Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine.

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.

Dual role of USP30 in controlling basal pexophagy and mitophagy.

USP30 is an integral protein of the outer mitochondrial membrane that counteracts PINK1 and Parkin-dependent mitophagy following acute mitochondrial depolarisation. Here, we use two distinct mitophagy reporter systems to reveal tonic suppression by USP30, of a PINK1-dependent component of basal mitophagy in cells lacking detectable Parkin. We propose that USP30 acts upstream of PINK1 through modulation of PINK1-substrate availability and thereby determines the potential for mitophagy initiation. We further show that a fraction of endogenous USP30 is independently targeted to peroxisomes where it regulates basal pexophagy in a PINK1- and Parkin-independent manner. Thus, we reveal a critical role of USP30 in the clearance of the two major sources of ROS in mammalian cells and in the regulation of both a PINK1-dependent and a PINK1-independent selective autophagy pathway.

The V-ATPase complex regulates non-canonical Atg8-family protein lipidation through ATG16L1 recruitment.

Conjugation of the Atg8 (autophagy related 8) family of ubiquitin-like proteins to phospholipids of the phagophore is a hallmark of macroautophagy/autophagy. Consequently, Atg8 family members, especially LC3B, are commonly used as a marker of autophagosomes. However, the Atg8 family of proteins are not found solely attached to double-membrane autophagosomes. In non-canonical Atg8-family protein lipidation they become conjugated to single membranes. We have shown that this process is triggered by recruitment of ATG16L1 by the vacuolar-type H+-translocating ATPase (V-ATPase) proton pump, suggesting a role for pH sensing in recruitment of Atg8-family proteins to single membranes.

Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation.

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.

USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.

New aspects of USP30 biology in the regulation of pexophagy.

Mitochondria and peroxisomes have a number of features in common: they each play interconnected roles in fatty acid and reactive oxygen species (ROS) metabolism and, once damaged, need to be removed by specialized autophagic mechanisms, termed mitophagy and pexophagy, respectively. Both processes can use ubiquitin as an initiating signal but whereas mitophagy has been extensively studied, pexophagy remains rather poorly understood. Our recent work, along with a new study from Kim and colleagues, has shed light on the molecular mechanism of pexophagy and the importance of reversible ubiquitination in its regulation. Collectively, these studies highlight the physiological role of the deubiquitinase USP30 in suppressing the turnover of peroxisomes. Abbreviations: ROS: reactive oxygen species; DUB: deubiquitinase or deubiquitylase; USP: ubiquitin specific protease; PINK1: PTEN induced kinase 1; CAT: catalase; KO: knock-out; SQSTM1/p62: sequestosome 1; LIR: LC3 interacting region; GFP: green fluorescent protein; RFP: red fluorescent protein; CRISPR: Clustered Regularly Interspaced Short Palendromic Repeat.

Calpain mobilizes Atg9/Bif-1 vesicles from Golgi stacks upon autophagy induction by thapsigargin.

CAPNS1 is essential for stability and function of the ubiquitous calcium-dependent proteases micro- and milli-calpain. Upon inhibition of the endoplasmic reticulum Ca2+ ATPase by 100 nM thapsigargin, both micro-calpain and autophagy are activated in human U2OS osteosarcoma cells in a CAPNS1-dependent manner. As reported for other autophagy triggers, thapsigargin treatment induces Golgi fragmentation and fusion of Atg9/Bif-1-containing vesicles with LC3 bodies in control cells. By contrast, CAPNS1 depletion is coupled with an accumulation of LC3 bodies and Rab5 early endosomes. Moreover, Atg9 and Bif-1 remain in the GM130-positive Golgi stacks and Atg9 fails to interact with the endocytic route marker transferrin receptor and with the core autophagic protein Vps34 in CAPNS1-depleted cells. Ectopic expression of a Bif-1 point mutant resistant to calpain processing is coupled to endogenous p62 and LC3-II accumulation. Altogether, these data indicate that calpain allows dynamic flux of Atg9/Bif-1 vesicles from the Golgi toward the budding autophagosome.