Showing papers on "Mitochondrial carrier published in 2000"

Dnm1p Gtpase-Mediated Mitochondrial Fission Is a Multi-Step Process Requiring the Novel Integral Membrane Component Fis1p

Abstract: Yeast Dnm1p is a soluble, dynamin-related GTPase that assembles on the outer mitochondrial membrane at sites where organelle division occurs. Although these Dnm1p-containing complexes are thought to trigger constriction and fission, little is known about their composition and assembly, and molecules required for their membrane recruitment have not been isolated. Using a genetic approach, we identified two new genes in the fission pathway, FIS1 and FIS2. FIS1 encodes a novel, outer mitochondrial membrane protein with its amino terminus exposed to the cytoplasm. Fis1p is the first integral membrane protein shown to participate in a eukaryotic membrane fission event. In a related study (Tieu, Q., and J. Nunnari. 2000. J. Cell Biol. 151:353–365), it was shown that the FIS2 gene product (called Mdv1p) colocalizes with Dnm1p on mitochondria. Genetic and morphological evidence indicate that Fis1p, but not Mdv1p, function is required for the proper assembly and distribution of Dnm1p-containing fission complexes on mitochondrial tubules. We propose that mitochondrial fission in yeast is a multi-step process, and that membrane-bound Fis1p is required for the proper assembly, membrane distribution, and function of Dnm1p-containing complexes during fission.

BNIP3 and Genetic Control of Necrosis-Like Cell Death through the Mitochondrial Permeability Transition Pore

Abstract: Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Abstract: Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182–1191

Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival

Abstract: Coupled cellular respiration requires that ATP and ADP be efficiently exchanged between the cytosol and the mitochondrial matrix. When growth factors are withdrawn from dependent cells, metabolism is disrupted by a defect in ATP/ADP exchange across the mitochondrial membranes. Unexpectedly, we find that this defect results from loss of outer mitochondrial membrane permeability to metabolic anions. This decrease in anion permeability correlates with the changes in conductance properties that accompany closure of the voltage-dependent anion channel (also known as mitochondrial porin). Loss of outer membrane permeability (i) results in the accumulation of stored metabolic energy within the intermembrane space in the form of creatine phosphate, (ii) is prevented by the outer mitochondrial membrane proteins Bcl-xL and Bcl-2, and (iii) can be reversed by growth factor readdition. If outer membrane impermeability persists, the disruption of mitochondrial homeostasis culminates in loss of outer mitochondrial membrane integrity, cytochrome c redistribution, and apoptosis. The recognition that outer membrane permeability is regulated under physiological conditions has important implications for the understanding of bioenergetics and cell survival.

Mdv1p is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division.

Abstract: Mitochondrial fission is mediated by the dynamin-related GTPase, Dnm1p, which assembles on the mitochondrial outer membrane into punctate structures associated with sites of membrane constriction and fission. We have identified additional nuclear genes required for mitochondrial fission, termed MDV (for mitochondrial division). MDV1 encodes a predicted soluble protein, containing a coiled-coil motif and seven COOH-terminal WD repeats. Genetic and two-hybrid analyses indicate that Mdv1p interacts with Dnm1p to mediate mitochondrial fission. In addition, Mdv1p colocalizes with Dnm1p in fission-mediating punctate structures on the mitochondrial outer membrane. Whereas localization of Mdv1p to these structures requires Dnm1p, localization of Mdv1p to mitochondrial membranes does not. This indicates that Mdv1p possesses a Dnm1p-independent mitochondrial targeting signal. Dnm1p-independent targeting of Mdv1p to mitochondria requires MDV2. Our data indicate that MDV2 also functions separately to regulate the assembly of Dnm1p into punctate structures. In contrast, Mdv1p is not required for the assembly of Dnm1p, but Dnm1p-containing punctate structures lacking Mdv1p are not able to complete division. Our studies suggest that mitochondrial fission is a multi-step process in which Mdv2p regulates the assembly of Dnm1p into punctate structures and together with Mdv1p functions later during fission to facilitate Dnm1p-dependent mitochondrial membrane constriction and/or division.

The Dynamin-Related Gtpase, Mgm1p, Is an Intermembrane Space Protein Required for Maintenance of Fusion Competent Mitochondria

Abstract: Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear. To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles. Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments. This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion. Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells. However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating. This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion. Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space. Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events. In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion.

Coenzyme Q is an obligatory cofactor for uncoupling protein function

Abstract: Uncoupling proteins (UCPs) are thought to be intricately controlled uncouplers that are responsible for the futile dissipation of mitochondrial chemiosmotic gradients, producing heat rather than ATP. They occur in many animal and plant cells and form a subfamily of the mitochondrial carrier family. Physiological uncoupling of oxidative phosphorylation must be strongly regulated to avoid deterioration of the energy supply and cell death, which is caused by toxic uncouplers. However, an H+ transporting uncoupling function is well established only for UCP1 from brown adipose tissue, and the regulation of UCP1 by fatty acids, nucleotides and pH remains controversial. The failure of UCP1 expressed in Escherichia coli inclusion bodies to carry out fatty-acid-dependent H+ transport activity inclusion bodies made us seek a native UCP cofactor. Here we report the identification of coenzyme Q (ubiquinone) as such a cofactor. On addition of CoQ10 to reconstituted UCP1 from inclusion bodies, fatty-acid-dependent H+ transport reached the same rate as with native UCP1. The H+ transport was highly sensitive to purine nucleotides, and activated only by oxidized but not reduced CoQ. H+ transport of native UCP1 correlated with the endogenous CoQ content.

Mitochondrial intermembrane junctional complexes and their role in cell death

Abstract: A mitochondrial complex comprising the voltage-dependent anion channel (outer membrane), the adenine nucleotide translocase (inner membrane) and cyclophilin-D (matrix) assembles at contact sites between the inner and outer membranes. Under pathological conditions associated with ischaemia and reperfusion the junctional complex ‘deforms’ into the permeability transition (PT) pore, which can open transiently, allowing free permeation of low Mr solutes across the inner membrane. This may be a critical step in the pathogenesis of lethal cell injury in ischaemia and reperfusion. Moreover, it is argued, the degree of pore opening may be an important determinant of the relative extent of apoptosis and necrosis under these conditions. In addition, mitochondria are the major sites of action of Bax and other apoptotic regulatory proteins of the Bcl-2 family. These proteins control a mitochondrial amplificatory loop in the apoptotic signalling pathway in which cytochrome c and other apoptogenic proteins of the mitochondrial intermembrane space are released into the cytosol. There are indications that the junctional complex, or components of it, may also mediate the action of Bax, but in a way that does not involve PT pore formation.

Gag3p, an Outer Membrane Protein Required for Fission of Mitochondrial Tubules

Abstract: Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth) Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane This association requires neither the DNM1 nor GAG2 gene products However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules

The role of the TIM8–13 complex in the import of Tim23 into mitochondria

Abstract: Tim8 and Tim13 are non-essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero-oligomeric complex. They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins. Here we show that the TIM8–13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23. The TIM8–13 complex binds to the N-terminal or intermediate domain of Tim23. It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation. The TIM8–13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria. The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr–Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness. It is demonstrated that import of human Tim23 is dependent on a high membrane potential. A mechanism to explain the pathology of MTS is discussed.

Tim18p Is a New Component of the Tim54p-Tim22p Translocon in the Mitochondrial Inner Membrane

Abstract: The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitive tim54-1 mutant. We identified a new gene, TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress the tim54-1 growth defect; the tim18::HIS3 disruption is synthetically lethal with tim54-1; Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa complex after blue native electrophoresis. We propose that Tim18p is a new component of the Tim54p-Tim22p machinery that facilitates insertion of polytopic proteins into the mitochondrial inner membrane.

Presence of a member of the mitochondrial carrier family in hydrogenosomes: conservation of membrane-targeting pathways between hydrogenosomes and mitochondria.

Abstract: A variety of phylogenetically diverse eukaryotes, including ciliates, fungi, and amoeboflagellates, lack typical eukaryotic organelles such as the mitochondrion and the peroxisome. Interestingly, these organisms often contain a double-membrane-bounded organelle called the hydrogenosome (32, 40). Organisms that contain this organelle live in microaerophilic habitats and rely on the hydrogenosome for fermentative carbohydrate metabolism. Similar to mitochondria, the hydrogenosome produces ATP, requiring its exchange with ADP from the cytosol. The hydrogenosome participates in carbohydrate metabolism, producing ATP, carbon dioxide, acetate, and molecular hydrogen from the fermentation of pyruvate. In this respect, hydrogenosomes can be regarded as the anaerobic equivalents of mitochondria as ATP generators in these cells. Although similar in many aspects, mitochondria and hydrogenosomes differ significantly in structure and function. The absence of cristae, DNA, F1F0 ATPase, respiratory-chain components, and cardiolipin and the presence of the enzymes hydrogenase and pyruvate-ferredoxin oxidoreductase in the hydrogenosome set it apart from the mitochondrion (17, 32). Based on the detection of enzymes typically present in anaerobic bacteria, it was proposed that the hydrogenosome originated from an endosymbiont related to the strict anaerobe Clostridium (31). Later, emerging similarities between mitochondria and hydrogenosomes led to the proposal that hydrogenosomes were converted mitochondria that lost their respiratory function as a result of movement into anaerobic habitats (6). Unfortunately, the lack of hydrogenosomal DNA has precluded a direct analysis of the origin of hydrogenosomes similar to that carried out using mitochondrial DNA which demonstrated that mitochondria evolved from an endosymbiont of the α-proteobacterial family (14). Recently, molecular analyses of hydrogenosomal heat shock proteins from Trichomonas vaginalis (5, 11, 16, 41) have demonstrated a close phylogenetic relationship between the nuclear genes encoding these proteins and their mitochondrial counterparts, suggesting a common symbiotic origin for the two organelles. The latest theory is one based on the metabolic force behind the symbiotic event—the hydrogen hypothesis (29). This hypothesis propounds that an α-proteobacterium which produced molecular hydrogen and carbon dioxide established a symbiosis with a methanogenic archaeon that utilized these as sources of energy. As these gases became depleted with changes in the early Earth's atmosphere, the host (the archaeon) would have become dependent on its symbiotic partner for its needs. This association further led to the transfer of genes from the symbiont to the host and to its successful establishment as an organelle. This ancient cell is proposed to be ancestral to eukaryotes that have diverged into a respiratory (mitochondria-containing) or a fermentative (hydrogenosome-containing) fate, depending on their habitats. A critical step in the evolution of the ancestral endosymbiont to an organelle would be the evolution of membrane proteins to allow communication between the organelle and its host cell. Hydrogenosomes and mitochondria undergo biogenesis by binary fission, followed by translocation of the nuclear-encoded proteins required for their functions (37, 40). Hence, membrane proteins would be essential not only for intracellular transport of substrates and products but also for translocation of host-encoded proteins during organelle biogenesis. These membrane proteins would have evolved at the time of or shortly prior to DNA transfer from symbiont to host nucleus. One of the first such membrane proteins that would have evolved in the case of ATP-producing organelles would be an ADP-ATP exchanger that would provide ATP to the cytosol (6). The evolution of this translocator would not be complete without developing a translocation machinery and organelle-targeting signals, all of which would involve a series of rare mutations. Therefore, the presence of phylogenetically-related membrane proteins and the use of similar translocation pathways for mitochondria and hydrogenosomes would reveal their evolution from a common progenitor if indeed a single ancestor gave rise to them as proposed (4, 29). Previous studies have indicated that common import signals are used for targeting proteins to the matrix of mitochondria and hydrogenosomes (4, 15, 50); however, studies comparing membrane translocation pathways have not been reported. Here, we report the characterization of the first membrane protein isolated from T. vaginalis hydrogenosomes and demonstrate that it is a member of the mitochondrial carrier family (MCF) (23). Within this group of MCF proteins, phylogenetic analysis indicates that the hydrogenosomal protein has a common origin with ADP-ATP carrier (AAC) proteins. In vivo and in vitro translocation analyses with hydrogenosomes and mitochondria demonstrate that AACs from the two organelles utilize similar translocation pathways and rely on internal signals for specific membrane targeting. These data reveal the presence of conserved membrane carriers in hydrogenosomes and mitochondria and indicate the coevolution of membrane protein-targeting pathways in the two organelles.

Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane.

Abstract: Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease

Abstract: Three cytosolic and one plasma membrane-bound 5′-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5′-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5′- and 2′(3′)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith–Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

Two Intermembrane Space Tim Complexes Interact with Different Domains of Tim23p during Its Import into Mitochondria

Abstract: Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p–Tim13p and Tim9p–Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p–Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH2-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p–Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p–Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

A Mitochondrial Division Apparatus Takes Shape

Abstract: Mitochondria are dynamic structures that divide and fuse continually throughout the life of a cell. Mitochondrial division is needed to maintain a full complement of mitochondria when cells divide, but also when they differentiate, increase in size or respond to changes in their environment. Despite

Targeting and insertion of nuclear-encoded preproteins into the mitochondrial outer membrane.

Abstract: Most mitochondrial proteins are synthesized in the cytosol as preproteins with a cleavable presequence and are delivered to the import receptors on the mitochondria by cytoplasmic import factors. The proteins are then imported to the intramitochondrial compartments by the import systems of the outer and inner membranes, TOM and TIM. Mitochondrial outer membrane proteins are synthesized without a cleavable presequence and most of them contain hydrophobic transmembrane domains, which, in conjunction with the flanking segments, function as the mitochondria import signals. Some of the proteins are inserted into the outer membrane by the TOM machinery; the import signal probably arrests further translocation and is released from the translocation channel to the lipid bilayer. The other proteins are inserted into the membrane by a novel pathway independent of the TOM machinery. This article reviews recent developments in the biogenesis of mitochondrial outer membrane proteins. BioEssays 22:364–371, 2000. © 2000 John Wiley & Sons, Inc.

Role of MMM1 in maintaining mitochondrial morphology in Neurospora crassa.

Abstract: Mmm1p is a protein required for maintenance of mitochondrial morphology in budding yeast. It was proposed that it is required to mediate the interaction of the mitochondrial outer membrane with the actin cytoskeleton. We report the cloning and characterization of MMM1 of the filamentous fungus Neurospora crassa, an organism that uses microtubules for mitochondrial transport. Mutation of the mmm-1 gene leads to a temperature-sensitive slow growth phenotype and female sterility. Mutant cells harbor abnormal giant mitochondria at all stages of the asexual life cycle, whereas actin filament-depolymerizing drugs have no effect on mitochondrial morphology. The MMM1 protein has a single transmembrane domain near the N terminus and exposes a large C-terminal domain to the cytosol. The protein can be imported into the outer membrane in a receptor-dependent manner. Our findings suggest that MMM1 is a factor of general importance for mitochondrial morphology independent of the cytoskeletal system used for mitochondrial transport.

Mitochondrial localization of AtOXA1, an arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane.

Abstract: ;Components of some protein complexes present in the inner membrane of mitochondria are encoded in both nuclear and mitochondrial genomes, and correct sorting and assembly of these proteins is necessary for proper respiratory function. Recent studies in yeast suggest that Oxa1p, a protein conserved between prokaryotes and eukaryotes, is an essential factor for protein sorting and assembly into membranes. We previously identified AtOXA1 ,a nArabidopsis homologue of OXA1 by functional complementation of a yeast oxa1 ‐ mutant. In this study, we investigated the genomic organization of AtOXA1 and localization of the AtOXA1 protein. Characterization of the AtOXA1 genomic region indicated that the gene consists of 10 exons and is located on chromosome V. A database search also revealed another gene coding for a putative protein homologous to AtOXA1 on chromosome II. Transient expression of a green fluorescent protein (GFP) fusion in suspension-cultured tobacco cells showed that AtOXA1 is targeted into mitochondria by its N-terminal presequence. Antibodies raised against AtOXA1 recognized a 38-kDa intrinsic protein of the inner mitochondrial membrane. Thus, localization of AtOXA1 in the mitochondrial inner membrane, together with our previous complementation experiment in yeast, suggested that it is a functional homologue of Oxa1p.

Isolation and characterization of cDNAs encoding mitochondrial uncoupling proteins in wheat: wheat UCP genes are not regulated by low temperature.

Abstract: Uncoupling proteins (UCP) found in the inner mitochondrial membrane of mammals dissipate the proton electrochemical gradient across the inner membrane to produce heat rather than synthesize ATP. Using PCR-based methods, we isolated two novel cDNA clones, WhUCP1a and WhUCP1b, that encode the mitochondrial uncoupling protein of wheat (Triticum aestivum L.). The cDNA clones each contain one ORF which can code for a protein of 286 amino acids with a predicted molecular mass of about 30.5 kDa, although three amino acid substitutions are found between them. The deduced amino acid sequences each possess three typical mitochondrial carrier signature domains and six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. Southern analysis suggested that the WhUCP1 gene may be present in as many as three copies in the wheat genome, and also that WhUCP proteins may be encoded by a small multigene family. Northern analysis revealed that the steady-state level of the WhUCP1 mRNA is quite low. Quantitative RT-PCR clearly showed that expression of the WhUCP1 gene in wheat seedlings is insensitive to low temperature. Our data suggest that WhUCP1 might have functions other than low temperature-induced thermogenesis, although WhUCP1 possesses all the typical features reported for known UCPs.

Uncoupling Proteins: Do They Have a Role in Body Weight Regulation?

Abstract: Several members of the mitochondrial carrier protein family are classified as uncoupling proteins. In contrast to the uncoupling protein specific to brown adipose tissue (UCP1), the physiological role of skeletal muscle uncoupling proteins (UCP2 and UCP3) in weight regulation seems more closely associated with the regulation of lipids as fuel substrate than as mediators of adaptive thermogenesis.