Palmitoylation-dependent association with CD63 determines the localization of Syt VII. (A) Mistargeting of CD63 affects the localization of Syt VII. NRK cells were transduced with adenovirus expressing Lamp1-RFP, Syt VII–YFP or Syt VII (C/S)–YFP, and CFP-CD63 or CFP-CD63 (Y/A) and imaged by live confocal microscopy. Syt VII–YFP and CFP-CD63 colocalize with Lamp1-RFP (top). CFP-CD63 (Y/A) lacking a functional lysosomal targeting motif localizes to the plasma membrane together with Syt VII–YFP (middle) but not with Syt VII (C/S), which is detected in the Golgi (bottom; Video 4) and (Video 5). Bars, 6 µm. (B and C) Syt VII coimmunoprecipitates with CD63 or CD63 (Y/A) only when palmitoylation sites are intact. NRK cells were transduced with adenovirus expressing FLAG-CD63 (B) or FLAG-CD63 (Y/A) (C) and Syt VII–YFP or Syt VII (C/S)–YFP, lysed in 1% Brij-97, and immunoprecipitated with anti-FLAG antibodies. The lysates and immunoprecipitates were subjected to immunoblot analysis with anti-GFP, anti-FLAG, or antiactin antibodies. Ponceau S staining of the immunoglobulin light chain was used as a loading control in the immunoprecipitates. Coimmunoprecipitation was observed between FLAG-CD63 or FLAG-CD63 (Y/A) and Syt VII–YFP, but not with the palmitoylation-defective Syt VII (C/S)–YFP.