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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Cell Signal. 2015 Aug 29;27(12):2444–2451. doi: 10.1016/j.cellsig.2015.08.017

Figure 4. Gβ binding-deficient mutant PAQR3(NΔ20) inhibits VSV-G transport from the Golgi to the PM.

Figure 4

A, HEK293 cells were transfected with mCherry-VSV-G alone (top row) or together with GFP-PAQR3 (middle row) or GFP-PAQR3(NΔ20) (bottom row), incubated for 24 h and then transferred to the non-permissive temperature of 39 °C for 16 h. Cells were then shifted to 20 °C for 2 h (after 1 h of which cyclohexamide was added) at which temperature VSV-G localizes to and is retained at the Golgi. Cells were then transferred to the permissive temperature of 32 °C for 2 h for VSV-G transport to the PM. Cells were then fixed and stained with anti-VSV-G followed by Alexa 594-conjugated secondary antibody to better visualize mCherry-VSV-G. PAQR3 and PAQR3-(NΔ20) were detected by intrinsic GFP fluorescence. Bar, 10 μm. B, HEK293 cells were transfected with GFP-VSV-G alone (top row) or together with HA-KDELr (bottom row) and the VSV-G transport assay was performed as described in A. After 2 h incubation at the permissive temperature of 32 °C, cells were fixed and stained with anti-HA antibody to visualize KDELr. GFP-VSV-G was visualized by intrinsic GFP fluorescence. C, Shown is a bar graph quantitating the localization of VSV-G in the absence or presence of co-expressed PAQR3, PAQR3(NΔ20) or KDELr. VSV-G localization in individual cells was scored as predominantly PM (PM), both PM and Golgi (PM/Golgi), or predominantly Golgi (Golgi). Values depicted are the means (+/− S.D., vertical bars) for 3 separate experiments. 100 cells were counted in each experiment. Asterisks indicate statistical significance (p<0.001, t-test) of the indicated bar (*, PM; **, PM/Golgi; ***, Golgi) compared to the corresponding bar of the mCh-VSV-G only sample (first set of bars). D, HEK293 cells were transfected with mCherry-VSV-G together with pcDNA3, GFP-PAQR3, GFP-PAQR3(NΔ20) or HA-KDELr, as indicated, and cells were cultured as described above for A and B. After 2 h at 32 °C to allow transport of VSV-G to the PM, cell surface proteins were biotinylated and isolated as described under Materials and Methods. VSV-G in total cell lysates (upper panel) and cell surface biotinylated VSV-G (lower panel) were detected by immunoblotting with an anti-VSV-G antibody