Extended Data Fig. 3: NSP6 undergoes homodimerization through the 1–157 region.

a, HeLa cells expressing C-terminal or N-terminal Flag-tagged NSP6 immunostained with anti-Flag antibody and an antibody against a luminal epitope of TGN46 after permeabilization with digitonin and subsequently with Triton-X-100 (see Methods). b, Model of the amphipathic helix of NSP6 (left panel) according to HELIQUEST (see Methods). Apolar residues are in yellow, polar residues and glycine have been given different colours. The arrow indicates the hydrophobic moment (µH = 0.409). Numbers indicate amino acid positions of the NSP6 protein. Right panel, model of the F220Q/T222W NSP6 mutant helix (µH = 0.191). Mutations that abolish the amphipathic character of the helix are in red. c, HeLa cells untransfected (left panel) or expressing Myc-NSP6 were immunostained for VAP-A or for Myc. Insets show the Myc-NSP6 signal. The number indicates the fraction of VAP-A associated with the NSP6 structures. Mean ± SD, N = 3, n = 74. d, HeLa cells expressing GFP-NSP6 F220Q/T222W mutant. e, f, g, Cell lysates (input) and immunoprecipitates (IP, with anti-HA or anti-Flag antibodies) from HeLa cells, untransfected or expressing the indicated NSPs were analysed by western blot with anti-HA, anti-Flag or anti-GFP antibodies as appropriate. Images are representative of three independent experiments. h, Fluoromicrographs of HeLa cells expressing GFP-NSP6(1–157) alone or with mCherry-NSP6. Scale bar, a, c, d, h, 10 µm.