Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy
Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy
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DOI：
10.1073/pnas.1506593112
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发表时间：
2015-05-26
影响因子：
11.1
通讯作者：
Harper, J. Wade
中科院分区：
文献类型：
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作者：
Ordureau, Alban;Heo, Jin-Mi;Harper, J. Wade
PTEN诱导的推定激酶蛋白1（PINK 1）和泛素（UB）连接酶PARKIN直接损伤线粒体进行线粒体自噬。PINK 1促进PARKIN募集到线粒体外膜（OMT），用于具有典型和非典型UB链的OMT蛋白的泛素化。PINK 1磷酸化PARKIN的UB样结构域中的Ser 65（S65）和UB本身中的保守Ser，但PARKIN激活和线粒体质量控制期间这些事件的时间顺序和相对重要性仍然知之甚少。使用“UBS 65 A替代”，我们发现PARKIN磷酸化和激活以及MOM蛋白质组上的Lys残基的遍在化类似地发生，无论UB替代在S65上磷酸化的能力如何。相反，多聚泛素（poly-UB）链的合成，PARKIN在细胞上的保留，以及线粒体自噬在UBS 65 A替代细胞中减少。类似的实验研究了单个UB链连接类型的作用，揭示了K6和K63链连接在线粒体自噬中的重要性，但PINK 1对K63链的磷酸化并没有增强体外与候选线粒体自噬受体视神经磷酸酶（OPTN）、多价螯合体-1（p62）和核点蛋白52（NDP 52）的结合。总线粒体的平行反应监测蛋白质组学显示，当PARKIN不能构建UB链时，p-S65-UB不存在，
The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using "UBS65A-replacement," we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UBS65A-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and