The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.

• Keywords
• COX, COX4, OXPHOS, biogenesis interdependency, cI, cIV, cIV assembly, complex I, complexome profiling, knock-out, mitochondria, mitochondrial protein synthesis,
• MeSH
• Glycolysis MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mitochondrial Diseases metabolism   MeSH
• Mitochondrial Proteins biosynthesis   MeSH
• Oxidative Phosphorylation MeSH
• Protein Subunits metabolism   MeSH
• Protein Biosynthesis * MeSH
• Electron Transport Complex IV metabolism   MeSH
• Oxygen Consumption MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• COX4I1 protein, human MeSH Browser
• Mitochondrial Proteins MeSH
• Protein Subunits MeSH
• Electron Transport Complex IV MeSH

Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.

• Keywords
• AAA complex, AFG3L2, YME1L, mitochondria, protease,
• MeSH
• ATPases Associated with Diverse Cellular Activities genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Metalloendopeptidases genetics   metabolism   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria metabolism   ultrastructure   MeSH
• Cell Proliferation genetics   physiology   MeSH
• ATP-Dependent Proteases genetics   metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AFG3L2 protein, human MeSH Browser
• ATPases Associated with Diverse Cellular Activities MeSH
• Metalloendopeptidases MeSH
• Mitochondrial Proteins MeSH
• ATP-Dependent Proteases MeSH
• YME1L1 protein, human MeSH Browser

Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.

• Keywords
• Cytochrome c oxidase, Doxycycline, Leigh syndrome, Pulse-chase, Respiratory supercomplexes, SURF1(−/−) mouse knockout,
• MeSH
• Species Specificity MeSH
• Fibroblasts metabolism   pathology   MeSH
• Leigh Disease genetics   metabolism   pathology   MeSH
• Humans MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Organ Specificity MeSH
• Electron Transport Complex IV genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Mitochondrial Proteins MeSH
• Electron Transport Complex IV MeSH
• Surf-1 protein MeSH Browser

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

• MeSH
• Apoptosis MeSH
• ATPases Associated with Diverse Cellular Activities MeSH
• Gene Knockdown Techniques MeSH
• GTP Phosphohydrolases metabolism   MeSH
• Humans MeSH
• Metalloendopeptidases metabolism   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins MeSH
• Mitochondria metabolism   MeSH
• NADH, NADPH Oxidoreductases metabolism   MeSH
• Cell Proliferation * MeSH
• ATP-Dependent Proteases metabolism   MeSH
• Peptide Hydrolases metabolism   MeSH
• Protein Isoforms metabolism   MeSH
• Electron Transport Complex I MeSH
• Electron Transport Complex IV metabolism   MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae cytology   metabolism   MeSH
• Electron Transport * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATPases Associated with Diverse Cellular Activities MeSH
• GTP Phosphohydrolases MeSH
• Metalloendopeptidases MeSH
• Mitochondrial Proteins MeSH
• NADH, NADPH Oxidoreductases MeSH
• NDUFB6 protein, human MeSH Browser
• OPA1 protein, human MeSH Browser
• ATP-Dependent Proteases MeSH
• Peptide Hydrolases MeSH
• Protein Isoforms MeSH
• Electron Transport Complex I MeSH
• Electron Transport Complex IV MeSH
• Saccharomyces cerevisiae Proteins MeSH
• YME1 protein, S cerevisiae MeSH Browser
• YME1L1 protein, human MeSH Browser