Giorgio Valentina, Bisetto Elena, Soriano Maria Eugenia, Dabbeni-Sala Federica, Basso Emy, Petronilli Valeria, Forte Michael A, Bernardi Paolo, Lippe Giovanna
Department of Biomedical Sciences and the Consiglio Nazionale delle Ricerche Institute of Neuroscience, University of Padova, I-35121 Padova, Italy.
J Biol Chem. 2009 Dec 4;284(49):33982-8. doi: 10.1074/jbc.M109.020115. Epub 2009 Sep 29.
Blue native gel electrophoresis purification and immunoprecipitation of F(0)F(1)-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F(1) sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.
通过蓝色非变性凝胶电泳从牛心线粒体中纯化F(0)F(1)-ATP合酶并进行免疫沉淀，结果显示亲环蛋白（CyP）D与该复合物相关联。用膜通透性双功能试剂3,3'-二硫代双丙酸二甲酯（DTBP）处理完整线粒体，可使CyPD与ATP合酶的侧柄交联，而未观察到与F(1)亚基、ATP合酶天然抑制蛋白IF1以及ATP/ADP载体之间的相互作用。ATP合酶与CyPD的相互作用对酶催化具有功能影响，并受到磷酸盐（增加CyPD结合并降低酶活性）和环孢素（Cs）A（降低CyPD结合并增加酶活性）的调节。用CsA处理MgATP亚线粒体颗粒或完整线粒体，可使CyPD从膜上解离，并激活该酶维持的ATP水解和合成。在CyPD缺失的线粒体中未检测到CsA的作用，该线粒体显示出比野生型线粒体更高的ATP合酶比活性。CyPD结合的调节似乎独立于IF1，IF1与ATP合酶的结合不受CsA处理的影响。这些发现表明，CyPD与ATP合酶侧柄的结合调节了该复合物的活性。
