Figure 3: TREX1 is a tail-anchored protein.

(a) Schematic of fluorescently tagged TREX1 constructs used for fluorescence protease protection assay. (b) Confocal images of living HeLa cells co-expressing GFP-TREX1 and mCherry-TREX1. Proteinase K treatment after selective plasma membrane permeabilization leads to rapid dissipation of cytosolic GFP fluorescence, while mCherry resists proteolysis. Scale bar, 20 μm. (c) Schematic of TREX1 constructs containing a C-terminal bovine opsin fragment with two N-linked glycosylation sites (glyc) used for glycosylation reporter assay. (d) In vitro translated WT FLAG-TREX1-wt-glyc, but not mutant FLAG-TREX1-D272fs-glyc lacking the transmembrane domain, is glycosylated in the presence of microsomal membranes (memb.) as visualized by anti-FLAG immunoblotting. Glycosylation of WT TREX1 is removed by endoglycosidase H (endo H). (e) Electron microscopy of immunogold-labelled GFP-TREX1 in HeLa cells shows expression of TREX1 (black dots marked by arrows) along the outer nuclear membrane and the cytosolic site of the ER membrane. White arrowheads indicate the inner nuclear membrane. Scale bar, 200 nm. (f) Formation of organized smooth ER consisting of lamellar stacks, branching tubules or whorls in cells overexpressing GFP-TREX1. Scale bar, 200 nm. (g) Magnification of the inset in (f) shows an even distribution of GFP-TREX1 at the extraluminal site of the ER. Scale bar, 100 nm. co, no lysate control; cy, cytoplasm; nu, nucleus.