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[Preprint]. 2026 May 24:2026.05.21.726867. [Version 1] doi: 10.64898/2026.05.21.726867

Figure 1: TGIF is an essential protein localized to the cis-Golgi.

Figure 1:

(A) Schematic illustration of TGIF domains predicted using Interpro, Marcoil and N-coils. The N-terminal domain (NTD: M1-L1101) contains a predicted RGPR-like domain (a.a. 8-222) with the remaining predicted to be intrinsically disordered. N-coils and Marcoils predict different start and end positions of the coiled-coil domain (grey) (indicated with black arrows). The C-terminal domain is shown in blue. The Rab6 binding region is predicted to be between (a.a. 1124-1554). (B) Alphafold predicted model of dimeric TGIF coiled-coil domain (a.a. 1102-2179). Six long stretches of alpha-helices are broken by 5 short unstructured breaks leading to a folded confirmation. N and C-termini are indicated with arrows (C) Alphafold predicted model of the TGIF N-terminal domain (a.a. 1-1101) and the first coiled-coil segment (a.a. 1102-1332). The NTD is made up of tighly packed alpha helices. This is a low confidence prediction with pIDDT scores between 50 and 70. (D) Construct shown in (C) rotated 90°. (E) Alphafold predicted model of TGIF tail domain as a dimer with the last coiled-coil segment. The CTD is formed from four short alpha helices which together form a H shape. (B-E) One monomer is shown in dark blue and the other shown in grey. (F) Immunofluorescence of TGIF-Ty parasites showing colocalization between TGIF (anti-Ty; green) and the cis-Golgi markers mGRASP - mCherry (magenta, top) and Sec13-EmGFP (magenta, bottom) during interphase and replication. Parasite periphery is indicated using anti-IMC3 antibodies (yellow). Scale bars are 5μm. Representative images from three experimental replicates (N = 47 vacuoles). Images are single z-slices. (G) Immunofluorescence of TGIF-Ty parasites after IAA treatment for 1, 2, 4, 6, and 8 hours. Anti-Ty (green) was used to visualize TGIF and an anti-MLC1 antibody (grey) indicate parasite periphery. Scale bars are 5μm. Representative images from two experimental replicates (N > 20 vacuoles per condition). Images are maximum intensity projection of z-stacks. (H) Western blot with anti-Ty (TGIF) and anti-tubulin (loading control) on TGIF-Ty parasite lysates harvested after 6 hours of IAA treatment, along with control parasites treated with ethanol. (I) Plaque assay of TGIF-Ty parasites grown for 8 days with ethanol (control) or IAA (knockdown). No plaques form upon TGIF knockdown.