Figure 5.

Selectivity of USP30-Dependent Buffering of MOM Ubiquitylation in iNeurons with and without Depolarization

(A) Workflow for analysis of USP30-dependent mitochondrial ubiquitylation.

(B) WT or USP30^−/− iNeurons were left untreated or depolarized with 10 μM antimycin A/5 μM oligomycin A as indicated and mitochondria immunoblotted with the indicated antibodies.

(C) Time course for quantified Ub chain linkage types in iNeurons with or without USP30 in response to depolarization as in (B). TMT intensities were normalized to untreated separately for each genotype. The inset shows the number of peptides used for quantification. Error bars represent SEM.

(D) Ubiquitylation kinetics for individual diGLY sites (and protein level in black) in TOMM5, TOMM20, and TOMM40 for WT and USP30^−/− iNeurons in response to depolarization as in (B).

(E) Log₂ ratio under untreated conditions of diGLY peptide intensities for USP30^−/− versus WT cells. Legend displays the color code for mitochondrial and non-mitochondrial diGLY sites.

(F) Ubiquitylation kinetics for individual diGLY sites (and corresponding protein level in black) in MOM-localized Parkin targets for WT and USP30^−/− iNeurons in response to depolarization as in (B).

(G) Time course for Ub phosphorylation on S65 in iNeurons with or without USP30 in response to depolarization as in (B).

See also Figure S5 and Table S3.