Karunadharma Pabalu P, Basisty Nathan, Chiao Ying Ann, Dai Dao-Fu, Drake Rachel, Levy Nick, Koh William J, Emond Mary J, Kruse Shane, Marcinek David, Maccoss Michael J, Rabinovitch Peter S
*Department of Pathology, Department of Environmental Health and Biostatistics, Department of Radiology, and Department of Genome Sciences, University of Washington, Seattle, Washington, USA; and The Scripps Research Institute, Jupiter, Florida, USA.
*Department of Pathology, Department of Environmental Health and Biostatistics, Department of Radiology, and Department of Genome Sciences, University of Washington, Seattle, Washington, USA; and The Scripps Research Institute, Jupiter, Florida, USA
FASEB J. 2015 Aug;29(8):3582-92. doi: 10.1096/fj.15-272666. Epub 2015 May 14.
The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [(2)H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman's ρ = 0.57; P < 10(-4)), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions.
线粒体呼吸链（RC）产生细胞内大部分ATP，需要严格的质量控制机制。为了在体内检测RC亚基蛋白质稳态，我们在不同条件下，通过液相色谱-串联质谱联用代谢性[（2）H3]-亮氨酸重同位素标记法测量了小鼠体内RC蛋白的半衰期（HLs）。我们研究了对照饮食或两种饮食方案（热量限制或雷帕霉素）之一喂养的年轻和年老小鼠的7种组织/组分，这两种饮食方案改变了蛋白质周转（总共42种条件）。我们观察到各组织间平均HL有6.5倍的差异，所有条件下有11.5倍的差异。将每种条件下的平均HL标准化后发现，相对HL在各条件下保持一致（斯皮尔曼相关系数ρ = 0.57；P < 10^(-4)），但亚基间高度异质性，总体平均范围为7.3倍，每个复合物内为2.2至4.6倍。为了确定调节这种保守分布的因素，我们进行了统计分析，以研究HL与亚基特性之间的相关性。HL与在线粒体内的定位、进化起源、蛋白质编码位置以及泛素化水平显著相关。这些发现挑战了复合物中所有亚基以可比速率周转的观点，并表明在不同细胞条件下，存在共同规则控制RC蛋白亚基的差异蛋白水解。
