![Figure 4. EFR3 is palmitoylated at a conserved N-terminal Cys-rich motif. (A) Alignment of N terminus of EFR3 orthologues in several species reveals a conserved Cys-rich motif. Red, Cys residues; blue, basic residues; green, hydrophobic residues. (B) EFR3A-FLAG and EFR3B-FLAG are palmitoylated at an N-terminal Cys-rich motif. Recombinant EFR3A-FLAG (wild type [WT] or C6S/C7S/C8S/C9S quadruple mutant, denoted C6–9S) or EFR3B-FLAG (wild type or C5S/C7S/C8S triple mutant, denoted C5,7,8S) were immunoprecipitated from lysates of HeLa cells metabolically labeled with alkynyl palmitate (alk-16) or vehicle and transfected with the appropriate EFR3-FLAG construct. The samples were subsequently labeled with biotin-azide using click chemistry, treated with or without hydroxylamine (NH2OH) to cleave thioester linkages, and analyzed by Western blotting, using streptavidin or anti-FLAG antibody. (C) Confocal imaging of live HeLa cells transfected with wild-type EFR3A-tdTomato, C6–9S EFR3A-tdTomato, or C5,7,8S EFR3B-tdTomato. Bars, 20 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/199/6/10.1083_jcb.201206095/5/jcb_201206095_fig4.jpeg?Expires=1771874881&Signature=ch-~WG~V1b~SJB6d4M0gnjkLryAtmFMFd-yBk6dYvkv8FMxifeXtTE0JIziB7E~5qFxFVrg4e2azQ4-fLIXoUavcxohPMbwy8SDavJp3StxA~3oyQ98j3amjbruU7-xOQmNhBSlC3Ytwz-IsWU-J6mjDRTzFmUeRXNz0CnOjB-Nf4IHLyEPi3oLxLvONCBI4Ebbzih59iEx2vrvEgVuYmlXXGhl8LoDKJRBZFMYEQNihFV0h1pQDuJp71WFxbH~pm9NEGWBmmSDNnH6fhODdaHbVLAFiB-uHs4BVnh8FJwLXJLDtgMOen8C~tVVV6DyaowktSYENsLEJ1gpKFmAUbg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
EFR3 is palmitoylated at a conserved N-terminal Cys-rich motif. (A) Alignment of N terminus of EFR3 orthologues in several species reveals a conserved Cys-rich motif. Red, Cys residues; blue, basic residues; green, hydrophobic residues. (B) EFR3A-FLAG and EFR3B-FLAG are palmitoylated at an N-terminal Cys-rich motif. Recombinant EFR3A-FLAG (wild type [WT] or C6S/C7S/C8S/C9S quadruple mutant, denoted C6–9S) or EFR3B-FLAG (wild type or C5S/C7S/C8S triple mutant, denoted C5,7,8S) were immunoprecipitated from lysates of HeLa cells metabolically labeled with alkynyl palmitate (alk-16) or vehicle and transfected with the appropriate EFR3-FLAG construct. The samples were subsequently labeled with biotin-azide using click chemistry, treated with or without hydroxylamine (NH2OH) to cleave thioester linkages, and analyzed by Western blotting, using streptavidin or anti-FLAG antibody. (C) Confocal imaging of live HeLa cells transfected with wild-type EFR3A-tdTomato, C6–9S EFR3A-tdTomato, or C5,7,8S EFR3B-tdTomato. Bars, 20 µm.
