Supplementary Materials

Structure of Parkin Reveals Mechanisms for Ubiquitin Ligase Activation

Jean-François Trempe, Véronique Sauvé, Karl Grenier, Marjan Seirafi, Matthew Y. Tang, Marie Ménade, Sameer Al-Abdul-Wahid, Jonathan Krett, Kathy Wong, Guennadi Kozlov, Bhushan Nagar, Edward A. Fon, Kalle Gehring

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

Download Supplement
  • Materials and Methods
  • Supplementary Text
  • Figs. S1 to S12
  • Table S1
  • References

Images, Video, and Other Other Media

Movie S1
Time-lapse imaging of parkin recruitment to the mitochondria after treatment with CCCP in HeLa cells transfected with GFP-parkin. Live cell imaging was initiated at 24 hours post-transfection and images were acquired every minute post-CCCP treatment. Cells were visualized using a filter that detects GFP. Images were acquired using a 20× objective for a total of 90 minutes and merged together to create a movie file. The movie shows a rapid and progressive recruitment of GFPparkin wild-type at the mitochondria after the addition of CCCP. At the beginning of the movie GFP-parkin wild-type is expressed throughout the cell. At 40 minutes, the distribution of GFP-parkin wild-type became more punctuate and gradually co-localized to the mitochondria. The W403A started recruiting at an earlier time point (30 min), while the C431S showed no recruitment.
Movie S2
Single cell time-lapse imaging of parkin recruitment to the mitochondria after treatment with CCCP in HeLa cells transfected with GFP-parkin mutants. To visualize mitochondria, cells were transduced with CellLight® mitochondria-RFP. Live cell imaging was initiated at 24 hours post-transfection and images were acquired every minute post-CCCP treatment. Cells were visualized using filters that detect GFP or RFP. Fluorescent images were merged to create a movie file. Images were acquired using a 20× objective for a total of 70 minutes. The movie shows a rapid and progressive recruitment of GFP-parkin wild-type at the mitochondria after the addition of CCCP. At the beginning of the movie GFP-parkin wild-type is expressed throughout the cell. At 40 minutes, the distribution of GFP-parkin wild-type becomes more punctuate and is colocalized at the mitochondria (seen as the merging of green and red colours to create yellow colour). The W403A started recruiting at an earlier time point (30 min), while the C431S showed no recruitment.