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© 2019 The Authors., The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here, we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated in arthropods a paralog protein (SLIMP) that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM and preventing mitochondrial DNA (mtDNA) accumulation. Thus, mitochondrial translation is directly coupled to mtDNA levels by a network based upon a profound structural modification of an animal ARS., This work was supported by grants SVP2014-068398 and BIO2015-64572 from the Spanish Ministry of Economy and Competitiveness (to L.R.d.P.).

We disrupted the three conserved disulfide bonds in Xenopus β3 (C127−C144, C154-C170, C191-C248), one at a time, using Cys to Ala/Ser mutations and coexpression with relatively ouabain-resistant (C113Y) Xenopus α1 in oocytes. We assessed function with two-microelectrode voltage clamp in 1µM ouabain to silence endogenous Na/K pumps. Single C127S/A or C154S, or double C127S+C154S β3 mutants yielded steady Na/K pump currents (saturating [Ko]) and transient Na charge movements (without Ko) like those of C113Y α1 coexpressed with wild-type β3, suggesting that neither assembly, trafficking, nor Na/K pump function require intact first and/or second disulfide bonds. In contrast, steady and transient pump currents were both ≥10 fold smaller in Na/K pumps with disrupted third disulfide bond after C113Y α1 coexpression with C191A/S β3. Similarly, in outside-out excised patches, palytoxin-induced pump-channel currents were ≥10 fold smaller for C113Y α1 Na/K pumps coexpressed with β3 C191S than with wild-type β3. Because in uninjected oocytes or oocytes expressing C113Y α1 alone, neither steady nor transient pump current could be detected, and palytoxin elicited no measurable current in outside-out patches, the third disulfide bond appears necessary for efficient Na/K-ATPase assembly and trafficking to the cell membrane but is not essential for assembled pump function. We disrupted salt bridges linking the αTM7-TM8 loop and β-exodomain in Na/K-ATPase structures (α1 E901-β3 K219, α1 E911-β3 R262), by coexpressing E901R(C113Y) or E911R(C113Y) α1 with wild-type β3. In both mutants, steady Na/K pump currents (at saturating [Ko]) and transient Na charge movements were like wild type; but the E901R α1 mutation selectively enhanced apparent Ko affinity ∼2 fold. These results suggest that some β-exodomain structural alterations can be tolerated; but α-β subunit interface interactions closely regulate Ko binding/occlusion steps, though not the slow step that occludes/deoccludes the third Na ion. [HL36783]

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.