Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3–Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol–cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.

Kgd4 is a novel subunit of the mitochondria! a-ketoglutarate dehydrogenase complex (KGDH). In yeast, the protein is present in two forms of unknown origin, as there is only one open reading frame and no alternative splicing. Here, we show that the two forms of Kgd4 derive from one mRNA that is translated by employing two alternative start sites. The standard, annotated AUG codon gives rise to the short form of the protein, while an upstream UUG codon is utilized to generate the larger form. However, both forms can be efficiently imported into mitochondria and stably incorporate into KGDH to support its activity. Translation of the long variant depends on sequences directly upstream of the alternative initiation site, demonstrating that translation initiation and its efficiency are dictated by the sequence context surrounding a specific codon. In summary, the two forms of Kgd4 follow a very unusual biogenesis pathway, supporting the notion that translation initiation in yeast is more flexible than it is widely recognized.

Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na+/H+ antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na+/H+ antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by Delta pH, but no longer respond to either an electrochemical potential (psi) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na+/H+ exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na+/H+ antiporters.