FIGURE 3.

Mapping the topology of GOAT by selective permeabilization of the plasma membrane and indirect immunofluorescence. A, mouse GOAT bearing a C-terminal 3xFLAG tag is localized to the ER in HeLa cells, as seen by co-localization with co-transfected CFP-KDEL, an ER marker; images are a confocal projection. B, selective permeabilization experiments mapping the positions of internal V5 epitopes. Two dishes of HeLa cells were transfected with a GOAT cDNA bearing an internal V5 or Myc epitope tag; all constructs have a C-terminal 3xFLAG tag as an internal control. Selective permeabilization with digitonin revealed cytosolic but not lumenal epitopes, whereas full permeabilization with Triton X-100 (TX-100) revealed all accessible epitopes. Lumenal epitopes were thus visible with Triton X-100 only. As a control, two dishes were stained for proteins with known cytosolic and lumenal epitopes and permeabilized identically; in this case, we used endogenous SSR1 (cytosolic) and transfected CFP-KDEL (lumenal). Identical exposures and image normalization for both permeabilizations ensure fair side-by-side comparison. C, V5 signal strength in indirect immunofluorescence of V5-tagged constructs from Figs. 3B and 4D (see “Experimental Procedures”).