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线粒体F1-ATP酶α或β亚基中DELSEED环N端边缘的突变在缺乏中央γ转子的情况下会增强ATP水解。

Mutations on the N-terminal edge of the DELSEED loop in either the α or β subunit of the mitochondrial F1-ATPase enhance ATP hydrolysis in the absence of the central γ rotor.

作者信息

La Thuy, Clark-Walker George Desmond, Wang Xiaowen, Wilkens Stephan, Chen Xin Jie

机构信息

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York, USA.

出版信息

Eukaryot Cell. 2013 Nov;12(11):1451-61. doi: 10.1128/EC.00177-13. Epub 2013 Sep 6.

Abstract

F(1)-ATPase is a rotary molecular machine with a subunit stoichiometry of α(3)β(3)γ(1)δ(1)ε(1). It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α(3)β(3) core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F(1)-ATPase have suggested that the α(3)β(3) core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F(1)-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ(o)) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F(1) complexes retained 21.7 to 44.6% of the native F(1)-ATPase activity. The γ-less F(1) subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ(o) conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α(3)β(3) subcomplex.

摘要

F(1)-ATP酶是一种旋转分子机器,亚基化学计量比为α(3)β(3)γ(1)δ(1)ε(1)。由于三个催化位点之间有效的协同作用,它具有强大的ATP水解活性。据信,中央γ转子决定了α(3)β(3)核心中催化位点的顺序构象变化,以实现协同作用。然而,最近对嗜热芽孢杆菌PS3 F(1)-ATP酶的研究表明,α(3)β(3)核心可以内在地进行单向协同催化(T. Uchihashi等人,《科学》333:755 - 758,2011)。这种不依赖γ的ATP水解模式的机制尚不清楚。在这里,一种独特的遗传筛选使我们能够鉴定出α和β亚基中的特定突变,这些突变在没有γ的情况下刺激线粒体F(1)-ATP酶的ATP水解。我们发现,α亚基中的F446I突变和β亚基中的G419D突变通过缺乏γ的乳酸克鲁维酵母突变体中线粒体DNA丢失(ρ(o))来抑制细胞死亡。体外ATP酶分析表明,突变体而非野生型无γ F(1)复合物保留了天然F(1)-ATP酶活性的21.7%至44.6%。无γ F(1)亚复合物被组装,但在体外结构和功能不稳定。α亚基中的Phe446和β亚基中的Gly419都位于两个亚基中DELSEED环的N端边缘。这两个位点的突变可能增强了催化所需构象变化向相邻α或β亚基的传递,从而在ρ(o)条件下实现强大的ATP水解和细胞存活。这项工作可能有助于我们理解α(3)β(3)亚复合物进行ATP水解所需的结构元件。

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