title: Proteomic analysis of F1F0-ATP synthase super-assembly in mitochondria of cardiomyoblasts undergoing differentiation to the cardiac lineage.
abstract: Mitochondria are essential organelles with multiple functions, especially in energy metabolism. An increasing number of data highlighted their role for cellular differentiation processes. We investigated differences in ATP synthase supra-molecular organization occurring in H9c2 cardiomyoblasts in the course of cardiac-like differentiation, along with ATP synthase biogenesis and maturation of mitochondrial cristae morphology. Using BN-PAGE analysis combined with one-step mild detergent extraction from mitochondria, a significant increase in dimer/monomer ratio was observed, indicating a distinct rise in the stability of the enzyme super-assembly. Remarkably, sub-stoichiometric mean values for ATP synthase subunit e were determined in both parental and cardiac-like H9c2 by an MS-based quantitative proteomics approach. This indicates a similar high proportion of complex molecules lacking subunit e in both cell types, and suggests a minor contribution of this component in the observed changes. 2D BN-PAGE/immunoblotting analysis and MS/MS analysis on single BN-PAGE band showed that the amount of inhibitor protein IF1 bound within the ATP synthase complexes increased in cardiac-like H9c2 and appeared greater in the dimer. In concomitance, a consistent improvement of enzyme activity, measured as both ATP synthesis and ATP hydrolysis rate, was observed, despite the increase of bound IF1 evocative of a greater inhibitory effect on the enzyme ATPase activity. The results suggest i) a role for IF1 in promoting dimer stabilization and super-assembly in H9c2 with physiological IF1 expression levels, likely unveiled by the fact that the contacts through accessory subunit e appear to be partially destabilized, ii) a link between dimer stabilization and enzyme activation.
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, Udine, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Bisetto
authors.forename: Marina
authors.initials: M
authors.lastname: Comelli
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Salzano
authors.forename: Paola
authors.initials: P
authors.lastname: Picotti
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.forename: Giovanna
authors.initials: G
authors.lastname: Lippe
authors.forename: Irene
authors.initials: I
authors.lastname: Mavelli
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D015151:Immunoblotting; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D021122:Protein Subunits; D040901:Proteomics; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D021122:Protein Subunits; D000255:Adenosine Triphosphate; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases