Alan 2017 MiP2017
Mitochondrial F1FO ATP synthase is the key enzyme of the oxidative phosphorylation apparatus, responsible for the production of up to 90% of the cellular ATP. The mammalian enzyme is composed of 17 structural subunits – two of the recently characterized ones (MLQ and DAPIT) are specific for higher eukaryotes. MLQ, also termed 6.8 kDa proteolipid (MP68), is nuclearly encoded protein with 58 amino acids, containing one putative membrane domain. The complete structure of MLQ, its stoichiometry within the enzyme, or its exact localization in the FO domain is as of yet unknown.
To clarify the biological role of MLQ, we created a MLQ knock-out model in the HEK293 cells by the CRISPR/Cas9 technology. We demonstrate defective biogenesis of the fully assembled ATP synthase in the absence of MLQ. While mitochondrially encoded subunits FO-a and A6L are synthesized and assembled into the complex, MLQ is required for their stabilization in the holoenzyme. In its absence, FO-a and A6L dissociate and are degraded as confirmed by pulse chase experiments. As a result, at the steady state, incomplete enzyme lacking the proton channel formed by subunit FO-a is present in MLQ -/- mitochondria. At the functional level, we observed significantly reduced rates of ADP stimulated respiration in MLQ -/- cells, while the hydrolytic activity of the ATP synthase was preserved. Secondary to the ATP synthase deficiency we observed decreased mRNA and protein levels of the mitochondrially encoded subunits of cytochrome c oxidase (CIV). This led to the reduced levels of COX holoenzyme and decrease in ATP synthase independent state 3 (FCCP) respiration.
We will discuss the molecular role of MLQ in F1FO ATP synthase assembly as well as the functional impact of MLQ KO on the cellular energetics. Moreover, we will report on progress of FO-a and A6L subunits degradation mechanism in MLQ KO cells.
Labels: MiParea: Respiration, Genetic knockout;overexpression
Organism: Human Tissue;cell: Kidney
Coupling state: ET
Affiliations and support
- Inst Physiol, Czech Acad Sciences, Prague, Czech Republic. - [email protected]
- The project is supported by the Czech Science Foundation (16-01813S).