PrimPol is required for replication reinitiation after mtDNA damage.

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells.

Proteostasis is crucial for life and maintained by cellular chaperones and proteases. One major mitochondrial protease is the ClpXP complex, which is comprised of a catalytic ClpX subunit and a proteolytic ClpP subunit. Based on two separate observations, we hypothesized that ClpX may play a leading role in the cellular function of ClpXP. Therefore, we analyzed the effect of ClpX overexpression on a myoblast proteome by quantitative proteomics. ClpX overexpression results in the upregulation of markers of the mitochondrial proteostasis pathway, known as the "mitochondrial unfolded protein response" (UPRmt). Although this pathway is described in detail in Caenorhabditis elegans, it is not clear whether it is conserved in mammals. Therefore, we compared features of the classical nematode UPRmt with our mammalian ClpX-triggered UPRmt dataset. We show that they share the same retrograde mitochondria-to-nucleus signaling pathway that involves the key UPRmt transcription factor CHOP (also known as Ddit3, CEBPZ or GADD153). In conclusion, our data confirm the existence of a mammalian UPRmt that has great similarity to the C. elegans pathway. Furthermore, our results illustrate that ClpX overexpression is a good and simple model to study the underlying mechanisms of the UPRmt in mammalian cells.

Binding to G-quadruplex RNA activates the mitochondrial GTPase NOA1.

NOA1 is an evolutionary conserved, nuclear encoded GTPase essential for mitochondrial function and cellular survival. The function of NOA1 for assembly of mitochondrial ribosomes and regulation of OXPHOS activity depends on its GTPase activity, but so far no ligands have been identified that regulate the GTPase activity of NOA1. To identify nucleic acids that bind to the RNA-binding domain of NOA1 we employed SELEX (Systemic Evolution of Ligands by EXponential Enrichment) using recombinant mouse wildtype NOA1 and the GTPase mutant NOA1-K353R. We found that NOA1 binds specifically to oligonucleotides that fold into guanine tetrads (G-quadruplexes). Binding of G-quadruplex oligonucleotides stimulated the GTPase activity of NOA1 suggesting a regulatory link between G-quadruplex containing RNAs, NOA1 function and assembly of mitochondrial ribosomes.

Nitric oxide-associated protein 1 (NOA1) is necessary for oxygen-dependent regulation of mitochondrial respiratory complexes.

In eukaryotic cells, maintenance of cellular ATP stores depends mainly on mitochondrial oxidative phosphorylation (OXPHOS), which in turn requires sufficient cellular oxygenation. The crucial role of proper oxygenation for cellular viability is reflected by involvement of several mechanisms, which sense hypoxia and regulate activities of respiratory complexes according to available oxygen concentrations. Here, we focus on mouse nitric oxide-associated protein 1 (mNOA1), which has been identified as an important component of the machinery that adjusts OXPHOS activity to oxygen concentrations. mNOA1 is an evolutionary conserved GTP-binding protein that is involved in the regulation of mitochondrial protein translation and respiration. We found that mNOA1 is located mostly in the mitochondrial matrix from where it interacts with several high molecular mass complexes, most notably with the complex IV of the respiratory chain and the prohibitin complex. Knock-down of mNOA1 impaired enzyme activity I+III, resulting in oxidative stress and eventually cell death. mNOA1 is transcriptionally regulated in an oxygen-sensitive manner. We propose that oxygen-dependent regulation of mNOA1 is instrumental to adjusting OXPHOS activity to oxygen availability, thereby controlling mitochondrial metabolism.

Hypoxic transcription gene profiles under the modulation of nitric oxide in nuclear run on-microarray and proteomics.

BACKGROUND: Microarray analysis still is a powerful tool to identify new components of the transcriptosome. It helps to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis. RESULTS: We identified 196 genes that were significantly regulated by hypoxia, 85 genes affected by nitric oxide and 292 genes induced by the cotreatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to all treatments but with different levels of expression in each group. We observed that 162 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia-regulated targets by NO. CONCLUSION: By eliminating the interference of steady state mRNA in gene expression profiling, we obtained a smaller number of significantly regulated transcripts in our study compared to published microarray data and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signaling.

NOA1, a novel ClpXP substrate, takes an unexpected nuclear detour prior to mitochondrial import.

The mitochondrial matrix GTPase NOA1 is a nuclear encoded protein, essential for mitochondrial protein synthesis, oxidative phosphorylation and ATP production. Here, we demonstrate that newly translated NOA1 protein is imported into the nucleus, where it localizes to the nucleolus and interacts with UBF1 before nuclear export and import into mitochondria. Mutation of the nuclear localization signal (NLS) prevented both nuclear and mitochondrial import while deletion of the N-terminal mitochondrial targeting sequence (MTS) or the C-terminal RNA binding domain of NOA1 impaired mitochondrial import. Absence of the MTS resulted in accumulation of NOA1 in the nucleus and increased caspase-dependent apoptosis. We also found that export of NOA1 from the nucleus requires a leptomycin-B sensitive, Crm1-dependent nuclear export signal (NES). Finally, we show that NOA1 is a new substrate of the mitochondrial matrix protease complex ClpXP. Our results uncovered an unexpected, mandatory detour of NOA1 through the nucleolus before uptake into mitochondria. We propose that nucleo-mitochondrial translocation of proteins is more widespread than previously anticipated providing additional means to control protein bioavailability as well as cellular communication between both compartments.

Postnatal cardiomyocyte growth and mitochondrial reorganization cause multiple changes in the proteome of human cardiomyocytes.

Fetal (fCM) and adult cardiomyocytes (aCM) significantly differ from each other both by structure and biochemical properties. aCM own a higher mitochondrial mass compared to fCM due to increased energy demand and show a greater density and higher degree of structural organization of myofibrils. The energy metabolism in aCM relies virtually completely on ß-oxidation of fatty acids while fCM use carbohydrates. Rewinding of the aCM phenotype (de-differentiation) arises frequently in diseased hearts spurring questions about its functional relevance and the extent of de-differentiation. Yet, surprisingly little is known about the changes in the human proteome occurring during maturation of fCM to aCM. Here, we examined differences between human fetal and adult hearts resulting in the quantification of 3500 proteins. Moreover, we analyzed mitochondrial proteomes from both stages to obtain more detailed insight into underlying biochemical differences. We found that the majority of changes between fCM and aCM were attributed to growth and maturation of cardiomyocytes. As expected, adult hearts showed higher mitochondrial mass and expressed increased levels of proteins involved in energy metabolism but relatively lower copy numbers of mitochondrial DNA (mtDNA) per total cell volume. We uncovered that the TFAM/mtDNA ratio was kept constant during postnatal development despite a significant increase of mitochondrial protein per mtDNA in adult mitochondria, which revises previous concepts.

Noncovalent, site-specific biotinylation of histidine-tagged proteins.

Site-specific conjugation of proteins to surfaces, spectroscopic probes, or other functional units is a key task for implementing biochemical assays. The streptavidin-biotin interaction has proven a highly versatile tool for detection, quantification, and functional analysis of proteins. We have developed an approach for site-specific reversible biotinylation of recombinant proteins through their histidine tag using biotin conjugated to the multivalent chelator trisnitrilotriacetic acid (BTtris-NTA). Stable binding of BTtris-NTA to His-tagged proteins was demonstrated, which is readily reversed by addition of imidazole, enabling versatile conjugation schemes in solution as well as at interfaces. Gel filtration experiments revealed that His-tagged proteins bind to streptavidin doped with BTtris-NTA in a 2:1 stoichiometry. Furthermore, an increased binding affinity toward His-tagged proteins was observed for BTtris-NTA linked to streptavidin compared to tris-NTA in solution and on surfaces. These results indicate an efficient cooperative interaction of two adjacent tris-NTA moieties with a single His-tag, yielding an extremely tight complex with a lifetime of several days. We demonstrate several applications of BTtris-NTA including multiplexed capturing of proteins to biosensor surfaces, cell surface labeling, and Western blot detection. The remarkable selectivity of the His-tag-specific biotinylation, as well as the highly stable, yet reversible complex provides the basis for numerous further applications for functional protein analysis.