RT Journal Article SR Electronic T1 USP30 sets a trigger threshold for PINK1–PARKIN amplification of mitochondrial ubiquitylation JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202000768 DO 10.26508/lsa.202000768 VO 3 IS 8 A1 Rusilowicz-Jones, Emma V A1 Jardine, Jane A1 Kallinos, Andreas A1 Pinto-Fernandez, Adan A1 Guenther, Franziska A1 Giurrandino, Mariacarmela A1 Barone, Francesco G A1 McCarron, Katy A1 Burke, Christopher J A1 Murad, Alejandro A1 Martinez, Aitor A1 Marcassa, Elena A1 Gersch, Malte A1 Buckmelter, Alexandre J A1 Kayser-Bricker, Katherine J A1 Lamoliatte, Frederic A1 Gajbhiye, Akshada A1 Davis, Simon A1 Scott, Hannah C A1 Murphy, Emma A1 England, Katherine A1 Mortiboys, Heather A1 Komander, David A1 Trost, Matthias A1 Kessler, Benedikt M A1 Ioannidis, Stephanos A1 Ahlijanian, Michael K A1 Urbé, Sylvie A1 Clague, Michael J YR 2020 UL http://www.life-science-alliance.org/content/3/8/e202000768.abstract AB 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.