Characterization of the mitochondrial GlutamyltRNAGln amidotransferase (GatCAB) as a new model for mitochondrial translation disorders.

Abstract

Mitochondrial OXPHOS diseases are provoked by dysfunction of the OXPHOS system, showing heterogeneity from a genetic, biochemical and clinical perspective. This complexity is partly due to the involvement of proteins encoded in two genomes located in different cellular compartments, nuclear and mitochondrial DNA (mtDNA). mtDNA encodes part of the mtRNAs translation machinery, including 22 tRNAs. All these are charged by their corresponding aminoacyl-tRNA synthetases (ARS2), except mt-Gln-tRNAGln whose synthesis is carried out through an indirect pathway. The mt-tRNAGln is charged with glutamic acid (Glu) by a non-discriminating mitochondrial glutamyl-tRNA synthetase (EARS2), then the Glu is converted to Gln by a Glutamyl-tRNAGln amidotransferase, using free glutamine as an amide donor, yielding Gln- tRNAGln. This mitochondrial amidotransferase activity lies in the GatCAB complex, which is formed by three subunits: GatA (QSRL1), GatB (GATB), and GatC (GATC). Patients with mutations in these genes presented with severe cardiomyopathy and lactic acidosis, which underscores the importance of the GatCAB complex as an essential component in the translational machinery of mitochondrial protein synthesis. We have generated Knockout (KO) lines for all subunits using the genomic editing system CRISPR/Cas9 in HEK293T cells. We measured the levels of the GatCAB subunits and mitochondrial proteins by western blot. We studied the oxygen consumption levels using a Clark electrode and detected the GatCAB complex by Blue Native-western blotting. In all cases, KO cells show reduced levels of mitochondrial proteins, decreased oxygen consumption and an absence of the GatCAB complex compared to wildtype cells. In the QRSL1 and GATC KO cells the levels of all subunits diminish, but in GATB KO cells the levels of subunits A and C remain stable. Possibly, QRSL1 and GATC could form a stable but nonfunctional dimer, to which GATB would join. GATB has two potential ATG codons at the start of its sequence separated by 12 nucleotides. From over 90 clones analyzed, we only obtained one KO, by generating an upstream ORF, and two cell lines with very low expression of GATB. The latter present a duplicated region at the beginning of GATB which changes the reading frame from the first ATG; suggesting the downstream ATG as an alternative translation start codon for those ribosome small subunits that do not assemble on the first one, yielding a GATB protein missing 4 amino acids. Characterization of the molecular mechanisms that lead to the synthesis of mt-Gln-tRNAGln and its role in the physiology of the cell could allow us to comprehend the pathological manifestation of the defects in these genes and propose possible therapeutic avenues.