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 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.

Highlights d ATG8 can undergo alternative conjugation to phosphatidylserine in cells d ATG8-PS occurs during non-canonical autophagy via singlemembrane ATG8 conjugation d ATG8-PS can be induced by LAP, influenza A, and lysosomal ionic imbalance d ATG8-PS undergoes differential delipidation by ATG4 isoforms

Highlights d Subtractive CRISPR screen identifies genes involved in noncanonical LC3 lipidation d v-ATPase regulates LC3 lipidation at erroneously neutral compartments d RALGAP complex involved in M2 proton channel induced LC3 lipidation d ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinson's Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. 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 most 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. In this model, 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 and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

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.

29 30 Autophagy is a fundamental catabolic process essential for development, 31 homeostasis and proper immune function 1 . During autophagy, a cascade of 32 ATG proteins target intracellular cargoes for lysosomal degradation and 33 recycling 2 . This pathway utilises a unique post-translational modification, the 34 conjugation of ATG8 proteins to phosphatidylethanolamine (PE) at 35 autophagosomes, which modulates cargo selection and maturation. 36 ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway 37 involving Single Membrane ATG8 Conjugation (SMAC) to endolysosomal 38compartments, which plays a key role in phagocytosis and other processes 3 . It 39 has been widely assumed that SMAC involves the same lipidation of ATG8 to 40 PE, but this has yet to be formally tested. Here, we show that ATG8 undergoes 41 alternative lipidation to phosphatidylserine (PS) during non-canonical 42 autophagy/SMAC. Using mass spectrometry, we find that activation of SMAC, 43 by pharmacological agents 4,5 , or during non-canonical autophagy processes such 44 as LC3-associated phagocytosis 6,7 and Influenza A virus infection 8 , induces the 45 covalent conjugation of ATG8 to PS, as well as PE. This alternative lipidation 46 event is dependent on the ATG16L1 WD40 domain, and occurs at PS enriched 47 endolysosomal membranes. Importantly, we find that the ATG8-PS and ATG8-48 PE adducts are differentially delipidated by isoforms of the ATG4 family, 49 indicating significant molecular distinctions and mechanisms between these two 50 species. 51Together, these results provide an important new insight into autophagy 52 signalling, revealing an alternative form of the hallmark ATG8-lipidation event, 53Main 60 61 A defining feature of autophagy is the lipidation of ATG8, a family of ubiquitin-like 62 proteins including mammalian LC3s (A/B/B2/C) and GABARAPs 63 (GABARAP/L1/L2) 9 . Nascent pro-ATG8 is first primed by a cysteine protease, 64 ATG4, to expose a conserved aromatic-Gly motif at its C-terminus 10 . A ubiquitin-65 like conjugation system, comprised of ATG7 (E1-like), ATG3 (E2-like) and 66 ATG16L1/ATG12/ATG5 (E3-like), then drives the covalent ligation of this glycine to 67 a lipid, phosphatidylethanolamine (PE), via an amide bond to its headgroup (Extended 68 Data Fig. 1a) 11,12 . This is a unique post-translational modification that recruits ATG8 69 to autophagosomal membranes, where it plays an important role in cargo loading and 70 autophagosome maturation 9,13 . The associated relocalisation of ATG8s, and the 71 characteristic protein bandshift between the unlipidated (ATG8-I) and lipidated 72 (ATG8-II) forms, are widely used to define and assay autophagy-related processes 73 14,15 . 74 75A second phospholipid, phosphatidylserine (PS), also bears an amino group in its 76 head moiety (Extended Data Fig. 1b), which can be conjugated to ATG8 in vitro 16 . 77However, in vivo, ATG8 lipidation is reported to occur exclusively to PE, in both 78 yeast 11 and mammalian cells 16 . The mechanism underlying cellular...

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.