A cox25mutant in the BY4741 background created by replacement of the fullCOX25gene with aKANMXcassette was obtained from Open Biosystems/Thermo Scientific (Huntsville, AL). matrix. Cox25 is an essential component of the complexes made up of newly synthesized Cox1, Ssc1, Mss51, and Cox14. In addition, Cox25 is also found to interact with Shy1 and Cox5 in a complex that does not contain Mss51. These results suggest that once Ssc1-Mss51 are released from the Cox1 stabilization complex, Cox25 Z-LEHD-FMK continues to interact with Cox14 and Cox1 to facilitate the formation of multisubunit COX assembly intermediates. Keywords:Cytochrome Oxidase, Mitochondria, Mitochondrial Metabolism, Protein Assembly, Translation Regulation, Yeast, COX14, COX25, MSS51, SSC1 == Introduction == Eukaryotic cells rely on the mitochondrial respiratory chain and oxidative phosphorylation system for aerobic ATP production. Cytochromecoxidase (COX)3is a heme A-copper terminal oxidase. It is the last enzyme of the respiratory chain and plays fundamental functions both in electron transfer from reduced cytochromecto molecular oxygen and in proton Z-LEHD-FMK pumping through the inner mitochondrial membrane to contribute to the generation of a proton gradient in the intermembrane space that is subsequently used by the F1F0-ATP synthase to drive synthesis of ATP. COX biogenesis is usually complicated by its dual genetic origin, with subunits (11 in yeast and 13 in mammals) encoded both in the organelle and in the nucleus. In most cases, the three subunits forming the catalytic core of the enzyme (subunits 13) are encoded in the mitochondrial DNA. In the yeastSaccharomyces cerevisiae, Z-LEHD-FMK COX assembly requires the assistance of at least 30 nuclear gene products acting at all stages of the assembly process (1,2). COX assembly requires the accumulation of its constitutive subunits in a defined stoichiometric ratio. Previous studies led to the notion of two mechanisms responsible for the concerted accumulation of COX subunits in yeast mitochondria. First, most unassembled COX subunit 1 and the other highly hydrophobic core subunits 2 and 3 are very efficiently post-translationally degraded (3). Second, Cox1 is usually subject to assembly-controlled translational auto-regulation (49). This kind of translational regulation was initially found to operate in the assembly of photosynthetic complexes in chloroplasts from the algaChlamydomonas reinardthii(10,11) and in higher plants (12) and termed control GRIA3 by epistasis of synthesis. A distinctive characteristic of these organellar translational auto-regulatory systems is the involvement of ternary factors, mRNA-specific translational activators, whose availability would be regulated by the specific gene products. In the case of yeast COX, the ternary factor is Mss51, a specific translational activator ofCOX1mRNA (49). Mss51 acts around the 5-UTR ofCOX1mRNA to promote translation initiation (4,7) and additionally acts on a target in the protein coding sequence ofCOX1mRNA, perhaps to promote elongation (4). During Cox1 synthesis around the mitoribosomes, Mss51 and newly synthesized Cox1 form a transient complex (4,6) that is stabilized by Cox14 (6), the mitochondrial hsp70 chaperone Ssc1, and its co-chaperone Mdj1 (8). Following Cox1 synthesis, the Ssc1-Mss51-Cox1-Cox14 complex remains stable until Cox1 proceeds to downstream assembly steps. We have postulated that these interactions down-regulate Cox1 synthesis when COX assembly is usually impaired by trapping Mss51 and limiting its availability forCOX1mRNA translation (6,8). The C-terminal residues of Cox1 have recently been shown to be essential for Mss51 sequestration and to stabilize the Mss51-Cox14 conversation (9). We have shown that when Mss51 is usually released from the complex, it is still in a very stable binary complex with Ssc1 (8). According to this Z-LEHD-FMK model, the release of Mss51-Ssc1 from the post-translational complex and Mss51 availability for Cox1 synthesis (8) probably occur when Cox1 acquires its prosthetic groups or interacts with other COX subunits, a step possibly catalyzed by Shy1, a protein involved in maturation and/or assembly of Cox1 (6,13,14). Coa1 could also participate in Cox1 maturation. Coa1 has been proposed to stabilize the Cox1-Ssc1-Mss51-Cox14 complex prior to its conversation with Shy1.