Figure 2.

The inactive FGD1 mutant blocks exit of VSVG from the Golgi complex in HeLa cells. Cells were transfected with wild-type FGD1 fused with GFP (GFP-FGD1; A, D, G, and J), the inactive GFP-FGD1-AS mutant (B, E, H, and J), or the active GFP-FGD1-dbdel mutant (C, F, I, and J) and infected with VSV. The cells were then fixed directly after the 40 and 20°C temperature block (to accumulate VSVG in the Golgi complex; A–C), or after 32°C for 60 min (to activate Golgi-to-plasma-membrane transport of VSVG) in the absence (D–I) and presence (G–I) of tannic acid (0.5%; to inhibit fusion of VSVG-positive post-Golgi carriers [PGCs] with the plasma membrane). The cells were then stained with an anti-VSVG antibody and examined under confocal microscopy. (A–C) Expression of all FGD1 isoforms allows accumulation of VSVG within the Golgi complex with the 20°C block. (D–F) On release of the 20°C block VSVG appeared at the plasma membrane of nontransfected cells (e.g., E, arrows) and of cells expressing GFP-FGD1 (D) or GFP-FGD1-dbdel (F). In GFP-FGD1-AS–transfected cells, VSVG remained mainly within the Golgi complex (E, asterisks). (G–I) On release of the 20°C block in the presence of tannic acid, numerous PGCs (G–I, arrows) were seen in nontransfected cells (e.g., H, arrowheads), and in cells expressing GFP-FGD1 (G) or GFP-FGD1-dbdel (I). In GFP-FGD1-AS–transfected cells (H, asterisk), most of the VSVG remained within the Golgi complex. (J) Quantification of PGCs per cell (mean ± SD; n = 30 cells), as illustrated in G–I. A reduction in VSVG carrier formation is seen upon expression of the inactive GFP-FGD1-AS mutant. Scale bars, (A, C, D, and F) 22 μm, (B and E) 30 μm, (G–I) 20 μm.