Fig. 4: The central disordered region (IDR1) in TUG is necessary and sufficient to mediate TUG localization to the ERGIC.

a Schematic of TUG variants used for expression in TUG KO HeLa cells. All the proteins also contained a C-terminal mCherry tag to aid the visualization of the proteins in living cells. b Images of TUG KO HeLa cells transfected with full-length TUG (Tug-mCherry; left) or with a variant where the central IDR, IDR1 was deleted (Tug-IDR1∆-mCherry; right). Although full-length TUG forms punctate structures, the mutant is present in a diffuse distribution. Also note that the mutant is excluded from the nucleus. c Images of TUG KO HeLa cells transfected with IDR1 alone, tagged with mCherry (IDR1-mCherry; left), or with IDR2 alone, also tagged with mCherry (IDR2-mCherry; right). Although IDR1 forms punctate structures in cells, IDR2 is present in a diffuse distribution. In (b) and (c), a minimum of three independent transfections were performed for Tug-mCherry and IDR1-mCherry, three independent transfections for IDR2-mCherry and two independent transfections for Tug-IDR1∆-mCherry. d Images of TUG KO HeLa cells transfected with IDR1-mCherry and treated with 1,6-hexanediol and imaged before (left) or 150 sec. after 1,6-hexanediol treatment (right). Note that the punctate structures containing IDR1-mCherry continue to be present after 1,6-hexanediol treatment. The structures before and after are not necessarily at the same position, due to shift and refocusing during imaging after drug treatment. The treatment with 1,6-hexanediol was done three times, with similar results each time. e Images of TUG KO HeLa cells transfected with IDR1-mCherry (left) and mCherry-tagged IDR from FUS protein (right). Note the differences in the distribution of the two IDRs in cells. mCherry-tagged FUS IDR was transfected in TUG KO HeLa cells in two independent experiments, with similar results. f Images from TUG KO HeLa cells co-transfected with IDR1-mCherry (red) and Em-ERGIC53 (green). Note the colocalization of the two proteins in peripheral punctate structures. g Images from TUG KO HeLa cells co-transfected with IDR1-mCherry (red) and Stx12-GFP (green). Note the partial colocalization of the two proteins in punctate structures. Representative images for colocalization of IDR1-mCherry were from three independent experiments with Em-ERGIC53 (f) and two independent experiments with Stx12-GFP (g).