Fig. 5: TgVIP1 localization and FFAT motif interaction.
a, Diagram of the predicted protein TgME49_233220 that we renamed TgVIP1 showing the approximate position of the signal sequence, FFAT motifs with sequences and the transmembrane domain. In ToxoDB, two transmembrane domains are predicted but the first one overlaps with the signal sequence. * indicates FFAT motif with higher score. TM, transmembrane domain; aa, amino acids. b, IFA. HeLa cells infected for 24 h with parental parasites, fixed and immunostained for GFP (green), to test the specificity of the antibody. Single z-slices are shown. c, IFA. HeLa cells infected for 24 h with TgVIP1 tagged at the C-terminus with mEmerald, fixed and either immunostained for GFP or imaged for native mEmerald signal. The exposure times and brightness and contrast changes for immunostained GFP are the same as for cells infected with parental parasites in b. Exposure times for the native mEmerald signal were longer due to a weaker signal. Single z-slices are shown. The white arrows indicate TgVIP1–GFP staining on the PVM. The yellow arrows indicate possible dense granules. Ab, antibody. d, ImmunoEM. HFF cells infected with TgVIP1–GFP for 24 h and immunostained for GFP. DG, dense granules; hcell, host cell. e, IFA. HFF (Hs68) cells were infected with parasites expressing TgVIP1–mCherry for 24 h, fixed and immunostained for VAPA (magenta) and mCherry (cyan). Single z-slices are shown. The boxed region is enlarged, and the white arrows indicate places where both VAPA and TgVIP1–mCherry signals are detected at the PVM. Line scan analysis with fluorescence intensities of the magenta (VAPA) and cyan (TgVIP1–mCherry) channels along the arrow in the inset image is shown. f, Pull-downs of VAP family members by peptides containing FFAT motifs. Peptides were generated with the FFAT motif of OSBP-related protein 1 (ORP1), a known VAP family interacting protein, (positive control), a random sequence (negative control) or the putative FFAT motif 1 or 2 from TgVIP1. The FFAT motif from the positive control and the higher-scoring TgVIP1 motif are shown in bold. The second, lower-scoring motif of TgVIP1 is underlined. The peptides were bound to streptavidin and incubated with HeLa cell lysate. The lysate and samples incubated with the beads were loaded on an SDS-PAGE gel, transferred to a PVDF membrane and immunostained with antibodies to VAPA, VAPB, MOSPD2 and the negative control actin. Lys, HeLa lysate; Neg, negative control peptide; Pos, positive control peptide; M1, the first and lower-scoring putative FFAT motif of TgVIP1; M2, the second and higher-scoring putative FFAT motif of TgVIP1.
