Fig. 2.

Myristoylation is required for proper intracellular localization of C-t-PAK2-myc. Indirect immunofluorescence confocal micrographs of COS-7 cells transfected with plasmids allowing expression of Gly-213-C-t-PAK2-myc, the nonmyristoylatable Ala-213-C-t-PAK2-myc chimeras, or vector alone at 12–14 h after transfection. (A) Gly-213-C-t-PAK2-myc localizes to the plasma membrane and membrane ruffles, whereas the Ala-213-C-t-PAK2-myc chimera remains cytosolic. Shown are C-t-PAK2-myc chimeras (anti-myc, green) and nuclei (Hoechst dye 33258 staining, blue). (B) Confirmation of Gly-213-C-t-PAK2-myc localization to plasma membrane ruffles as indicated by colocalization with actin (red). The merged images are presented in Right. Yellow indicates apparent colocalization of the green and red signals. (C) The N-terminal 14 amino acids of C-t-PAK2 encompassing the myristoylation signal and a short polybasic domain are sufficient to localize EGFP to membranes. COS-7 cells were transfected with plasmids expressing chimeric EGFPs in which the first 14 amino acids of C-t-PAK2 (Gly-213-N15-EGFP or the corresponding nonmyristoylatable mutant Ala-213-N15-EGFP) were inserted after the initiator methionine or vector alone. Images were obtained 12–14 h after transfection. EGFP was detected either by live cell fluorescence microscopy (Live) or indirect immunofluorescence (Fixed) by confocal microscopy. (Scale bars, 10 μm.)