Loss-of-function studies show that the human mitochondrial YME1L protease ensures cell proliferation, maintains normal cristae morphology and complex I activity, acts in an antiapoptotic manner, protects mitochondria from accumulation of oxidatively damaged membrane proteins, and is involved in proteolytic regulation of respiratory chain biogenesis.

The biogenesis of eukaryotic COX (cytochrome c oxidase) requires several accessory proteins in addition to structural subunits and prosthetic groups. We have analysed the assembly state of COX and SCO2 protein levels in various tissues of six patients with mutations in SCO2 and SURF1. SCO2 is a copper-binding protein presumably involved in formation of the Cu(A) centre of the COX2 subunit. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated that COX holoenzyme is reduced to 10-20% in skeletal muscle and brain of SCO2 and SURF1 patients and to 10-30% in heart of SCO2 patients, whereas liver of SCO2 patients' contained normal holoenzyme levels. The steady-state levels of mutant SCO2 protein ranged from 0 to 20% in different SCO2 patient tissues. In addition, eight distinct COX subcomplexes and unassembled subunits were found, some of them identical with known assembly intermediates of the human enzyme. Heart, brain and skeletal muscle of SCO2 patients contained accumulated levels of the COX1.COX4.COX5A subcomplex, three COX1-containing subcomplexes, a COX4.COX5A subcomplex and two subcomplexes composed of only COX4 or COX5A. The accumulation of COX1.COX4.COX5A subcomplex, along with the virtual absence of free COX2, suggests that the lack of the Cu(A) centre may result in decreased stability of COX2. The appearance of COX4.COX5A subcomplex indicates that association of these nucleus-encoded subunits probably precedes their addition to COX1 during the assembly process. Finally, the consequences of SCO2 and SURF1 mutations suggest the existence of tissue-specific functional differences of these proteins that may serve different tissue-specific requirements for the regulation of COX biogenesis.

Mammalian CcO (cytochrome c oxidase) is a hetero-oligomeric protein complex composed of 13 structural subunits encoded by both the mitochondrial and nuclear genomes. To study the role of nuclear-encoded CcO subunits in the assembly and function of the human complex, we used stable RNA interference of COX4, COX5A and COX6A1, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)-293 cells. Knockdown of Cox4, Cox5a and Cox6a resulted in reduced CcO activity, diminished affinity of the residual enzyme for oxygen, decreased holoCcO and CcO dimer levels, increased accumulation of CcO subcomplexes and gave rise to an altered pattern of respiratory supercomplexes. An analysis of the patterns of CcO subcomplexes found in both knockdown and overexpressing cells identified a novel CcO assembly intermediate, identified the entry points of three late-assembled subunits and demonstrated directly the essential character as well as the interdependence of the assembly of Cox4 and Cox5a. The ectopic expression of the heart/muscle-specific isoform of the Cox6 subunit (COX6A2) resulted in restoration of both CcO holoenzyme and activity in COX6A1-knockdown cells. This was in sharp contrast with the unaltered levels of COX6A2 mRNA in these cells, suggesting the existence of a fixed expression programme. The normal amount and function of respiratory complex I in all of our CcO-deficient knockdown cell lines suggest that, unlike non-human CcO-deficient models, even relatively small amounts of CcO can maintain the normal biogenesis of this respiratory complex in cultured human cells.

Sco1 and Sco2 are mitochondrial copper-binding proteins involved in the biogenesis of the Cu(A) site in the cytochrome c oxidase (CcO) subunit Cox2 and in the maintenance of cellular copper homeostasis. Human Surf1 is a CcO assembly factor with an important but poorly characterized role in CcO biogenesis. Here, we analyzed the impact on CcO assembly and tissue copper levels of a G132S mutation in the juxtamembrane region of SCO1 metallochaperone associated with early onset hypertrophic cardiomyopathy, encephalopathy, hypotonia, and hepatopathy, assessed the total copper content of various SURF1 and SCO2-deficient tissues, and investigated the possible physical association between CcO and Sco1. The steady-state level of mutant Sco1 was severely decreased in the muscle mitochondria of the SCO1 patient, indicating compromised stability and thus loss of function of the protein. Unlike the wild-type variant, residual mutant Sco1 appeared to migrate exclusively in the monomeric form on blue native gels. Both the activity and content of CcO were reduced in the patient's muscle to approximately 10-20% of control values. SCO1-deficient mitochondria showed accumulation of two Cox2 subcomplexes, suggesting that Sco1 is very likely responsible for a different posttranslational aspect of Cox2 maturation than Sco2. Intriguingly, the various SURF1-deficient samples analyzed showed a tissue-specific copper deficiency similar to that of SCO-deficient samples, suggesting a role for Surf1 in copper homeostasis regulation. Finally, both blue native immunoblot analysis and coimmunoprecipitation revealed that a fraction of Sco1 physically associates with the CcO complex in human muscle mitochondria, suggesting a possible direct relationship between CcO and the regulation of cellular copper homeostasis.