Figure S2.
Additional details of the Rab2-Syd Co-IP interaction, the predicted structure of RUFY, and RUFY-DLIC Co-IP results. (A) Co-IP experiment performed on lysates from HEK cells transfected with constructs encoding epitope-tagged Drosophila proteins, illustrating the detergent sensitivity of the Rab2Q65L:Syd interaction. Western blot of eluates probed against myc showing coprecipitation of myc-Syd in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of HA-Rab2Q65L when immunoprecipitating against HA. In lanes 1–2, the experiment was performed in the presence of 0.1% saponin and 0.25% Triton X-100, and proteins were eluted from the anti-HA beads by boiling in SDS-PAGE loading buffer. In lanes 3–4, the experiment was performed only in the presence of 0.1% saponin, and proteins were eluted with 100 mM NaOH. In lanes 5–6, the same anti-HA beads that were eluted with 100 mM NaOH were boiled in SDS-PAGE loading buffer to elute the remaining protein. (B) Co-IP of myc-Syd-N2 by HA-Rab2, HA-Rab2S20N, and HA-Rab2Q65L. Compared with the experiment using full-length myc-Syd shown in Fig. 2 B, the amount of transfecting DNA-encoding HA-Rab2S20N was increased to match the higher expression levels of HA-Rab2 and HA-Rab2Q65L. (C) Top, expected structure of Syd homodimer assembled from three separate AlphaFold predictions mapped onto the domain architecture of Syd. Middle, alignment of the RH2 domain from Drosophila Syd, human JIP3 and JIP4, and the cnidarian (Hydra vulgaris) JIP3/4 ortholog. Small dots in alignment indicate residue identity to Syd-RH2. The predicted locations of Helix 1, Helix 2, and the intervening loop from the AlphaFold model in Fig. 2 H are shown together with a helical propensity estimation (bottom). Also indicated are the location of the residues mutated to alanines in D (large colored dots below alignment) and Fig. 2 I (red triangles), the C-terminal extent of the Syd-N4 (Syd1-485) truncation, and the partially conserved DLIC-like motif involved in autoinhibition (Singh et al., 2024). (D) Left, Co-IP of WT myc-Syd-N2 and three different sets of myc-Syd-N2 alanine substitution mutants by HA-Rab2Q65L. The position of the mutations is indicated in C. Right, Quantification of the anti-myc immunosignal from eluted WT and mutated myc-Syd-N2 (n = three independent experiments). ANOVA, followed by Tukey’s test. (E) The effect of different levels of LRRK2 activity on Co-IP of full-length myc-Syd by HA-Rab2Q65L. Endogenous HEK cell LRRK2 activity was inhibited by treatment of cells with 2µM MLi-2 for 2 h before lysis, or increased by co-transfection with a constitutively active LRRK2G2019S mutant. (F) Structure of an RUFY dimer predicted using AlphaFold 3, and the RUFY domain architecture. (G) Co-IP of V5-tagged DLIC with HA-tagged Syd-N4 (Syd1-485) and RUFY. Source data are available for this figure: SourceData FS2.

Additional details of the Rab2-Syd Co-IP interaction, the predicted structure of RUFY, and RUFY-DLIC Co-IP results. (A) Co-IP experiment performed on lysates from HEK cells transfected with constructs encoding epitope-tagged Drosophila proteins, illustrating the detergent sensitivity of the Rab2Q65L:Syd interaction. Western blot of eluates probed against myc showing coprecipitation of myc-Syd in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of HA-Rab2Q65L when immunoprecipitating against HA. In lanes 1–2, the experiment was performed in the presence of 0.1% saponin and 0.25% Triton X-100, and proteins were eluted from the anti-HA beads by boiling in SDS-PAGE loading buffer. In lanes 3–4, the experiment was performed only in the presence of 0.1% saponin, and proteins were eluted with 100 mM NaOH. In lanes 5–6, the same anti-HA beads that were eluted with 100 mM NaOH were boiled in SDS-PAGE loading buffer to elute the remaining protein. (B) Co-IP of myc-Syd-N2 by HA-Rab2, HA-Rab2S20N, and HA-Rab2Q65L. Compared with the experiment using full-length myc-Syd shown in Fig. 2 B, the amount of transfecting DNA-encoding HA-Rab2S20N was increased to match the higher expression levels of HA-Rab2 and HA-Rab2Q65L. (C) Top, expected structure of Syd homodimer assembled from three separate AlphaFold predictions mapped onto the domain architecture of Syd. Middle, alignment of the RH2 domain from Drosophila Syd, human JIP3 and JIP4, and the cnidarian (Hydra vulgaris) JIP3/4 ortholog. Small dots in alignment indicate residue identity to Syd-RH2. The predicted locations of Helix 1, Helix 2, and the intervening loop from the AlphaFold model in Fig. 2 H are shown together with a helical propensity estimation (bottom). Also indicated are the location of the residues mutated to alanines in D (large colored dots below alignment) and Fig. 2 I (red triangles), the C-terminal extent of the Syd-N4 (Syd1-485) truncation, and the partially conserved DLIC-like motif involved in autoinhibition (Singh et al., 2024). (D) Left, Co-IP of WT myc-Syd-N2 and three different sets of myc-Syd-N2 alanine substitution mutants by HA-Rab2Q65L. The position of the mutations is indicated in C. Right, Quantification of the anti-myc immunosignal from eluted WT and mutated myc-Syd-N2 (n = three independent experiments). ANOVA, followed by Tukey’s test. (E) The effect of different levels of LRRK2 activity on Co-IP of full-length myc-Syd by HA-Rab2Q65L. Endogenous HEK cell LRRK2 activity was inhibited by treatment of cells with 2µM MLi-2 for 2 h before lysis, or increased by co-transfection with a constitutively active LRRK2G2019S mutant. (F) Structure of an RUFY dimer predicted using AlphaFold 3, and the RUFY domain architecture. (G) Co-IP of V5-tagged DLIC with HA-tagged Syd-N4 (Syd1-485) and RUFY. Source data are available for this figure: SourceData FS2.

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