Figure 1.

Parkin-K2 regulation at mitochondria.A, MS/MS spectrum of triply charged m/z 646.3459 ion corresponding to the K2 ubiquitinated peptide FQGGKKEELIGIAYNR. The SILAC labeling ratio (L, Parkin; H, Vector) is indicated. The predicted Parkin Ub sites in K2, K581, and K582 are shown. B, AlphaFold model of human K2 (RMSD between K2 and K3, 1.2 Å). The position of K581 and K582 is indicated. Inset, sequence alignment of K family proteins. C, PC3 cells expressing Parkin were reconstituted with K2-WT or K2-DM and imaged by confocal microscopy. TOM20 was a mitochondrial marker. Merged images are shown. The scale bar represents 10 μm. Inset, magnification of indicated areas. The scale bar represents 5 μm. D, parental or Parkin-expressing PC3 cells (Parkin-PC3) were transfected with FLAG-vector (V) or FLAG-K2, and immune complexes precipitated with an antibody to FLAG were analyzed for expression of FLAG (left) or ubiquitin (Ub, right) by Western blotting. E, FLAG-K2 immune complexes as in (D) were analyzed with an antibody to total Ub or K63 or K48 Ub by Western blotting. F, PC3 cells with Doxy-induced conditional expression of Parkin were reconstituted with GFP-K2-WT or GFP-K2-DM, and immune complexes precipitated with IgG or an antibody to GFP were analyzed for reactivity with GFP (left), Ub (middle), or K48 Ub (right) by Western blotting. G, PC3 cells expressing vector (V), WT Parkin, or Parkin S65A or C431S mutant were analyzed by Western blotting. Bottom, densitometric quantification of protein bands. H, PC3 cells as in (D) were analyzed by cycloheximide (CHX) block and release followed by Western blotting at the indicated time intervals. I, Parkin-expressing PC3 cells were reconstituted with K2-WT or K2-DM and analyzed by CHX block and release and Western blotting. Doxy, doxycycline; MS/MS, tandem mass spectroscopy; SILAC, stable isotope labeling by amino acids in cell culture.