The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.The S100 proteins comprise a multigene family of low-molecular-weight EF-hand calcium binding and zinc binding proteins (5, 13, 16, 24, 33). To date, 19 different S100 proteins have been assigned to this protein family, and they show different degrees of similarity, ranging from 25 to 56% identity at the amino acid level. With S100B, S100P, and S100Z being the exceptions, the majority of the S100 genes are clustered on human chromosome 1q21 (33). Most S100 proteins serve as calcium sensor proteins that, upon activation, regulate the function and/or subcellular distribution of specific target proteins (13, 33, 47), and they are characterized by common structural motifs, including two low-affinity (K_D [equilibrium dissociation constant] of ∼10 μM to 100 μM) helix-loop-helix calcium binding domains (EF hands) that are separated by a hinge region and flanked by amino- and carboxy-terminal domains. The carboxy-terminal domain is variable among S100 proteins, and it typically is the site that is responsible for the selective interaction of each individual S100 protein with specific target proteins (30). S100 proteins are often upregulated in cancers, in inflammation, and in response to cellular stress (14, 16), suggesting that they function in cell responses to stress situations. Consistent with this hypothesis, stress situations were necessary to reveal phenotypes associated with the S100 knockout in mice (11, 14, 33, 56). Moreover, recent observations revealed a new function for the S100 protein family that included their ability to assist and regulate multichaperone complex-ligand interactions (41, 50, 51).One member of the S100 protein family, S100B, has attracted much interest in the past few years because, like other proteins implicated in neurodegeneration (e.g., amyloid, superoxide dismutase, and dual-specificity tyrosine phosphorylation-regulated kinase 1A), its gene is located within a segment of chromosome 21, which is trisomic in Down''s syndrome (DS). Its expression in the brain of mammals coincides with defined periods of central nervous system (CNS) maturation and cell differentiation (43). In oligodendrocyte progenitor cells (OPC), S100B expression is associated with differentiation, and S100B contributes to OPC differentiation in response to demyelinating insult (11). To understand the contribution of S100B to OPC differentiation, we searched for high-affinity S100B target proteins in this cell type by using far-Western analysis. A major and highly specific S100B target protein was identified, the mitochondrial ATAD3A protein.ATAD3A belongs to a new family of eukaryote-specific mitochondrial AAA⁺ ATPase proteins (17). In the human genome, two genes, Atad3A and Atad3B, are located in tandem on chromosome 1p36.33. The Atad3A gene is ubiquitous among multicellular organisms but absent in yeast. The Atad3B gene is specific to the human genome (27). ATAD3A is a mitochondrial protein anchored into the mitochondrial inner membrane (IM) at contact sites with the outer membrane (OM). Thanks to its simultaneous interaction with the two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the inner and outer membranes and controls diverse cell responses ranging from mitochondrial metabolism, cell growth, and mitochondrial fission 20a, 25). The ATAD3A protein has also been identified as a mitochondrial DNA binding protein (23) and as a cell surface antigen in some human tumors (20, 21). The plasma membrane localization of ATAD3A in tumor cells is suggestive that ATAD3A mitochondrial routing can be compromised in pathological situations such as cancer. To understand the functional response resulting from the interaction between S100B and ATAD3A, we first characterized the minimal interaction domain on ATAD3A for S100B binding using thermodynamic studies of wild-type and ATAD3A variants as well as via nuclear magnetic resonance (NMR) spectroscopy techniques. These studies allowed us to further refine the consensus S100B binding motif, which is conserved in several other S100B target proteins, including the p53 protein and several newly discovered target proteins associated with the cell translational machinery. We next analyzed the cellular interaction of S100B with truncated ATAD3A mutants that harbor the S100B binding domain but that are deficient for mitochondrial import. These studies revealed that S100B could assist ATAD3A mutant proteins during cytoplasmic processing by preventing dysfunctional aggregation events. Our results are discussed in light of the possible function of S100B in assisting the cytoplasmic processing of proteins for proper folding and subcellular localization.     

ATAD2B is a phylogenetically conserved nuclear protein expressed during neuronal differentiation and tumorigenesis

ATAD2 is an E2F target gene that is highly expressed in gastrointestinal and breast carcinomas. Here we characterize a related gene product, ATAD2B. Both genes are evolutionarily conserved, with orthologues present in all eukaryotic genomes examined. Human ATAD2B shows a high degree of similarity to ATAD2. Both contain an AAA domain and a bromodomain with amino acid sequences sharing 97% and 74% identity, respectively. The expression of ATAD2B was studied in the chicken embryo using a polyclonal antibody raised against a recombinant fragment of human ATAD2B. Immunohistochemistry revealed transient nuclear expression in subpopulations of developing neurons. The transient nature of the expression was confirmed by immunoblotting homogenates of the developing telencephalon. Cell fractionation was used to confirm the nuclear localization of ATAD2B in the developing nervous system: anti-ATAD2B recognizes a smaller band (approximately 160 kDa) in the nuclear fraction and a larger band (approximately 300 kDa) in the membrane fraction, suggesting that posttranslational processing of ATAD2B may regulate its transport to the nucleus. The expression of ATAD2B was also studied in human tumors. Oncomine and immunohistochemistry reveal ATAD2B expression in glioblastoma and oligodendroglioma; ATAD2B immunostaining was also elevated in human breast carcinoma. In tumors ATAD2B appears to be cytoplasmic or membrane bound, and not nuclear. Our observations suggest that ATAD2B may play a role in neuronal differentiation and tumor progression.     

ATAD3, a vital membrane-bound mitochondrial ATPase involved in tumor progression

ATAD3 (ATPase family AAA domain-containing protein 3) is a mitochondrial membrane bound ATPase whose function has not yet been discovered but its role is essential for the embryonic development. The ATAD3 gene exists since the pluri-cellular organisms with specialized tissues and remains unique until vertebrates. In primates and humans, two other genes have appeared (called ATAD3B and ATAD3C versus ATAD3A the ancestral gene). ATAD3 knock down in different non-transformed cell lines is associated with drastic changes in the mitochondrial network, inhibition of proliferation and modification of the functional interactions between mitochondria and endoplasmic reticulum. However, the analysis of the functions of ATAD3A and ATAD3B in different human cancer cell lines shows on the contrary that they can have anti-proliferative effects and induce chemoresistant properties. ATAD3 may therefore be implicated in an unknown but essential and growth-linked mitochondrial function existing since pluri-cellular -organization and involved in tumorigenesis.     

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.     

ATAD3, a vital membrane bound mitochondrial ATPase involved in tumor progression

ATAD3 (ATPase family AAA Domain-containing protein 3) is a mitochondrial membrane bound ATPase whose function has not yet been discovered but its role is essential for embryonic development. The ATAD3 gene has existed since the pluri-cellular organisms with specialized tissues and has remained unique until vertebrates. In primates and human, two other genes have appeared (called ATAD3B and ATAD3C versus ATAD3A the ancestral gene). ATAD3 knock-down in different non-transformed cell lines is associated with drastic changes in the mitochondrial network, inhibition of proliferation and modification of the functional interactions between mitochondria and endoplasmic reticulum. However, the analysis of the cellular properties of ATAD3A and ATAD3B in different human cancer cell lines shows on the contrary that they can present anti-proliferative and chemoresistant properties. ATAD3 may therefore be implicated in an unknown but essential and growth-linked mitochondrial function existing since pluri-cellular organization and involved in tumorigenesis.     

Expression analysis of ATAD3 isoforms in rodent and human cell lines and tissues

ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67 kDa, (ATAD3l; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the peri-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human.     

Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria

Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes.     

膜蛋白ATAD3A在线粒体质量控制中的作用

线粒体质量控制对于线粒体网络的稳态和线粒体功能的正常发挥具有重要意义。三磷酸腺苷酶家族蛋白3A(ATAD3A)是同时参与调节线粒体结构功能、线粒体动力学和线粒体自噬等重要生物学过程的线粒体膜蛋白之一。近期研究表明,ATAD3A既可与Mic60/Mitofilin和线粒体转录因子A (TFAM)等因子相互作用以维持线粒体嵴的形态和氧化磷酸化功能,又能与发动蛋白相关蛋白1 (Drp1)结合而正性/负性调节线粒体分裂,还可作为线粒体外膜转位酶（TOM）复合物和线粒体内膜转位酶（TIM）复合物之间的桥接因子而介导PTEN诱导激酶（PINK1）输入线粒体进行加工,显示出促自噬或抗自噬活性。本文对ATAD3A在调控线粒体质量控制中的作用及其机制进行了综述。     

The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.     

ATAD3B is a mitophagy receptor mediating clearance of oxidative stress‐induced damaged mitochondrial DNA

Mitochondrial DNA (mtDNA) encodes several key components of respiratory chain complexes that produce cellular energy through oxidative phosphorylation. mtDNA is vulnerable to damage under various physiological stresses, especially oxidative stress. mtDNA damage leads to mitochondrial dysfunction, and dysfunctional mitochondria can be removed by mitophagy, an essential process in cellular homeostasis. However, how damaged mtDNA is selectively cleared from the cell, and how damaged mtDNA triggers mitophagy, remain mostly unknown. Here, we identified a novel mitophagy receptor, ATAD3B, which is specifically expressed in primates. ATAD3B contains a LIR motif that binds to LC3 and promotes oxidative stress‐induced mitophagy in a PINK1‐independent manner, thus promoting the clearance of damaged mtDNA induced by oxidative stress. Under normal conditions, ATAD3B hetero‐oligomerizes with ATAD3A, thus promoting the targeting of the C‐terminal region of ATAD3B to the mitochondrial intermembrane space. Oxidative stress‐induced mtDNA damage or mtDNA depletion reduces ATAD3B‐ATAD3A hetero‐oligomerization and leads to exposure of the ATAD3B C‐terminus at the mitochondrial outer membrane and subsequent recruitment of LC3 for initiating mitophagy. Furthermore, ATAD3B is little expressed in m.3243A > G mutated cells and MELAS patient fibroblasts showing endogenous oxidative stress, and ATAD3B re‐expression promotes the clearance of m.3243A > G mutated mtDNA. Our findings uncover a new pathway to selectively remove damaged mtDNA and reveal that increasing ATAD3B activity is a potential therapeutic approach for mitochondrial diseases.     

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.     

ATAD3B is a human embryonic stem cell specific mitochondrial protein, re-expressed in cancer cells, that functions as dominant negative for the ubiquitous ATAD3A

Here we report on the identification of a human pluripotent embryonic stem cell (hESC) specific mitochondrial protein that is re-expressed in cancer cells, ATAD3B. ATAD3B belongs to the AAA+ ATPase ATAD3 protein family of mitochondrial proteins specific to multicellular eukaryotes. Using loss- and gain-of-function approaches, we show that ATAD3B associates with the ubiquitous ATAD3A species, negatively regulates the interaction of ATAD3A with matrix nucleoid complexes and contributes to a mitochondria fragmentation phenotype. We conclude that ATAD3B is a negative regulator of ATAD3A and may function as an adaptor of mitochondrial homeostasis and metabolism in hESCs and cancer cells.     

Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells.

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels (< 1 microg x mg(-1) membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins. Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.     

Mutations in MTO2 related to tRNA modification impair mitochondrial gene expression and protein synthesis in the presence of a paromomycin resistance mutation in mitochondrial 15 S rRNA

Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA.     

ATAD3 proteins: brokers of a mitochondria–endoplasmic reticulum connection in mammalian cells

In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)–mitochondria organizing network (ERMIONE) controls mitochondria–ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear‐encoded ATPases associated with diverse cellular activities (AAA+‐ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain‐containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial–ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE‐like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial–ER interaction and recognizable neurological syndromes. The primate‐specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties.     

Topological analysis of ATAD3A insertion in purified human mitochondria

ATAD3 is a mitochondrial inner membrane-associated protein that has been predicted to be an ATPase but from which no associated function is known. The topology of ATAD3 in mitochondrial membranes is not clear and subject to controversy. A direct interaction of the N-terminal domain (amino-acids 44–247) with the mtDNA has been described, but the same domain has been reported to be sensitive to limited proteolysis in purified mitochondria. Furthermore, ATAD3 has been found in a large purified nucleoid complex but could not be cross-linked to the nucleoid. To resolve these discrepancies we used two immunological approaches to test whether the N-terminal (amino-acids 40–53) and the C-terminal (amino-acids 572–586) regions of ATAD3 are accessible from the cytosol. Using N-terminal and C-terminal specific anti-peptide antibodies, we carried out back-titration ELISA measurements and immuno-fluorescence analysis on freshly purified human mitochondria. Both approaches showed that the N-terminal region of ATAD3A is accessible to antibodies in purified mitochondria. The N-terminal region of ATAD3A is thus probably in the cytoplasm or in an accessible intermembrane space. On the contrary, the C-terminal region is not accessible to the antibody and is probably located within the matrix. These results demonstrate both that the N-terminal part of ATAD3A is outside the inner membrane and that the C-terminal part is inside the matrix.     

Mdm12p, a Component Required for Mitochondrial Inheritance That Is Conserved between Budding and Fission Yeast

Saccharomyces cerevisiae cells lacking the MDM12 gene product display temperature-sensitive growth and possess abnormally large, round mitochondria that are defective for inheritance by daughter buds. Analysis of the wild-type MDM12 gene revealed its product to be a 31-kD polypeptide that is homologous to a protein of the fission yeast Schizosaccharomyces pombe. When expressed in S. cerevisiae, the S. pombe Mdm12p homolog conferred a dominant-negative phenotype of giant mitochondria and aberrant mitochondrial distribution, suggesting partial functional conservation of Mdm12p activity between budding and fission yeast. The S. cerevisiae Mdm12p was localized by indirect immunofluorescence microscopy and by subcellular fractionation and immunodetection to the mitochondrial outer membrane and displayed biochemical properties of an integral membrane protein. Mdm12p is the third mitochondrial outer membrane protein required for normal mitochondrial morphology and distribution to be identified in S. cerevisiae and the first such mitochondrial component that is conserved between two different species.     

Msp1/ATAD1 maintains mitochondrial function by facilitating the degradation of mislocalized tail‐anchored proteins

The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1^−/− mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity.     

Isolation and characterization of the putative nuclear modifier gene MTO1 involved in the pathogenesis of deafness-associated mitochondrial 12 S rRNA A1555G mutation

The human mitochondrial 12 S rRNA A1555G mutation has been found to be associated with aminoglycoside-induced and non-syndromic deafness. However, putative nuclear modifier gene(s) have been proposed to regulate the phenotypic expression of this mutation. In yeast, the mutant alleles of MTO1, encoding a mitochondrial protein, manifest respiratory-deficient phenotype only when coupled with the mitochondrial 15 S rRNA P(R)454 mutation corresponding to human A1555G mutation. This suggests that the MTO1-like modifier gene may influence the phenotypic expression of human A1555G mutation. Here we report the identification of full-length cDNA and elucidation of genomic organization of the human MTO1 homolog. Human Mto1 is an evolutionarily conserved protein that implicates a role in the mitochondrial tRNA modification. Functional conservation of this protein is supported by the observation that isolated human MTO1 cDNA can complement the respiratory deficient phenotype of yeast mto1 cells carrying P(R)454 mutation. MTO1 is ubiquitously expressed in various tissues, but with a markedly elevated expression in tissues of high metabolic rates including cochlea. These observations suggest that human MTO1 is a structural and functional homolog of yeast MTO1. Thus, it may play an important role in the pathogenesis of deafness-associated A1555G mutation in 12 S rRNA gene or mutations in tRNA genes.