Showing papers on "Mitochondrial carrier published in 1996"

Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22

Abstract: TRANSLOCATION of mitochondrial preproteins across the inner membrane is facilitated by the TIM machinery1–8 Tim23 binds to matrix targeting signals and initiates membrane potential-dependent import8 Tim23 and Tim17 are constituents of a translocation channel across the inner membrane6 Tim44 is associated with this channel at the matrix side6, and Tim44 recruits mitochondrial Hsp70 and its co-chaperone Mge1, which drive protein translocation into the matrix using ATP as an energy source9–14 Tim22 is a new component of the import machinery of mitochondria, which shares sequence similarity with both Tim23 and Tim17 Here we report that Tim22 is required for the import of proteins of the mitochondrial ADP/ ATP carrier (AAC) family into the inner membrane Members of the yeast AAC family are synthesized without matrix targeting signals15,16 Tim22 is in an assembly of high relative molecular mass that is distinct from the Tim23–Timl7 complex6 Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent ofTim22

VDAC, a Channel in the Outer Mitochondrial Membrane

Abstract: Proteins that form aqueous channels in membranes generate conduction pathways with a variety of shapes and sizes. Perhaps the largest channel-forming protein is the 2-MDa ryanodine receptor while the smallest may be gramicidin. However, the size of the conducting pathway is not correlated with the amount of protein mass needed to make up the structure, as demonstrated by the fact that some of the narrowest conducting pathways are produced by very large amounts of protein (e. g., 0.3 MDa for the Na+/ K+/Ca2+ channel family). In contrast, the focus of this review, the voltage-dependent anion channel (VDAC) of the mitochondrial outer membrane, produces one of the largest aqueous pathways from a single 30-kDa protein. VDAC also demonstrates that functional complexity does not seem to correlate well with the amount of protein used to form a channel. VDAC has a small amount of protein mass but displays complex behavior. It has two voltage-gating processes, can be controlled by metabolites and regulatory proteins, is able to form complexes with other proteins and enzymes, and responds to the protein concentration of the cytoplasm (Colombini, 1994). Thus, many functions are packed into a single, relatively small VDAC protein.

Internal targeting signal of the BCS1 protein: a novel mechanism of import into mitochondria.

Abstract: The BCS1 protein is anchored in the mitochondrial inner membrane via a single transmembrane domain and has an N(out)-C(in) topology. Unlike the majority of nuclear encoded mitochondrial preproteins, the BCS1 protein does not contain an N-terminal targeting sequence. A positively charged segment of amino acids which is located immediately C-terminal to the transmembrane domain acts as an internal targeting signal. In order to function, we postulate that this sequence co-operates with the transmembrane domain to form a tight hairpin loop structure. This loop is translocated across the inner membrane via the MIM/mt-Hsp70 machinery in a membrane potential-dependent manner. This novel mechanism of import and sorting of the BCS1 protein is proposed to represent a more general mechanism used by a number of inner membrane proteins.

Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins.

Abstract: Mdj1p,aDnaJhomologinthemitochondriaofSaccharomycescerevisiae,isinvolvedinthefoldingofproteins in the mitochondrial matrix. In this capacity, Mdj1p cooperates with mitochondrial Hsp70 (mt-Hsp70). Here, we analyzed the role of Mdj1p as a chaperone for newly synthesized proteins encoded by mitochondrial DNA and for nucleus-encoded proteins as they enter the mitochondrial matrix. A series of conditional mutants of mdj1wasconstructed.MutationsinthevariousfunctionaldomainsledtoapartiallossofMdj1pfunction.The mutant Mdj1 proteins were defective in protecting the tester protein firefly luciferase against heat-induced aggregationinisolatedmitochondria.Themitochondriallyencodedvar1proteinshowedenhancedaggregation after synthesis in mdj1 mutant mitochondria. Mdj1p and mt-Hsp70 were found in a complex with nascent polypeptide chains on mitochondrial ribosomes. Mdj1p was not found to interact with translocation intermediates of imported proteins spanning the two membranes and exposing short segments into the matrix, in accordancewiththelackofrequirementofMdj1pinthemt-Hsp70-mediatedproteinimportintomitochondria. Ontheotherhand,precursorproteinsintransitwhichhadfurtherenteredthematrixwerefoundinacomplex with Mdj1p. Our results suggest that Mdj1p together with mt-Hsp70 plays an important role as a chaperone formitochondriallysynthesizedpolypeptidechainsemergingfromtheribosomeandfortranslocatingproteins at a late import step.

Regulated protein degradation in mitochondria.

Abstract: Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.

Three genes for mitochondrial proteins suppress null-mutations in both Afg3 and Rca1 when over-expressed

Abstract: The AFG3 gene of Saccharomyces cerevisiae encodes a mitochondrial inner membrane protein with ATP-dependent protease activity. To gain more insight into the function of this protein, multi-copy suppressors of an afg3-null mutation were isolated. Three genes were found that restored partial growth on non-fermentable carbon sources, all of which affect the biogenesis of respiratory competent mitochondria: PIM1(LON) encodes a matrix-localized ATP-dependent protease involved in the turnover of matrix proteins; OXA1(PET1402) encodes a putative mitochondrial inner membrane protein involved in the biogenesis of the respiratory chain; and MBA1 encodes a mitochondrial protein required for optimal respiratory growth. All three genes also suppressed a null mutation in a related gene, RCA1, as well as in the combination of afg3- and rca1-null.

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

Abstract: 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.

Isolation and Analysis of the arg-13 Gene of Neurospora crassa

Abstract: Mutations in arg-13 result in slow growth in minimal medium and can suppress mutations in carbamyl phosphate synthase-aspartate carbamyl transferase within the pyrimidine pathway; the exact biochemical function of the gene product is unknown. To understand the role of arg-13 in arginine metabolism, cosmids rescuing growth in arg-13 mutants were cloned and mapped to the position of arg-13 on LG IR. Northern analysis showed the arg-13 message to contain ~2100 nt, although a 1.4kb genomic fragment truncated at the 5′ and 3′ ends of the gene encodes a shortened transcript that can rescue arg-13 function. Expression of mRNA arising from the mutant arg-13 gene is induced by arginine starvation, although wild type ( arg-13 + ) is not derepressed in minimal medium. The sequence of the arg-13 gene shows ARGl3 to be a member of the mitochondrial carrier superfamily with three repeats of a ~100-amino acid domain, six putative membrane spanning regions, and three copies of the mitochondrial carrier consensus pattern. This information plus available and new nutritional data are consistent with the hypothesis that arg-13 encodes a mitochondrial basic amino acid carrier whose existence was predicted based upon previous physiological, nutritional and biochemical data.

SHM1: A multicopy suppressor of a temperature‐sensitive null mutation in the HMG1‐like abf2 gene

Abstract: HM, an HMG1-like mitochondrial DNA-binding protein, is required for maintenance of the yeast mitochondrial genome when cells are grown in glucose. To better understand the role of HM in mitochondria, we have isolated several multicopy suppressors of the temperature-sensitive defect associated with an abf2 null mutation (lacking HM protein). One of these suppressors, SHM1, has been characterized at the molecular level and is described herein. SHM1 encodes a protein (SHM1p) that shares sequence similarity to a family of mitochondrial carrier proteins. On glycerol medium, where mitochondrial function is required for growth, shm1 deletion mutants are able to grow whereas shm1 abf2 double mutants are severely inhibited. These results suggest the SHM1p plays an accessory role to HM in the mitochondrion.

Anion channels of the inner membrane of mammalian and yeast mitochondria

Abstract: The inner membrane of yeast and mammalian mitochondria has been studiedin situ with a patch clamp electrode. Anion channels were found in both cases, although their behavior and regulation are different. In mammalian mitochondria, the principal channel is of around 100 pS conductance and opens mainly under depolarized membrane potentials. As no physiological compound able to alter its peculiar voltage dependence has yet been found, it is proposed that this channel may serve as a safeguard mechanism for recharging the mitochondrial membrane potential. Two other anion channels, each with a distinct conductance (one of approx. 45 pS, the second of at least a tenfold higher value) and kinetics are harbored in the yeast inner membrane. Matrix ATP was found to interact with both, but with a different mechanism. It is proposed that the 45 pS channel may be involved in the homeostatic mechanism of mitochondrial volume.

Apoptosis of cells lacking mitochondrial DNA

Abstract: The mitochondrial genome of animals encodes a few subcomponents of the respiratory chain complexes I, III and IV, whereas nuclear DNA encodes the overwhelming majority, both in quantitative and qualitative terms, of mitochondrial proteins. Complete depletion of mitochondrial DNA (mtDNA) can be achieved by culturing cells in the presence of inhibitors of mtDNA replication or mitochondrial protein synthesis, giving rise to mutant cells (ϱ∘ cells) which carry morphological near-to-intact mitochondria with respiratory defects. Such cells can be used to study the impact of mitochondrial respiration on apoptosis. ϱ∘ cells do not undergo cell death in response to determined stimuli, yet they conserve their potential to undergo full-blown apoptosis in many experimental systems. This indicates that mtDNA and associated functions (in particular mitochondrial respiration) are irrelevant to apoptosis execution. However, the finding that mtDNA-deficient mitochondria can undergo apoptosis does not argue against the involvement of mitochondria in the apoptotic process, since mitochondria from ϱ∘ cells conserve most of their functions including those involved in the execution of the death programme: permeability transition and release of one or several intermembrane proteins causing nuclear apoptosis.

Analysis of a 36.2 kb DNA sequence including the right telomere of chromosome VI from Saccharomyces cerevisiae.

Abstract: The nucleotide sequence of a 36·2-kb distal region containing the right telomere of chromosome VI was determined. Both strands of DNA cloned into cosmid clone 9965 and plasmid clone pEL174P2 were sequenced with an average redundancy of 7·9 per base pair, by both dye primer and dye terminator cycle sequencing methods. The G + C content of the sequence was found to be 37·9%. Eighteen open reading frames (ORFs) longer than 100 amino acids were detected. Four of these ORFs (9965orfR017, 9965orfF016, 9965orfR009 and 9965orfF003) were found to encode previously identified genes (YMR31, PRE4, NIN1 and HXK1, respectively). Six ORFs (9965orfR013, 9965orfF018, 9965orfF006, 9965orfR014, 9965orfF013 and 9965orfR020) were found to be homologous to hypothetical 121·4-kDa protein in the BCK 5′ region, Bacillus subtilis DnaJ protein, hypothetical Trp-Asp repeats containing protein in DBP3-MRPL27, putative mitochondrial carrier YBR291C protein, Salmonella typhimurium nicotinate-nucleotide pyrophosphorylase, and Escherichia coli cystathionine β-lyase, respectively. The putative proteins encoded by 9965orfF018, 9965orfR014 and 9965orfR020 were found to be, respectively, a new member of the family of DnaJ-like proteins, the mitochondrial carrier protein and cystathionine lyase. The nucleotide sequence reported here has been deposited in the DDBJ/GenBank/EMBL data library under Accession Number D44597.

Protein targeting to mitochondria

Abstract: Contents. List of Contributors. Preface (F.U. Hartl). Targeting Signals for Protein Import into Mitochondria and other Subcellular Organelles (G. von Heijne). Protein Transport into Mitochondria: Cytosolic Factors which operate during and after Translation in Protein Trafficking (L. Estey and M.G. Douglas). Presequence Binding Proteins as Cytosolic Import-Stimulation Factors in Mitochondrial Protein Import (K. Mihara and T. Omura). Molecular Mechanisms of Protein Translocation into and across the Mitochondrial outer Membrane (R. Lill, A. Mayer, H. Steiner, G. Kispal, and W. Neupert). Targeting and Insertion of Proteins into the Mitochondrial outer Membrane (G.C. Shore, H.M. McBride, D.G. Millar, N.A.E. Steenaart, and M. Nguyen). Targeting and Translocation of Preproteins by the TOMs of the Mitochondrial Receptor Complex (P. Keil, A. Hordinger, and N. Planner). Mitochondrial Import of Cytochrome C (M.E. Dumont). Translocation of Preproteins across the Mitochondrial inner Membrane: TIMs and HSP70 (M. Meijer, A. Maarse, M. Kubrich, and N. Planner). Unraveling the Protein Translocation Machinery in the Mitochondrial inner Membrane (N.G. Kronidou and M. Horst). Proteolytic Processing of Mitochondrial Precursor Proteins (W.A. Fenton and F. Kalousek). Sorting of Proteins to the Mitochondrial Intermembrane Space (R.A. Stuart, H. Folsch, A. Gruhler, and W. Neupert). Energetics of Mitochondrial Protein Import and Intramitochondrial Protein Sorting (S. Rospert). Export of Proteins from Mitochondria (R.O. Poyton, K.A. Sevarino, E.E. McKee, D.J.M. Duhl, V. Cameron, and B. Goehring). Protein Folding in Mitochondria (J. Hohfeld). Assembly of Multisubunit Complexes in Mitochondria (M. Prescott, R.J. Devenish, and P. Nagley). The Division and Inheritance of Mitochondria (M.P. Yaffe). Index.

Export of Proteins from Mitochondria

Abstract: Publisher Summary This chapter discusses the protein export, and its relationship(s) to the other transport pathways of mitochondria. Nuclear gene products are exported from the mitochondrial matrix to the inner membrane space by a process termed “conservative sorting.” Proteins that follow this pathway are translated in the cytosol and localized to the matrix by standard import pathways. Nuclear gene products may also be released from the mitochondrial matrix or intermembrane space into the cytosol. Thus, nuclear gene products may be exported to either the intermembrane space or to the cytosol. Several lines of evidence indicate that mitochondrially-encoded proteins are inserted into the inner mitochondrial membrane co-translationally. The most direct evidence comes from studies with cytochrome c oxidase presubunit II in yeast. The leader peptide of this preprotein is processed by Implp, which is located on the external surface of the inner membrane. Hence, processing is a convenient measure of insertion because the precursor becomes accessible to the protease only after its insertion into the inner membrane. Because presubunit II is processed co-translationally, it follows that it is also inserted into the membrane co-translationally.

Molecular Mechanisms of Protein Translocation into and Across the Mitochondrial Outer Membrane

Abstract: Publisher Summary This chapter summarizes the current knowledge on the molecular mechanisms underlying protein insertion into and translocation across the outer membrane. Recently, however, it became clear that both the outer membrane and the inner membrane contain individual protein translocation machineries each of which can transport preproteins. In addition, it could be demonstrated that matrix-targeted proteins are able to traverse both membranes in consecutive steps demonstrating the independence of the two machineries. Mitochondrial preproteins are believed to be maintained in a translocation competent state by cytosolic factors, for example, by members of the Hsp70 protein family and by presequence binding factors. Recognition and translocation across the outer membrane is mediated by a number of proteins organized in a large complex, the so-called mitochondrial receptor complex. Initial interaction with the mitochondrial outer membrane is mediated by protease-sensitive surface components of this complex. It is suggested that by using a fusion protein between the presequence of the β-subunit of F1-ATPase and cytochrome c heme lyase (CCHL), the translocation reaction into the matrix could be dissected into two both independent and consecutive steps.

Proteolytic Processing of Mitochondrial Precursor Proteins

Abstract: Publisher Summary This chapter discusses the proteolytic processing of mitochondrial precursor proteins. It is suggested that pattern of proteolytic maturation of imported mitochondrial proteins involves a hierarchy of cleavages by a limited number of mitochondrial peptidases. Most cleaved precursors, whether ultimately destined for the matrix, the inner membrane, or the intermembrane space, are acted on by mitochondrial processing peptidase (MPP) in its role as the general mitochondrial peptidase. A major subset of these, eventually localizing to either the matrix or the inner membrane, is cleaved specifically by mitochondrial intermediate peptidase only after MPP has exposed a suitable octapeptide at the amino-terminus of the intermediate. A few others, targeted to the intermembrane space (or that face of the inner membrane) by exposed sequences reminiscent of bacterial signal peptides, are cleaved by a localized protease, inner membrane peptidase, after the second targeting step is complete. In all cases examined, the proteolytic steps are not required for transport, but serve to generate mature amino-termini that permit protein folding, membrane insertion, and/or macromolecular complex assembly to produce the active enzymes or functional structures of mitochondria.

Chapter 33 Protein transport across the outer and inner membranes of mitochondria

Abstract: Publisher Summary The identification of quite a number of protein components involved in mitochondrial protein import has contributed significantly to understand the mechanism of this complex process. Mitochondrial preprotein import is a multi-step process, which is facilitated by the sequential and coordinated action of two separate import machineries located in the outer and inner mitochondrial membrane (Tom and Tim complex). The Tom machinery translocates the charged presequences of matrix targeted preproteins across the outer membrane but not the entire protein. Preproteins destined for the outer membrane and the intermembrane space can be imported and inserted by the Tom machinery independently of the inner membrane. The driving forces for these reactions are unknown; the free energies of membrane insertion and membrane association and folding could represent such forces. From the trans-site, the matrix targeting sequence is passed on to the inner membrane complex (Tim). The components on the inner membrane, which receive the presequence and probably initiate the transfer into the matrix remain to be identified.

Translocation of preproteins across the mitochondrial inner membrane: TIMs and HSP70

Abstract: Publisher Summary The view that the inner membrane possesses a structurally independent protein import system has been substantiated by the recent identification of three mitochondrial inner membrane proteins, which are essential components of the transport machinery. This chapter focuses on the properties of these translocase of the mitochondrial inner membrane (Tim)-proteins. The identification of the Tim-proteins now opens a broad area for characterization of the import apparatus. This will include the topology of the Tims, their mode of interaction with preproteins, and the specificity of the transport machinery. Of particular importance will be the analysis of the interaction of the Tims with each other and with mt-Hsp70 and partner proteins, as well as the identification of putative further components required for inner membrane transport. The process of mitochondrial protein uptake appears to be unidirectional and irreversible, that is, an imported protein is unable to move back into the cytosol. The conformational changes accompanying the import, folding, and assembly of proteins are by themselves usually not sufficient to drive an unidirectional transport. This is particularly obvious in the case ofprotein translocation across the mitochondrial inner membrane. The supply of two external energy sources, ATP and the membrane potential AY across the inner membrane, is needed to drive protein import. The chapter discusses that with some preproteins short segments may move back across the inner membrane when the function of the matrix Hsp70 is strongly impaired—for example, by ATP-depletion of the matrix, supporting the view of an energy-driven unidirectional transport.

Topogenesis of inner membrane proteins of mitochondria

Abstract: Proteins of the inner mitochondrial membrane display a diverse range of topological arrangements. These proteins, encoded either by the nuclear or mitochondrial genomes, contain sequence determinants, so-called topo- genic signals, which direct newly synthesized proteins to their final orientation in the membrane. The recent analysis of sorting pathways of a number of proteins highlights that these topogenic signals might act in different ways.

Protein Import Across the Inner Mitochondrial Membrane

Abstract: Most mitochondrial proteins are synthesized in the cytosol and must then be transported across the organelle membranes to reach their functional destination. Mitochondria contain two translocation systems, one in the outer membrane and one in the inner membrane (Glick et al., 1991; Pfanner et al., 1992). The system in the mitochondrial outer membrane (MOM complex) consists of at least six protein components most of which have been isolated, cloned and sequenced (for review see Kiebler et al., 1993).