Fig. 3: α-Syn’s N-terminus is required for mitochondrial localization and TOM40/TOM20 interaction.

A α-Syn structure PDB ID:1xq8 divided into 20 residue fragments, docked against TOM40 structure PDB ID: 7CK6 using AutoDockCrankPep. Image created with PyMOL. B Graph determining α-Syn residues with a higher binding affinity for TOM40. C Schematic representation of vectors used for generating wild-type (WT), mitochondria-targeted signal (MTS), and truncated (Δ1–33) overexpressing α-Syn SH-SY5Y stable cell lines (N N-terminal, NAC nonamyloidogenic component, C C-terminal). Representative immunoblot from WT, MTS, and Δ1-33 α-Syn whole-cell extracts (Ab: α-Syn 204, Biolegend). D Densitometry analysis indicates TOM40 protein level are specifically affected in WT (Lns 3,4) and MTS α-Syn (Lns 6–8) overexpressing models. E Immunofluorescence images confirming cellular localization of Flag α-Syn (Ab: Anti-DDDDK tag, Abcam) after 48 h Dox induction in WT α-Syn, MTS α-Syn, and Δ1-33 α-Syn. Scale bar = 10 µm. F Quantification of Manders’ colocalization coefficient M1 (Fraction of red channel = Flag α-Syn in colocalization with green channel = TOM20) or M2 (Fraction of green channel = TOM20 in colocalization with red channel = Flag α-Syn). G PLA images highlighting the interaction between α-Syn (Ab: α-Syn 3H2897, Santa Cruz), and TOM20 in mitochondria (green foci), co-stained with MitoTracker (red). Scale bar = 10 µm. H PLA analysis showing increased WT and MTS α-Syn interaction with TOM20. Scale bar = 10 µm. Data are presented as mean ± s.e.m. from three independent experiments; quantification of PLA foci (H) was derived from 50 cells. The statistical analyses used Student’s t-test (D), one-way ANOVA (F), and two-way ANOVA (H). ns non-significant (p > 0.05).