Mitochondrial translocation of APE1 relies on the MIA pathway

APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1's mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival

COA6 facilitates cytochrome c oxidase biogenesis as thiol-reductase for copper metallochaperones in mitochondria.

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6 are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulphide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis

The oxidation status of Mic19 regulates MICOS assembly.

The function of mitochondria depends on the proper organization of mitochondrial membranes. The morphology of the inner membrane is regulated by the recently identified mitochondrial contact site and cristae organizing system (MICOS) complex. MICOS mutants exhibit alterations in crista formation, leading to mitochondrial dysfunction. However, the mechanisms that underlie MICOS regulation remain poorly understood. MIC19, a peripheral protein of the inner membrane and component of the MICOS complex, was previously reported to be required for the proper function of MICOS in maintaining the architecture of the inner membrane. Here, we show that human and yeast MIC19 proteins undergo oxidation in mitochondria and require the mitochondrial intermembrane space assembly (MIA) pathway, which couples the oxidation and import of mitochondrial intermembrane space proteins for mitochondrial localization. Detailed analyses identified yeast Mic19 in two different redox forms. The form that contains an intramolecular disulfide bond is bound to Mic60 of the MICOS complex. Mic19 oxidation is not essential for its integration into the MICOS complex but plays a role in MICOS assembly and the maintenance of proper inner membrane morphology. These findings suggest that Mic19 is a redox-dependent regulator of MICOS function

Proteasome activity contributes to prosurvival response upon mild mitochondrial stress in Caenorhabditis elegans

Defects in mitochondrial function activate compensatory responses in the cell. Mitochondrial stress that is caused by unfolded proteins inside the organelle induces a transcriptional response (termed the “mitochondrial unfolded protein response” [UPRmt]) that is mediated by activating transcription factor associated with stress 1 (ATFS-1). The UPRmt increases mitochondrial protein quality control. Mitochondrial dysfunction frequently causes defects in the import of proteins, resulting in the accumulation of mitochondrial proteins outside the organelle. In yeast, cells respond to mistargeted mitochondrial proteins by increasing activity of the proteasome in the cytosol (termed the “unfolded protein response activated by mistargeting of proteins” [UPRam]). The presence and relevance of this response in higher eukaryotes is unclear. Here, we demonstrate that defects in mitochondrial protein import in Caenorhabditis elegans lead to proteasome activation and life span extension. Both proteasome activation and life span prolongation partially depend on ATFS-1, despite its lack of influence on proteasomal gene transcription. Importantly, life span prolongation depends on the fully assembled proteasome. Our data provide a link between mitochondrial dysfunction and proteasomal activity and demonstrate its direct relevance to mechanisms that promote longevity

Structural determinants of PINK1 topology and dual subcellular distribution

<p>Abstract</p> <p>Background</p> <p>PINK1 is a mitochondria-targeted kinase that constitutively localizes to both the mitochondria and the cytosol. The mechanism of how PINK1 achieves cytosolic localization following mitochondrial processing remains unknown. Understanding PINK1 subcellular localization will give us insights into PINK1 functions and how mutations in PINK1 lead to Parkinson's disease. We asked how the mitochondrial localization signal, the transmembrane domain, and the kinase domain participate in PINK1 localization.</p> <p>Results</p> <p>We confirmed that PINK1 mitochondrial targeting signal is responsible for mitochondrial localization. Once inside the mitochondria, we found that both PINK1 transmembrane and kinase domain are important for membrane tethering and cytosolic-facing topology. We also showed that PINK1 dual subcellular distribution requires both Hsp90 interaction with the kinase domain and the proteolysis at a cleavage site downstream of the transmembrane domain because removal of this cleavage site completely abolished cytosolic PINK1. In addition, the disruption of the Hsp90-PINK1 interaction increased mitochondrial PINK1 level.</p> <p>Conclusion</p> <p>Together, we believe that once PINK1 enters the mitochondria, PINK1 adopts a tethered topology because the transmembrane domain and the kinase domain prevent PINK1 forward movement into the mitochondria. Subsequent proteolysis downstream of the transmembrane domain then releases PINK1 for retrograde movement while PINK1 kinase domain interacts with Hsp90 chaperone. The significance of this dual localization could mean that PINK1 has compartmental-specific functions.</p

The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling

Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to null mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low

Screening for Toxic Amyloid in Yeast Exemplifies the Role of Alternative Pathway Responsible for Cytotoxicity

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species

Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole

TESTLoc: protein subcellular localization prediction from EST data

Abstract Background The eukaryotic cell has an intricate architecture with compartments and substructures dedicated to particular biological processes. Knowing the subcellular location of proteins not only indicates how bio-processes are organized in different cellular compartments, but also contributes to unravelling the function of individual proteins. Computational localization prediction is possible based on sequence information alone, and has been successfully applied to proteins from virtually all subcellular compartments and all domains of life. However, we realized that current prediction tools do not perform well on partial protein sequences such as those inferred from Expressed Sequence Tag (EST) data, limiting the exploitation of the large and taxonomically most comprehensive body of sequence information from eukaryotes. Results We developed a new predictor, TESTLoc, suited for subcellular localization prediction of proteins based on their partial sequence conceptually translated from ESTs (EST-peptides). Support Vector Machine (SVM) is used as computational method and EST-peptides are represented by different features such as amino acid composition and physicochemical properties. When TESTLoc was applied to the most challenging test case (plant data), it yielded high accuracy (~85%). Conclusions TESTLoc is a localization prediction tool tailored for EST data. It provides a variety of models for the users to choose from, and is available for download at http://megasun.bch.umontreal.ca/~shenyq/TESTLoc/TESTLoc.html</p