Mitochondrial cytochrome c oxidase (COX) is the primary site of cellular oxygen consumption and is essential for aerobic energy generation in the form of ATP. Human COX is a copper-heme A hetero-multimeric complex formed by 3 catalytic core subunits encoded in the mitochondrial DNA and 11 subunits encoded in the nuclear genome. Investigations over the last 50 years have progressively shed light into the sophistication surrounding COX biogenesis and the regulation of this process, disclosing multiple assembly factors, several redox-regulated processes leading to metal co-factor insertion, regulatory mechanisms to couple synthesis of COX subunits to COX assembly, and the incorporation of COX into respiratory supercomplexes. Here, we will critically summarize recent progress and controversies in several key aspects of COX biogenesis: linear versus modular assembly, the coupling of mitochondrial translation to COX assembly and COX assembly into respiratory supercomplexes.

Unlike in metazoans, plant microRNAs (miRNAs) undergo stepwise nuclear maturation before engaging cytosolic, sequence-complementary transcripts in association with the silencing effector protein ARGONAUTE1 (AGO1). Since their discovery, how and under which form plant miRNAs translocate to the cytosol has remained unclear, as has their sub-cellular AGO1 loading site(s). Here, we show that the N termini of all plant AGO1s contain a nuclear-localization (NLS) and nuclear-export signal (NES) that, in Arabidopsis thaliana (At), enables AtAGO1 nucleo-cytosolic shuttling in a Leptomycin-B-inhibited manner, diagnostic of CRM1(EXPO1)/NES-dependent nuclear export. Nuclear-only AtAGO1 contains the same 2'O-methylated miRNA cohorts as its nucleo-cytosolic counterpart, but it preferentially interacts with the miRNA loading chaperone HSP90. Furthermore, mature miRNA translocation and miRNA-mediated silencing both require AtAGO1 nucleo-cytosolic shuttling. These findings lead us to propose a substantially revised view of the plant miRNA pathway in which miRNAs are matured, methylated, loaded into AGO1 in the nucleus, and exported to the cytosol as AGO1:miRNA complexes in a CRM1(EXPO1)/NES-dependent manner.

Copper is essential for proper functioning of cytochrome c oxidases, and therefore for cellular respiration in eukaryotes and many bacteria. Here we show that a new periplasmic protein (PCuAC) selectively inserts Cu(I) ions into subunit II of Thermus thermophilus ba3 oxidase to generate a native CuA site. The purported metallochaperone Sco1 is unable to deliver copper ions; instead, it works as a thiol-disulfide reductase to maintain the correct oxidation state of the CuA cysteine ligands.