Ordureau Alban, Heo Jin-Mi, Duda David M, Paulo Joao A, Olszewski Jennifer L, Yanishevski David, Rinehart Jesse, Schulman Brenda A, Harper J Wade
Department of Cell Biology, Harvard Medical School, Boston, MA 02115;
Department of Structural Biology, Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105; and.
Proc Natl Acad Sci U S A. 2015 May 26;112(21):6637-42. doi: 10.1073/pnas.1506593112. Epub 2015 May 12.
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 "UB(S65A)-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 UB(S65A)-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 <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains.
PTEN诱导的假定激酶蛋白1（PINK1）和泛素（UB）连接酶PARKIN引导受损线粒体进行线粒体自噬。PINK1促进PARKIN募集到线粒体外膜（MOM），以便用典型和非典型UB链对MOM蛋白进行泛素化。PINK1使PARKIN的类泛素结构域中的Ser65（S65）以及泛素本身的保守Ser磷酸化，但这些事件在PARKIN激活和线粒体质量控制过程中的时间顺序和相对重要性仍知之甚少。使用“UB（S65A）替代”，我们发现，无论UB替代物在S65上被磷酸化的能力如何，PARKIN的磷酸化和激活以及一组MOM蛋白上赖氨酸残基的泛素化都以相似的方式发生。相比之下，在UB（S65A）替代细胞中，多聚泛素（poly-UB）链合成、PARKIN在MOM上的保留以及线粒体自噬都减少了。研究单个UB链连接类型作用的类似实验揭示了K6和K63链连接在线粒体自噬中的重要性，但PINK1对K63链的磷酸化在体外并未增强与候选线粒体自噬受体视黄醛结合蛋白（OPTN）、聚集体蛋白1（p62）和核点蛋白52（NDP52）的结合。对总线粒体进行的平行反应监测蛋白质组学显示，当PARKIN无法构建UB链时不存在p-S65-UB，并且在线粒体受损时，单体UB池中<0.16%的UB发生S65磷酸化。在体外将p-S65-UB和p-S65-PARKIN结合显示，未磷酸化的UB加速转移到PARKIN自身、其底物线粒体Rho GTP酶（MIRO）和UB上。我们的数据进一步定义了一种前馈线粒体泛素化途径，该途径涉及磷酸化后PARKIN的激活、MOM上的UB链合成、UB链磷酸化以及通过与磷酸化UB链结合进一步募集PARKIN并进行酶促扩增。
