This report aims to explore how Bcl-xL, a Bcl-2 family protein, regulates PINK1/Parkin-dependent mitophagy. Compared with the Hela cells expressing Parkin alone, co-expression of Bcl-xL significantly inhibited CCCP (Carbonyl cyanide 3- chlorophenylhydrazone)-induced mitochondrial Parkin accumulation and mitophagy. Western blotting analysis illustrated that over-expressed Bcl-xL inhibited CCCP-induced decrease of mitochondrial proteins in Parkin over-expressed cells. Fluorescence loss in photobleaching (FLIP) analyses demonstrated that Bcl-xL inhibited the CCCP-induced translocation of Parkin into mitochondria not by retrotranslocating Parkin from mitochondria to cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed in Hela cells that Bcl-xL physically bound with Parkin to form oligomer in cytoplasm, and that Bcl-xL also directly interacted with PINK1 on mitochondria. analysis for HEK293 T cells verified that endogenous Bcl-xL interacted with both endogenous Parkin and PINK1. Collectively, Bcl-xL inhibits PINK1/Parkin- dependent mitophagy by preventing the accumulation of Parkin on mitochondria via two regulation ways: directly binds to Parkin in cytoplasm to prevent the translocation of Parkin from cytoplasm to mitochondria and directly binds to PINK1 on mitochondria to inhibit the Parkin from cytoplasm to mitochondria by PINK1.

本报告旨在探讨Bcl-2家族蛋白Bcl-xL如何调节PINK1 / Parkin依赖性线粒体。与仅表达Parkin的Hela细胞相比，Bcl-xL的共表达显着抑制CCCP（碳氰化物3-氯苯基hydr）诱导的线粒体Parkin积累和线粒体吞噬。蛋白质印迹分析表明，过度表达的Bcl-xL抑制了CCCP诱导的帕金森州过度表达细胞中线粒体蛋白的减少。光漂白（FLIP）分析中的荧光损失表明，Bcl-xL不会通过将帕金蛋白从线粒体逆向转运到细胞质来抑制CCCP诱导的帕金酸转运到线粒体。荧光共振能量转移（FRET）成像显示Hela细胞中Bcl-xL与Parkin物理结合，在细胞质中形成寡聚物，并且Bcl-xL也直接与线粒体上的PINK1相互作用。HEK293 T细胞的分析证实内源性Bcl-xL与内源性Parkin和PINK1相互作用。Bcl-xL通过两种调节方式阻止线粒体上的Parkin积聚，从而抑制PINK1 / Parkin依赖性线粒体：直接结合至细胞质中的Parkin，以防止Parkin从细胞质转移至线粒体，并直接结合至线粒体PINK1，从而抑制线粒体通过PINK1将帕金从细胞质转移到线粒体。