Figure 3: GRASP depletion accelerates VSV-G trafficking through the Golgi membranes.

(a) VSV-G traffic to the cell surface is accelerated by GRASP depletion. HeLa cells stably expressing VSV-G-ts045-GFP under an inducible promoter were transfected with control (ctrl), GRASP55 (55), GRASP65 (65), or both GRASP55 and GRASP65 (55+65) RNAi. The cells were incubated with doxycycline at 40.5 °C for 16 h to induce VSV-G expression and shifted to 32 °C for 120 min to release the VSV-G from the ER. The cells were collected, and the VSV-G on the surface was stained with an antibody against its extracellular domain followed by incubation with fluorescently labelled secondary antibodies. Total VSV-G and surface-bound VSV-G were assessed by flow cytometry. The ratio of surface-to-total VSV-G normalized against control cells (mean±s.e.m.) are shown based on three independent experiments. (b) GRASP depletion accelerates VSV-G trafficking. HeLa cells transfected with indicated RNAi were infected with VSV-G ts045-GFP adenovirus and incubated at 40.5 °C for 16 h. Cells were shifted to 32 °C, incubated for indicated time period (chase), and lysed. Proteins were treated with EndoH and analysed by western blot for VSV-G-GFP. Arrows indicate the EndoH resistant form and arrowheads indicate the EndoH sensitive form of VSV-G. Molecular weight markers are indicated on the left. (c) Quantitation of (b) to indicate the percentage of VSV-G in EndoH resistant form. Note that the difference between the GRASP-depleted cells and control RNAi-treated cells is most dramatic after 15–45 min chase when VSV-G travels through the Golgi membranes. (d) GRASP depletion accelerates VSV-G trafficking through the Golgi membranes. HeLa cells transfected with indicated RNAi were infected with VSV-G ts045-GFP adenovirus and incubated at 40.5 °C for 16 h. Cells were shifted to 15 °C for 2 h and then to 20 °C for 0 and 10 min followed by immunofluorescence microscopy. The co-localization of VSV-G with GalT was quantified with ImageJ. Shown are representative results (mean±s.e.m. from 20 different cells) from three independent experiments.