Extended Data Fig. 6. Potential TG binding mutant retains function in vivo.
(a) Western blot analysis of whole-cell lysates from strains expressing C-terminal 13xmyc tagged seipin from endogenous or PGK1 promoter. (b) Western blot analysis of fractions from size-exclusion chromatography of Triton X-100 solubilized membrane extracts carrying potential TG binding mutant C260L S266L T269I with C-terminal 13xmyc. (c) Growth of yeast strain sei1∆ carrying plasmids with C-terminally GFP-tagged SEI1 from yeast (WT), or indicated mutant on synthetic medium ± 100 µg/ml terbinafine. (d) Localization of seipin WT-GFP and C260L S266L T269I-GFP mutant expressed from plasmids in sei1∆ cells. Size bar, 5 µm (e) Analysis of LD morphology using BODIPY staining. Seipin mutants with C-terminal 13xmyc tag were expressed from PGK1 promoter. Size bar = 5 µm. (f,g) Quantification of experiment in panel d. n=3 biologically independent experiments.