Figure 7.

A rab6 and p50 binding fragment of BicD inhibits rab6A-GTP-directed Golgi-to-ER transport. (A) HeLa GalNAc-T2^GFP stably transfected cells were injected with an expression vector encoding mycBicD-C. After 24 h, the same cells were injected again with DNA encoding rab6A-GTP. Seven hours later, cells were fixed and stained with antibodies directed to myc-tag and rab6. Each injected cell was then scored for the level of BicD-C and rab6 protein and analyzed for the extent of Golgi-to-ER transport of GalNAc-T2^GFP. Rab6-GTP-expressing cells are represented by diamonds on the x-axis, BicD-C-expressing cells by squares on the y-axis, and double-injected coexpressing cells by circles. Using the same phenotypic classification of GalNAc-T2^GFP distribution as shown in Figure 4, an intact Golgi localization is represented by open symbols, partial GalNAc-T2^GFP in the ER by gray symbols, and full ER localization by black symbols. Note that exogenous expression of BicD-C alone does not affect Golgi integrity. (B-D) Representative image of HeLa GalNAc-T2^GFP cells (C) stained for rab6 (A) and BicD-C (D). Microinjected cells are labeled with asterisks in each panel. In the coexpressing BicD-C/rab6A-GTP cell (arrowhead), GalNAc-T2^GFP return to the ER is inhibited compared with the cell expressing rab6A-GTP alone, which shows the characteristic accumulation of GalNAc-T2^GFP in the ER (arrow). This figure is representative of three independent experiments. Bar, 10 μm.