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Distance-dependent cellular palmitoylation of de-novo-designed sequences and their translocation to plasma membrane subdomains

¹ Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain
² Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain

View larger version (27K): [in a new window]   Fig. 1. Illustration of the linker-GFP construct with the location of the consensus myristoylation sequence and the positions where the mutations were introduced (A), expression of the recombinant proteins in COS-7 cells (B) and fatty acylation of the GFP chimeras (C). By recursive PCR, the triplet AGS repeated nine times was created following a consensus N-myristoylation sequence and fused in frame to the GFP sequence. Mutations were introduced at Gly2, Cys3, Ser9, Ser15 and Ser21 in various combinations, as described in the Materials and Methods (A). The different linker-GFP constructs were inserted in a pCDNA3 vector that was used to transfect COS-7 cells (B). Transfected COS-7 cells were starved for 1 hour in DMEM without serum and were then metabolically labeled for 4 hours with either [3H]-myristic acid (Myr) or [3H]-palmitic acid (Palm). Cell lysates were immunoprecipitated with an anti-GFP antibody, analyzed by SDS-PAGE and exposed to a film as described in the Materials and Methods. Identical results were obtained in two independent metabolic labeling experiments.

View larger version (48K): [in a new window]   Fig. 2. Subcellular localization of the various constructs characterized in the present work as visualized by laser confocal microscopy. COS-7 cells were transfected with the various constructs, and the fluorescence was analyzed 36 hours after transfection. GFP fluorescence was visualized by confocal microscopy at an excitation wavelength of 488 nm. Bar, 50 µm.

View larger version (24K): [in a new window]   Fig. 3. Subcellular fractionation of COS-7 cells expressing the various GFP constructs. Transfected COS-7 were lysed and after clarification of the cellular debris by centrifugation were fractionated into supernatant (S) and pellet (P) fractions by ultracentrifugation for 16 hours at 200,000 g as described in the Materials and Methods. The fractions were subjected to SDS-PAGE, analyzed by western blot with an antibody against GFP, and the resulting bands were quantified using UVIband V97 software. These results are representative of a minimum of five independent fractionations, with less than 5% variation among different experiments.

View larger version (20K): [in a new window]   Fig. 4. Sucrose flotation gradients in the presence of Triton X-100. COS-7 cells transfected with the GFP-tagged constructs were extracted in the presence of Triton X-100 at 4°C and subjected to centrifugation on a 40:30:5% sucrose gradient as described in the Materials and Methods. After centrifugation, the gradient tubes were divided into 12 equal aliquots collected from the bottom and analyzed by SDS-PAGE and western blotting. The amount of protein in each sample was determined using a micro-Lowry method (upper panel; average distribution). Similar results were obtained in four independent experiments.

View larger version (55K): [in a new window]   Fig. 5. Colocalization studies of linker-GFP, G2A-GFP and C3S-GFP with caveolin 1. COS-7 cells were transfected with the linker-GFP (upper panels), G2A-GFP (middle panels) or C3S-GFP (lower panels) constructs and analyzed 36 hours post-transfection (A). The GFP fluorescence (left panels) was obtained after excitation at 488 nm, whereas the Cy3 fluorescence (middle panels) was obtained after excitation at 543 nm. The right panels show the merge of both fluorescence signals. Bar, 50 µm. The physical interaction of caveolin-1 with the GFP chimeras was analyzed in pull-down experiments (B). COS-7 cells transfected with the linker-GFP construct, C3S-GFP construct or mock-transfected were lysed and immunoprecipitated with anti-caveolin-1 antibodies (left panel) or with anti-GFP antibodies (right panel) as described in the Materials and Methods. The immunoprecipitated samples were then analyzed (immunodetected, I.D.) with anti-GFP, anti-caveolin-1 or anti-Gq antibodies as outlined in the figure.

View larger version (69K): [in a new window]   Fig. 6. Colocalization studies of linker-GFP, G2A-GFP and C3S-GFP with the Golgi marker BODIPY-TR-ceramide (A) and cycloheximide treatment of the linker-GFP and C3S-GFP chimeras (B). (A) COS-7 cells were transfected with the linker-GFP (upper panels), G2A-GFP (middle panels) or C3S-GFP (lower panels) constructs and, 36 hours post-transfection, they were incubated with the Golgi apparatus marker BODIPY-Texas Red-ceramide (1.5 µM in DMEM). The GFP fluorescence (left panels) was obtained after excitation at 488 nm whereas the Texas Red fluorescence (middle panels) was obtained after excitation at 543 nm. Right panels show the merge of both fluorescence signals. Bar, 50 µm. (B) Changes induced in the localization of the linker-GFP and C3S-GFP mutants upon treatment with 100 µg/ml cycloheximide for 2 hours. The treatment was performed 24 hours post-transfection.

View larger version (32K): [in a new window]   Fig. 7. In vivo changes in the subcellular localization of the linker-GFP construct upon cholesterol depletion using ß-methyl cyclodextrin and changes in the subcellular localization of various GFP chimeras upon incubation with 2-bromopalmitate. COS-7 cells were transfected with the linker-GFP plasmid and incubated with 10 mM ß-methyl cyclodextrin for 0, 10, 20 or 30 minutes (A-D) at 36 hours after transfection. Arrows in D denote changes in the plasma membrane fluorescence. COS-7 cells were also transfected with the linker-GFP, G2A-GFP, C3S-GFP, C3S/S9C-GFP, C3S/S15C-GFP or C3S/S21C-GFP plasmids and, 36 hours post-transfection, incubated with 25 µM 2-bromopalmitate for 16 hours (E-J). Bar, 50 µm.