Open Access

Mitochondria contain their own protein synthesis machinery with mitoribosomes that are similar to prokaryotic ribosomes. The thirteen proteins encoded in the mitochondrial genome are members of the respiratory chain complexes that generate a proton gradient, which is the electromotoric force for ATP synthesis. NOA1 (Nitric Oxide Associated Protein-1) is a nuclear encoded GTPase that positively influences mitochondrial respiration and ATP production. Although a role in mitoribosome assembly was assigned to NOA1 the underlying molecular mechanism is poorly understood. This work shows that the multi-domain protein NOA1 serves multiple purposes for the function of mitochondria. NOA1 is a dual localized protein that makes a detour through the nucleus before mitochondrial import. The nuclear shuttling is mediated by a nuclear localization signal and the now identified nuclear export signal. SELEX (Systemic Evolution of Ligands by Exponential Enrichment) analysis revealed a G-quadruplex binding motif that characterizes NOA1 as ribonucleoprotein (RNP). G-quadruplex binding was coupled to the GTPase activity and increased the GTP hydrolysis rate. The sequence of localization events and the identification of NOA1 being a RNP lead to the discussion of an alternative import pathway for RNPs into mitochondria. The short-lived NOA1 contains ClpX recognition motifs and is specifically degraded by the mitochondrial matrix protease ClpXP. NOA1 is the first reported substrate of ClpXP in higher eukaryotes and augments the contribution of the ClpXP protease for mitochondrial metabolism. To assess the direct action of NOA1 on the mitoribosome co-sedimentation assays were performed. They showed that the interaction of NOA1 and the mitoribosome is dependent on the GTPase function and the nascent peptide chain. In vitro, NOA1 facilitated the membrane insertion of newly translated and isotope labeled mitochondrial translation products into inverted mitochondrial inner membrane vesicles. In conclusion, NOA1 is a G-quadruplex-RNP that acts as mitochondrial membrane insertion factor for mtDNA-encoded proteins. This thesis provides a comprehensive model of the molecular function of NOA1 and is the basis for future research. The identification of NOA1 as ClpXP substrate is a major contribution to the field of mitochondrial research.

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.