No abstract

Mitochondria play key roles in activating apoptosis in mammalian cells. Bcl-2 family members regulate the release of proteins from the space between the mitochondrial inner and outer membrane that, once in the cytosol, activate caspase proteases that dismantle cells and signal efficient phagocytosis of cell corpses. Here we review the extensive literature on proteins released from the intermembrane space and consider genetic evidence for and against their roles in apoptosis activation. We also compare and contrast apoptosis pathways in Caenorhabditis elegans, Drosophila melanogaster, and mammals that indicate major mysteries remaining to be solved.

Localization of the dynamin-related GTPase Drp1 to mitochondria relies on the mitochondrial fission factor Mff.

Differential localization to the inner and outer mitochondrial membranes regulates PINK1 stability and function.

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagyrelevant sites, including the ubiquitin-and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN's UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.A s a cell survival pathway, autophagy selectively frees the cytosolic compartment from bulky protein aggregates, invading bacteria or damaged organelles such as mitochondria and peroxisomes (1, 2). In this context, the posttranslational modifier ubiquitin (Ub) has been widely recognized as a selective signal driving autophagy of such cellular components and cargoes (3, 4). Recently, ubiquitin itself has been discovered to be phosphorylated to promote autophagic clearance of damaged mitochondria (mitophagy; reviewed in refs. 5 and 6). Ser/Thr kinase PINK1 phosphorylates S65 of Ub, which is critical for two steps of this process: allosteric activation of the E3 Ub ligase Parkin and recruitment of the autophagic machinery, including autophagy receptors (7)(8)(9)(10)(11)(12)(13)(14).Autophagy receptors function as decoders for the various ubiquitin signals on cargoes, linking cargoes to autophagosomal membranes (4); however, the basis of their individual recruitment to cargo as well as their distinct and cooperative functions in cargo sequestration are still poorly understood. The autophagy receptors optineurin (OPTN) and p62 are first activated by protein kinases to effectively target autophagic membranes or their polyUb cargo (15-17). TANK-binding kinase 1 (TBK1) phosphorylates OPTN on S177, thereby enhancing LC3-binding affinity and autophagic clearance of cytosolic Salmonella (15). Activity and specificity of TBK1 are defined by adaptor proteins; these recruit TBK1 to microdomains on ubiquitinated Salmonella or mitochondria, thereby facilitating its local clustering and activation (18), where it in turn can phosphorylate autophagy receptors (15). It is relevant to stress that a number of mutations in both OPTN and TBK1 have been identified in patients suffering from amyotrophic lateral sclerosis (ALS) and...

Summary The Bcl-2 family member Bax translocates from the cytosol to mitochondria where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by pro-survival Bcl-2 proteins, but how they inhibit Bax remains controversial, because they neither co-localize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-xL in detergents and cell free MOMP activity, but unexpectedly induce Bax accumulation on mitochondria. Fluorescence Loss in Photobleaching (FLIP) reveals constant retrotranslocation of wt Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on pro-survival Bcl-2 family proteins and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-xL inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.

Summary Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.

The role of fission and autophagy in mitochondrial proteostasis is unclear. Burman et al. report that Drp1-mediated mitochondrial fission limits the spread of PINK1-activated Parkin ubiquitination to restrain mitophagy near sites of regional damage and allow the selective removal of subdomains.