechanism by which mitochondrial shape is established and maintained, we screened yeast mutants for those defective in mitochondrial morphology. One of these mutants, mmm1, is temperature-sensitive for the external shape of its mitochondria. At the restrictive temperature, elongated mitochondria appear to quickly collapse into large, spherical organelles. Upon return to the permissive temperature, wild-type mitochondrial structure is restored. The morphology of other cellular organelles is not affected in mmm1 mutants, and mmm1 does not disrupt normal actin or tubulin organization. Cells disrupted in the MMM1 gene are inviable when grown on nonfermentable carbon sources and show abnormal mitochondrial morphology at all temperatures. The lethality of mmm1 mutants appears to result from the inability to segregate the aberrant-shaped mitochondria into daughter cells. Mitochondrial structure is therefore important for normal cell function. Mmm1p is located in the mitochondrial outer membrane, with a large carboxyl-terminal domain facing the cytosol. We propose that Mmm1p maintains mitochondria in an elongated shape by attaching the mitochondrion to an external framework, such as the cytoskeleton.
239 citations
TL;DR: After synthesis in the cytosol, most mitochondrial proteins must traverse mitochondrial membranes to reach their functional location and play an essential mechanistic role at various steps of this process.
197 citations
TL;DR: It is proposed that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp.
Abstract: The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp70 (mhsp70) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[gamma-thio]triphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp70 and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.
169 citations
TL;DR: It is suggested that these domains of Mas22p on either side of the outer membrane function as a relay system for transferring the basic targeting sequences of precursor proteins into the mitochondria.
Abstract: We have cloned the gene encoding the protein Mas22p, which spans the outer membrane of yeast mitochondria. Cells that completely lack Mas22p are inviable. The plasmid-borne MAS22 gene suppresses several defects resulting from the deletion of one or more of the mitochondrial protein import receptors. Defects of Mas20p-deficient cells are explained by the reduced level of Mas22p in these mutants. Mas22p has one acidic domain in the cytosol and a second acidic domain in the mitochondrial intermembrane space. We suggest that these domains of Mas22p on either side of the outer membrane function as a relay system for transferring the basic targeting sequences of precursor proteins into the mitochondria.
145 citations
TL;DR: It is shown that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions, and helps to facilitate assembly of mitochondriatically encoded subunits of the ATP synthase complex.
Abstract: Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix. Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria. The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro. In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions. Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex. By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation. Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra-molecular complexes.
142 citations
TL;DR: It is proposed that MIM17 is an essential component of the preprotein import machinery of the mitochondrial inner membrane of Saccharomyces cerevisiae.
110 citations
TL;DR: The electrical membrane potential and ATP are needed to drive protein tranlocation through the MIM import machinery.
110 citations
TL;DR: The RCA1 gene has been cloned by complementation of an rca1 mutant with a yeast genomic library and the sequence of the encoded product shows Rca1 protein to be a new member of a recently described family of ATPases.
TL;DR: The authors' observations raise the possibility that Sms1p and Mas6p act together to translocate proteins across the inner membrane, suggesting that SMS1p is a new inner membrane import component.
Abstract: MAS6 encodes an essential inner membrane protein required for mitochondrial protein import in the yeast Saccharomyces cerevisiae (Emtage and Jensen, 1993). To identify new inner membrane import components, we isolated a high-copy suppressor (SMS1) of the mas6-1 mutant. SMS1 encodes a 16.5-kDa protein that contains several potential membrane-spanning domains. The Sms1 protein is homologous to the carboxyl-terminal domain of the Mas6 protein. Like Mas6p, Sms1p is located in the mitochondrial inner membrane and is an essential protein. Depletion of Sms1p from cells causes defects in the import of several mitochondrial precursor proteins, suggesting that Sms1p is a new inner membrane import component. Our observations raise the possibility that Sms1p and Mas6p act together to translocate proteins across the inner membrane.
TL;DR: It is demonstrated that Yta10p, a mitochondrial homologue of a conserved family of putative ATPases in Saccharomyces cerevisiae, is essential for this proteolytic process of membrane‐associated polypeptides in mitochondria.
TL;DR: It is concluded that MIM17 and MIM23 are integral parts of a preprotein translocation channel and cooperate with MIM44 and hsp70 at the same protein import site.
TL;DR: Analysis of PMP47- dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelle.
Abstract: Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320-322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47-dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.
TL;DR: It is shown that an RNA with 120 nucleotides, the sequence of which is identical to that of the nuclear‐encoded 5SRNA, exists in bovine mitochondria, although the mitochondrial genome encodes no 5S RNA gene.
TL;DR: The cloning of a cDNA encoding a new member of the highly conserved mammalian 70-kDa heat shock protein (hsp 70) family termed PBP74 is reported, and a variety of immunological and biochemical approaches are used both in vitro and in vivo to demonstrate that it is imported into and resides in mitochondria.
Abstract: The cloning of a cDNA encoding a new member of the highly conserved mammalian 70-kDa heat shock protein (hsp 70) family termed PBP74 was recently reported. Critical to an understanding of the function of this new hsp 70 is delineating its subcellular localization. Here we use a variety of immunological and biochemical approaches both in vitro and in vivo to demonstrate that PBP74 is imported into and resides in mitochondria. By confocal immunofluorescence microscopy PBP74 is detected in mitochondria, colocalizing with the mitochondrial 60-kDa heat shock protein. To address the inherent problem of serological cross-reactivity among the hsp70 family members, an influenza virus hemagglutinin epitope tag was introduced into the PBP74 cDNA. The epitope-tagged PBP74 protein transiently expressed in L cells localized to mitochondria. Moreover, deletion of the N-terminal 46-amino acid presequence results in a cytosolic localization of the epitope-tagged protein. Cell fractionation studies demonstrated PBP74 in purified mitochondria in a protease-protected location. After coupled transcription-translation the precursor of PBP74 is imported into isolated yeast mitochondria, where it becomes processed to the mature protein. According to a subfractionation of the mitochondria, the imported protein was found to be localized in the matrix space. Import in vitro is time- and temperature-dependent, requires matrix ATP, and is abolished upon depletion of the membrane potential across the mitochondrial inner membrane. Similarly, in mammalian cells PBP74 is synthesized as a pre-protein that requires membrane potential-dependent import into mitochondria for its maturation. Taken together, our data demonstrate that PBP74 is a mammalian mitochondrial hsp70.
TL;DR: It is suggested that these are dynamic structures, with channel conductances altered by the presence of ATP, and that ligand-mediated conformational changes in the porin-adenine nucleotide translocator complexes may be a general mechanism in signal transduction.
Abstract: Complexes made up of the kinases, hexokinase and glycerol kinase, together with the outer mitochondrial membrane voltage-dependent anion channel (VDAC) protein, porin, and the inner mitochondrial membrane protein, the adenine nucleotide translocator, are involved in tumorigenesis, diabetes mellitus, and central nervous system function. Identification of these two mitochondrial membrane proteins, along with an 18 kD protein, as components of the peripheral benzodiazepine receptor, provides independent confirmation of the interaction of porin and the adenine nucleotide translocator to form functional contact sites between the inner and outer mitochondrial membranes. We suggest that these are dynamic structures, with channel conductances altered by the presence of ATP, and that ligand-mediated conformational changes in the porin-adenine nucleotide translocator complexes may be a general mechanism in signal transduction.
TL;DR: Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established.
Abstract: Mitochondrial protein targeting includes both intramitochondrial sorting of pro- teins 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 pas- sage 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 mito- chondrial 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 ultrastructral data and recent biochemical findings we propose that mitochon- drial 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. o 1994 Wiley-Liss, Inc.
TL;DR: It is postulate that the mitochondrial metabolism in intact cells is not regulated by free ADP, but induced by substrates wf kinases such as glucose or creatine, which can be the signal that stimulates the mitochondria metabolism transmitted by the mitochondrial creatine kinase.
TL;DR: Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration.
Abstract: Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.
TL;DR: The transport of nuclear-encoded proteins from the cytosol into mitochondria is mediated by targeting (signal) sequences present on precursor forms, but the presence of a mitochondrial targeting sequence alone is not sufficient for specific targeting to the organelle and further to the various subcompartments.
Book•
01 Jul 1994
TL;DR: This book contains studies utilizing the techniques of biochemistry, physiology, molecular biology and genetics to reveal the structure and function of mitochondrial transport systems.
Abstract: Mitochondrial transport systems are essential to mitochondrial function and therefore to energy homeostasis within the cell The book contains studies utilizing the techniques of biochemistry, physiology, molecular biology and genetics to reveal the structure and function of mitochondrial transport systems It is divided into the following six sections: proton translocation - the uncoupling protein and the ATPase; carriers and transporters; mitochondrial ion channel; structure of the outer mitochondrial membrane channel, VDAC; VDAC, peripheral kinases and energy utilization; mitochondrial channels in humans and relationship to disease
TL;DR: 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.
Abstract: 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.
01 Jan 1994
TL;DR: The chapter discusses that the majority of mitochondrial proteins are encoded by the nucleus, synthesized on cytoribosomes and imported into the mitochondrion, which contains their own DNA, which is circular and double stranded.
Abstract: Publisher Summary This chapter describes the mitochondrion as an intracellular organelle found in virtually all eukaryotic cells, where it plays a major role in cellular ATP production. It consists of four compartments; the inner and outer membranes and two soluble fractions, the matrix and the intermembrane space. The inner membrane and matrix are associated with most of the functional activities of the mitochondria, including those involved with the tricarboxylic acid (TCA) cycle, fatty acid oxidation and ATP generation. The inner membrane is folded forming cristae giving it a much larger surface area than the outer membrane. The main function of the mitochondrion is ATP synthesis. The enzymes for the TCA cycle and β-oxidation of fatty acids are situated in the mitochondrial matrix where dicarboxylic, tricarboxylic, and fatty acids are oxidized generating NADH and FADH2. The chapter discusses that the majority of mitochondrial proteins are encoded by the nucleus, synthesized on cytoribosomes and imported into the mitochondrion. However, mitochondria contain their own DNA (mtDNA), which is circular and double stranded.
TL;DR: The sequences are presented of cDNAs encoding five related proteins from the nematode worm, Caenorhabditis elegans, that can be recognised as the homologues of the ADP/ATP, phosphate and oxoglutarate/malate carrier proteins that have been found in the inner membranes of mitochondria in other species.
Abstract: The sequences are presented of cDNAs encoding five related proteins from the nematode worm, Caenorhabditis elegans. Three of them can be recognised as the homologues of the ADP/ATP, phosphate and oxoglutarate/malate carrier proteins that have been found in the inner membranes of mitochondria in other species. These carrier proteins, and the uncoupling protein from the mitochondria in mammalian brown adipose tissue, have common features in their primary and secondary structures, and are members of the same protein super-family. Members of this super-family have polypeptide chains approximately 300 amino acid long that consist of three tandem related sequences of about 100 amino acids. The tandem repeats from the different proteins are inter-related, and each repeat is probably folded into a common secondary structural motif consisting of two hydrophobic stretches of amino acids with the potential to form membrane spanning ex-helices, linked by an extensive hydrophilic region. The common characteristic feat...
01 Jan 1994
TL;DR: This chapter discusses the genetic aspects of mitochondrial myopathies, which are the major producers of cellular adenosine triphosphate (ATP) by the process of oxidative phosphorylation (OXPHOS).
Abstract: Publisher Summary This chapter discusses the genetic aspects of mitochondrial myopathies. Mitochondria are the major producers of cellular adenosine triphosphate (ATP) by the process of oxidative phosphorylation (OXPHOS). The mitochondrial OXPHOS system encompasses five multiple subunit enzyme complexes plus the adenine nucleotide translocator (ANT), all of which are embedded in the mitochondrial inner membrane. Four of these five OXPHOS enzyme complexes contain polypeptide subunits encoded by both the nucleus and the mitochondria. The two promoters, light strand promoter (LSP) and heavy-strand promoter (HSP), of mtDNA transcription are adjacent to each other within the displacement loop (D-loop). Inverted sequences located upstream from both promoters bind mitochondrial transcription factor (mtTFl). MtTFl is required in addition to the mitochondrial RNA (mtRNA) polymerase for efficient transcription. The genetic code of the mtDNA differs from nucleus and virtually all other organisms. The mtDNA genetic code is highly degenerate, so that only 22 tRNAs are required for translation. When uridine is in the wobble position, all 4 members of a codon family can be read by 1 mitochondrial tRNA.
TL;DR: The results suggest a possible functional interaction between mitochondrial carrier protein and malate dehydrogenase, and the highly purified protein displayed all the properties of the dicarboxylate carrier.
TL;DR: A strong requirement for ATP is found on both sides of the mitochondrial inner membrane, suggesting that one or more previously undetected mitochondrial proteins outside the inner membrane utilize ATP to promote efficient pre-F1 beta import.
Patent•
01 Jun 1994
TL;DR: In this article, small RNAs encoded within the nucleus of mammalian cells that specifically import to the mitochondria were identified as potential carriers for import of biological molecules, including metabolites and proteins into the mitochondrial compartment.
Abstract: The invention relates to small RNAs encoded within the nucleus of mammalian cells that specifically import to the mitochondria. The RNAs bind to several nucleolar peptides and thus provide potential carriers for import of biological molecules, including metabolites and proteins, into the mitochondrial compartment. Mitochondrial dysfunction in several maternally inherited human diseases may be correctable employing linkage of mitochondrial import signal to mitochondrial tRNA sequences expressed from nuclear trans-genes without requirement for direct genetic transformation of mitochondria.