Figure 8.

PINK1^G411A does not discriminate between the common (WT) and the minor (TVLN) confirmation of Ub. (A) Mitochondrial fractions from HEK293T cells – PINK1^G411 (G), PINK1^A411 (A), or PINK1^S411 (S) – were used for kinase reactions with recombinant FLAG-tagged Ub as the substrate. Supernatants were analyzed and Ub phosphorylation was determined with anti-p-S65-Ub or anti-FLAG antibody using Phos-tag gels (asterisk marks phosphorylated Ub). VDAC1 was used as a mitochondrial loading control. (B) Quantifications of the p-S65-Ub:Ub ratio from Phos-tag gels. Shown is the mean ± SD of three independent experiments. Statistical analysis was performed using two-way ANOVA and Tukey’s post-host test (*** p < 0.0005). (C) In vitro kinase reactions confirm that the Ub[TVLN] (Ub-CR) is a superior substrate for TcPINK1 compared to WT Ub. Recombinant TcPINK1 was incubated with equal amounts of His-tagged WT or Ub[TVLN] for 90 min. Note the substantial amounts of WT Ub that remain un-phosphorylated under these conditions. (D) Mitochondria from HEK293T WT PINK1 (labeled G) and PINK1^G411A (labeled A) cells were used for kinase reactions as in (A), with His-tagged WT Ub or Ub[TVLN] as substrate as in (C). HSPD1/HSP60 was used as a mitochondrial loading control, and silver staining to quantify total Ub. Note the strong phosphorylation of WT Ub by PINK1^G411A, while C-terminally retracted Ub[TVLN] was phosphorylated by both WT PINK1 and PINK1^G411A. (E) Quantification of in organello kinase reactions confirm an overall stronger and equally efficient phosphorylation of WT Ub and Ub[TVLN] by PINK1^G411A, compared to WT PINK1. Shown is the mean of p-S65-Ub:Ub normalized to PINK1 levels at 4 h reaction time ± SD of five experiments. Statistical analysis was performed using two-way ANOVA with Sidak’s post-hoc test (** p < 0.005).