Fig. 5.

NO confined near the Golgi apparatus by WT-eNOS-RFP delays the exocytotic pathway of VSVG trafficking. (A) VSVG transport to the plasma membrane was impaired in cells expressing WT-eNOS-RFP. BSC-1 cells, grown on 12-mm coverslips in a 12-well dish, were microinjected with a mixture of tsO45-VSVG-GFP plasmid DNA and WT-eNOS-RFP, RFP-eNOS-NLS, or RFP alone (control). After the microinjection, cells were incubated at 40°C for 2–3 h for the expression, then incubated at 4°C for 15 min for the protein folding in the presence of cyclohexamide (100 μg/ml). At this point, tsO45-VSVG-GFP protein is exclusively localized at ER. Then, incubation of cells at 32°C was started to chase the transport of tsO45-VSVG-GFP from ER to Golgi and to the surface of plasma membrane (40 min). After the incubation at 32°C for 40 min, cells were fixed in 4% paraformaldehyde and processed for the immunofluorescence of cell surface VSVG protein, using a mAb to the luminal/cell surface VSVG that was labeled with Alexa 647. The quantification of the surface VSVG protein was performed by measuring the fluorescent intensity of Alexa 647, which was normalized by total expression of VSVG in cells by measuring fluorescent intensity of GFP. ∗, P < 0.01. (B) The impaired VSVG transport in WT-eNOS-RFP-expressing cells was NO-dependent. To determine whether the decreased VSVG transport in cells expressing WT-eNOS-RFP is NO-dependent, cells microinjected with WT-eNOS-RFP and tsO45-VSVG-GFP were incubated at 40°C for 3 h in the presence or absence of l-NAME (100 μM). Temperature was then shifted to 32°C for 40 min to chase VSVG transport to the cell surface. ∗, P < 0.01. (C) S-nitrosylation of NSF was increased in cells expressing WT-eNOS-RFP. COS-7 cells, transfected with myc-NSF and WT-eNOS-RFP or RFP-eNOS-NLS by transient transfection, were used 48 h after transfection. As a control, cells were transfected with myc-NSF alone to check for endogenous S-nitrosylation. Cells were incubated in DMEM without serum for 6 h, followed by the incubation with DMEM containing ATP (100 μM) with or without 0.2% HgCl₂ for 1 h. To detect S-nitrosylation of NSF, the biotin-switch method developed by Jaffrey and colleagues (46, 47) was used. (Top) Purified S-nitrosylated protein was subjected to 10% SDS/PAGE and Western blot analysis by using myc antibody to detect NSF. (Middle and Bottom) eNOS expression and NSF expression were also determined in the starting lysate before the isolation of S-nitrosylated proteins. Below the blot is the densitometric ratio of nitrosylated NSF to total NSF in the starting lysates.