Zíková Alena, Schnaufer Achim, Dalley Rachel A, Panigrahi Aswini K, Stuart Kenneth D
Seattle Biomedical Research Institute, Seattle, WA, USA.
PLoS Pathog. 2009 May;5(5):e1000436. doi: 10.1371/journal.ppat.1000436. Epub 2009 May 15.
The mitochondrial F(0)F(1) ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F(0)F(1) ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F(1) subunits, three to F(0) subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F(1) alpha subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F(0)F(1)-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought.
线粒体F(0)F(1) ATP合酶是绝大多数真核生物中一种重要的多亚基蛋白复合体，但对于它在早期分化的真核病原体布氏锥虫中的组成和作用却知之甚少。我们通过亲和纯化、免疫沉淀和蓝色天然凝胶电泳相结合的方法纯化了F(0)F(1) ATP合酶，并对其组成和功能进行了表征。我们鉴定出了22种蛋白质，其中5种与F(1)亚基相关，3种与F(0)亚基相关，还有14种与动质体以外的蛋白质没有明显的同源性。对F(1)α亚基或两种新蛋白质之一的表达进行RNA干扰沉默表明，它们各自对于前循环期（昆虫阶段）细胞的存活至关重要，并且对于F(0)F(1)-ATP合酶复合体的结构完整性也很重要。在沉默这些蛋白质中的每一种的表达后，我们还观察到氧化磷酸化产生的ATP显著减少，而底物磷酸化并未受到严重影响。与早期报道相反，我们的前循环期布氏锥虫细胞即使在有葡萄糖存在的情况下对ATP合酶抑制剂寡霉素也很敏感。因此，这两种新蛋白质似乎对于功能复合体的结构组织至关重要，并且这些生物体中线粒体能量产生的调节比以前认为的更为复杂。
