Vevea Jason D, Alessi Wolken Dana M, Swayne Theresa C, White Adam B, Pon Liza A
Department of Pathology and Cell Biology, Columbia University.
Herbert Irving Comprehensive Cancer Center, Columbia University.
J Vis Exp. 2013 Jul 22(77):50633. doi: 10.3791/50633.
Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
线粒体在许多细胞过程中发挥作用，从能量代谢、钙稳态到细胞寿命控制和程序性细胞死亡。这些过程影响线粒体的氧化还原状态和ATP产生，并受其影响。在这里，我们描述了两种比率型、基因编码的生物传感器的用途，它们可以在活酵母细胞中以亚细胞分辨率检测线粒体氧化还原状态和ATP水平。线粒体氧化还原状态使用靶向线粒体基质的氧化还原敏感型绿色荧光蛋白（roGFP）进行测量。线粒体roGFP在GFP的第147和204位含有半胱氨酸，它们经历可逆的、依赖环境的氧化和还原，进而改变蛋白质的激发光谱。MitGO-ATeam是一种Förster共振能量转移（FRET）探针，其中F0F1-ATP合酶的ε亚基夹在FRET供体和受体荧光蛋白之间。ATP与ε亚基的结合导致蛋白质构象变化，使FRET供体和受体靠近，并允许荧光共振能量从供体转移到受体。
