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First published online 28 March 2006
doi: 10.1242/jcs.02878

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N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways

¹ Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
² Unidad de Citometría, Fundación CNIC, Instituto de Salud Carlos III, Melchor Fernández Almagro 3, 28040 Madrid, Spain

View larger version (27K): [in a new window]   Fig. 1. Wild-type and mutant NOS2-GFP chimeras. (A) The N-terminus of NOS2 was mutated by PCR, creating C3S, Myr, Myr/C3S and A2C mutant chimeras fused to GFP. We created both full-length NOS2 chimeras (residues 1-1144) and short chimeras (residues 1-94). The different NOS2-GFP constructs were inserted into a pCDNA3 vector that was used to transfect COS7 cells and the enzymatic activity (B), incorporation of radioactive palmitate and myristate (C) and protein expression (D) were determined. Enzymatic activity of NOS2 was determined using the Griess method as previously reported (Navarro-Lérida et al., 2004). Transfected COS7 cells were starved for 1 hour in DMEM without serum and were then metabolically labelled for 4 hours with either [3H]myristic acid (Myr) or [3H]palmitic acid (Palm). Cell lysate proteins were separated by SDS-PAGE and expression of the NOS2 chimeras was determined by western blotting with antibodies to GFF.

View larger version (79K): [in a new window]   Fig. 2. Subcellular localisation of the various constructs and laser confocal microscopy with the Golgi marker ß-cop. COS7 cells were transfected with the various NOS2-GFP constructs and the fluorescence was analysed 36 hours post transfection. The subcellular localisation of the full-length NOS2-GFP chimeras (A) and the (1-94)-NOS2 chimeras (B) was determined. Changes induced in the subcellular distribution of the Myr chimera when treated with 10 µM 2-Br-Palmitate were also determined (C). In order to determine the colocalisation of the Myr, Myr/C3S and A2C NOS2(1-94) chimeras with the Golgi marker ß-cop, double immunofluorescence staining was performed using a Cy3-conjugated secondary antibody. As a positive control, we used residues 1-55 of NOS3 fused to GFP, which is known to become myristoylated and palmitoylated (Navarro-Lérida et al., 2002). Cell nuclei were stained with Hoechst 33258 (blue). Bars, 50 µm.

View larger version (57K): [in a new window]   Fig. 3. Subcellular fractionation of COS7 cells expressing the various GFP constructs. (A) Transfected COS7 were lysed and after clarification of the cellular debris by centrifugation, fractionated into supernatant (S) and pellet (P) fractions by ultracentrifugation for 16 hours at 200,000 g as described in the Materials and Methods section. The fractions were subjected to a SDS-PAGE, analysed by western blot with an antibody against GFP and the resulting bands were quantified using UVIband V97 software. In addition, COS7 cells transfected with the tagged GFP constructs were extracted in the presence of Triton X-100 at 4°C and subjected to centrifugation on a 40:30:5% sucrose gradient. After centrifugation, the gradient tubes were divided into 12 equal aliquots collected from the bottom and analysed by SDS-PAGE and western blot (lower panels). (B) COS7 cells were transfected with the Myr, Myr (1-94) and A2C (1-94) NOS2-GFP. The bottom right panels are the merges of the three fluorescence signals. Bars, 50 µm.

View larger version (66K): [in a new window]   Fig. 4. Nitric oxide synthesis, subcellular targeting of the NOS2 chimeras at 25°C and in vivo recovery of the subcellular localisation of the wild-type and Myr NOS2 chimeras. (A) COS7 cells were transfected with the wild-type, Myr and C3S NOS2 chimeras and the cells were grown at both 37°C and 25°C. The NOS2 activity was determined at both temperatures using the Griess assay and was represented relative to the wild-type (relative units, RU) at 37°C (usually about 20 µM nitrites). At the same time, the cells grown at 25°C were fixed and analysed by laser confocal microscopy following the GFP fluorescence after excitation at 488 nm. (B) Areas in the plasma membrane with positive fluorescence for GFP (boxed) were selected in the wild-type, Myr (1-94) and A2C(1-94) NOS2 chimeras. The green fluorescence was bleached with the laser and the cell was allowed to recover the plasma membrane fluorescence for 30 minutes. The recovery is representative of three independent experiments. The ratio increment is depicted below. Bar, 50 µm.

View larger version (54K): [in a new window]   Fig. 5. Localisation of the sites of intracellular synthesis of ·NO in vivo, NOS2 activity in the presence of additional substrate and cofactor and effect of the NOS2 inhibitor 1400W and the ·NOS donor DETA-NONOate on the NOS2 fluorescence. (A) COS7 cells were transfected with the full-length wild-type NOS2-GFP and 36 hours post-transfection, the cells were incubated in vivo with 5 µM of the ·NO-sensitive probe diamino-Rhodamine-4M (DAR-4M) for 1 hour at 37°C. The location of the intracellular sites of ·NO synthesis is indicated in red. (B) COS7 cells transfected with the wild-type, C3S and Myr full-length NOS2-GFP chimeras were incubated with (shaded bars) or without (black bars) an excess of the NOS2 substrate L-Arg (10 mM) and the cofactor H4B (10 µM) and ·NO-synthesising activity was determined. (C) Changes in the immunofluorescence signal of the wild-type full length NOS2-GFP chimera after addition of 100 µM of the NOS inhibitor 1400W or 100 µM of the ·NO donor DETA-NONOate. Arrows indicate the plasma membrane areas where the NOS2 GFP fluorescence can be observed. Cell nuclei were stained with Hoechst 33258 (blue).

View larger version (25K): [in a new window]   Fig. 6. Agents that disrupt the ER-to-Golgi traffic affect NOS2 activity and subcellular distribution. COS7 cells were transfected with the full-length wild-type (A) and Myr (B) NOS2-GFP chimeras and 12 hours later increasing concentrations of brefeldin, monensin or megalomicin were added to the cell culture for 16 hours. After the treatment, the NOS2 activity was determined with the Griess assay. In a parallel experiment, COS7 cells transfected with their (1-94) counterparts were incubated with or without the desired drug for 4 hours, then starved for 1 hour in DMEM without serum and were then metabolically labelled for 4 hours with [3H]palmitic acid. Brefeldin A, monensin, megalomicin or DMSO (control) levels were maintained during the labelling period. The incorporation of radiolabelled palmitate was assessed both for wild-type NOS2(1-94)-GFP (A, bottom panel) and Myr NOS2(1-94) (B, left-hand panel, BFA only). The results shown are representative of two independent experiments.

View larger version (19K): [in a new window]   Fig. 7. Residues 1-33 of NOS2 are not part of the catalytic core of NOS2. (A) Absorbance spectrum of the recombinant NOS2 (33) expressed in E. coli. The contribution of the heme prosthetic group is centered around 390 nm whereas the contribution of the FAD and FMN flavins is centered around 450 nm. The insert shows a Coomassie Blue-stained gel with molecular weight markers (M), boiled (B) NOS2 and unboiled (U) NOS2, where the SDS-resistant dimers (D) can be partially observed as distinct form the monomers (M). Approximately 2 µg of purified protein were loaded per lane. Panel B show the catalytic activity of the recombinant NOS2(33) (delta-33) protein compared with the wild-type NOS2 (wt).

View larger version (46K): [in a new window]   Fig. 8. The subcellular distribution of the palmitoylated proteins PSD-95, GAP-43 and NOS2 is different in COS7 cells, C2C12 myotubes and H9C2 cardiac myocytes. (A) Both PSD-95-GFP and GAP-43-GFP constructs were transfected in COS7 cells and the subcellular distribution was analysed with the Golgi marker ß-cop labelled with a Cy3 secondary antibody (red). The right panels show the merges of the three fluorescence signals. Cell nuclei were stained with Hoechst 33258 (blue). (B) Mouse C2C12 myoblasts were converted into myotubes and a PSD-95 construct was transfected in the presence of a mixture of the cytokines LPS and IFN- that induce NOS2 expression. Inducible NOS is present to differing degrees in most of the cells with a patchy distribution (red staining). A magnified image of the boxed area in the merged image shows the colocalisation more clearly (right panel). (C) H9C2 rat cardiac myocytes were transfected with a PSD-95 construct in the presence of a mixture of the cytokines LPS and IFN that induce NOS2 expression. Bars, 50 µm.

View larger version (49K): [in a new window]   Fig. 9. The non-palmitoylated C3S is able to form dimers. COS7 cells were cotransfected with full-length NOS2 with either the wild-type or the C3S (1-94)-NOS2-GFP chimera. Antibodies that recognise the C-terminus of NOS2 or the GFP tag were used independently to immunoprecipitate the complexes. The immunodetection (western blot) was performed using both anti-GFP antibodies (upper panel) and anti-NOS2 antibodies (bottom panel). The (1-94) NOS2-GFP chimeras appear at 42 kDa whereas the NOS2 appears at 133 kDa.