Fig. 3: DHHC9 palmitoylates GLUT1 at Cys207 to maintain GLUT1 PM localization.
From: DHHC9-mediated GLUT1 S-palmitoylation promotes glioblastoma glycolysis and tumorigenesis
a In vitro palmitoylation analysis was performed by mixing purified WT DHHC9/GCP16 and DHHC9 C169S/GCP16 with purified WT GLUT1 or GLUT1 C207S in the presence of palmitoyl alkyne-CoA. GLUT1 palmitoylation levels were analyzed by click reaction and streptavidin bead pulldown, followed by immunoblotting. b In vitro palmitoylation analysis was performed by mixing purified WT DHHC9/GCP16 and DHHC9 C169S/GCP16 with purified WT GLUT1 or GLUT1 C207S in the presence of palmitoyl-CoA. The palmitoylation levels of GLUT1 were analyzed by APE assays. The top band indicates the palmitoylated GLUT1 (PEG-GLUT1). c Flag-tagged WT rDHHC9 or rDHHC9 C169S was reconstitutively expressed in GBM cells with the knockout of endogenous DHHC9. Immunoblotting was performed using indicated antibodies. d DHHC9-knockout U87 or T98G cells rescued with Flag-tagged WT rDHHC9 or rDHHC9 C169S was metabolically labeled with 50 μM of alkynyl PA for 4 h. Palmitoylation levels of GLUT1 were analyzed by click reaction and streptavidin bead pulldown, followed by immunoblotting. e APE assay was performed to analyze the GLUT1 palmitoylation in DHHC9-knockout U87 or T98G cells with reconstituted expression of Flag-tagged WT rDHHC9 or rDHHC9 C169S. The top band indicates the palmitoylated GLUT1 (PEG-GLUT1). f DHHC9-knockout U87 or T98G cells with or without reconstituted expression of Flag-tagged WT rDHHC9 or rDHHC9 C169S were incubated with 1 µg ml−1 of CTB conjugated with Alexa Fluor 594 for 5 min at 37 °C. GLUT1 cellular localization was visualized by immunofluorescent staining using the antibody against GLUT1 and the PM was marked by Alexa Fluor 594-conjugated CTB. PM plasma membrane. Scale bar, 20 μm. g Levels of GLUT1 in the PM and ICM fractions were analyzed by immunoblotting in DHHC9-knockout U87 or T98G cells rescued with Flag-tagged WT rDHHC9 or rDHHC9 C169S. ATP1A1, calnexin, and GAPDH served as the marker of the PM, ICM, and cytosol fraction, respectively. PM plasma membrane, ICM intracellular membrane, WCL whole-cell lysate. Representative results were obtained from at least three independent experiments with similar results. Source data are provided as a Source Data file.
