Quantitative Middle-Down MS Analysis of Parkin-Mediated Ubiquitin Chain Assembly.

Misregulation of the E3 ubiquitin ligase Parkin and the kinase PINK1 underlie both inherited and idiopathic Parkinson's disease-associated neurodegeneration. Parkin and PINK1 work together to catalyze the assembly of ubiquitin chains on substrates located on the outer mitochondrial membrane to facilitate autophagic removal of damaged mitochondria through a process termed mitophagy. Quantitative measurements of Parkin-mediated chain assembly, both and on mitochondria, have revealed that chains are composed of Lys6, Lys11, Lys48, and Lys63 linkages. The combinatorial nature of these chains is further expanded by the ability of PINK1 to phosphorylate individual subunits. The precise architecture of chains produced by the coordinated action of PINK1 and Parkin, however, are unknown. Here, we demonstrate that quantitative middle-down mass spectrometry using uniformly N-labeled ubiquitin variants as internal standards informs on the extent of chain branching. We find that Parkin is a prolific branching enzyme . Quantitative middle-down mass spectrometry also reveals that phospho-Ser65-ubiquitin (pSer65-Ub)-a key activator of Parkin-is not incorporated into chains to a significant extent. Our results suggest that Parkin-mediated chain branching is "on-pathway", and branch points are the principal targets of the deubiquitinase USP30.