Fig. 4: Mutagenesis analysis of the role of TRAK1 Site-1 and Site-2 in MIRO1 binding.

a Sequence alignment of a subgroup of TRAK1/2 from 183 vertebrate sequences (Supplementary Fig. 1a), highlighting conserved region 4 (CR4). Amino acids conserved in 85–97% and 97–100% of the sequences are shaded in light and dark blue, respectively. Residues W589 and L597, which were mutated to aspartate (W589D and L597D), are marked with stars. b, d Representative SDS-PAGE analyses of MIRO1_1-591 pulldown by wild-type (WT), W589D, and L597D variants of MBP-TRAK1_569-623 and MBP-TRAK1_{416-446,561-623} (n = 4). Pulldowns were performed in the presence of 1 mM CaCl₂ and 50 µM GTP. From left to right, the gel lanes correspond to the MIRO1_1-591 load control, and amylose pulldowns of MIRO1_1-591, MBP-TRAK1 constructs, and MIRO1_1-591 + MBP-TRAK1 constructs. c, e Densitometric quantification of the pulldowns. Colored bars (matching the gel contours in parts b and d) and brackets represent the mean ± SD for each pulldown condition. Data are normalized to the average of the corresponding WT MBP-TRAK1 construct. Statistical analyses were performed using a right-tailed one-way ANOVA with Tukey’s multiple comparisons test. P-values: part c (WT vs W589D p < 0.0001, WT vs L597D p < 0.0001, W589D vs L597D p = 0.9754), part e (WT vs W589D p < 0.0001, WT vs L597D p = 0.0019, W589D vs L597D p = 0.0001). P-values are also indicated in the figures, with ns for p > 0.05. Gels and quantifications are provided in the Source Data file.