Fig 2: vps35 genetically interacts with parkin. (A–F) Climbing ability was assessed at different ages in animals single and double heterozygous for null alleles of parkin (park25) and two independent null alleles of vps35, vps35MH20 (A–C) and vps35E42 (D–F). n = 38–48 (A), 42–75 (B), 36–54 (C), 50–63 (D), 63–80 (E) and 44–64 (F). (G) Survival analysis of single and double heterozygotes, alongside park25 homozygotes, exposed to 5 mm paraquat (PQ). n = 8–60 animals. (H) Analysis of dopaminergic neuron loss in the PPL1 cluster in 30-day-old animals. n = 17, 22 and 16, respectively. Histograms show mean ± SEM. Statistical analysis: ns = not significant, **P < 0.01, ***P < 0.001 and ****P < 0.0001; one-way ANOVA with Bonferroni's correction, or Log-rank test for survival.

Fig 3: GluN2A Ser-1459 phosphorylation enhances SNX27-retromer binding(A) ITC experiments comparing the binding of GluN2A C-terminal peptides, either WT (blue), or the S1459A phospho-deficient (orange), S1459E phospho-mimetic (green), or S1462E PDZ binding-deficient (purple, negative control) mutant, to the purified SNX27 PDZ domain protein. The thermodynamic parameters of binding are given in Table S1.(B) GST pull-down experiments from HEK293T cells co-transfected with plasmids encoding myc-SNX27 and GST alone or GST-GluN2A C-tails including WT, S1459A (SA), S1459E (SE), and the V1464E PDZbm mutant (PDZd). Total cell lysates (input) and bound proteins (pull-down) were resolved by SDS-PAGE and analyzed by western blotting with specific antibodies against myc, VPS35, VPS26, and GST.(C–E) Quantification of the levels of myc-SNX27 (C), VPS35 (D), and VPS26 (E) binding to GST-GluN2A C-tails. Data represent mean ± SEM of band intensities normalized to WT values (n = 3–4; from three independent experiments). *p < 0.05, ***p < 0.001, ****p < 0.0001 using one-way ANOVA with a Tukey’s multiple comparison test.(F) GST pull-down experiments revealed that the H112A mutation in myc-SNX27, which disrupts binding to PDZbm, failed to bind both WT and the S1459E phospho-mimetic GST-GluN2A C-tails in HEK293T cells.(G) HEK293T cells were co-transfected with plasmids encoding myc-SNX27, GST-GluN2A C-tails (WT or S1459A), together with pEGFP or pEGFP-tCaMKIIa (constitutively active truncated CaMKIIa). Cells were lysed and pulled down with GSH-Sepharose. Bound proteins and total lysates were analyzed by immunoblotting with specific antibodies against myc, GST, GluN2A pS1459, and GFP.(H) Quantification of the level of myc-SNX27 binding to GST-GluN2AC-tails. Data represent mean ± SEM of band intensities normalized to WT values(n = 3; from three independent experiments). *p < 0.05 using two-way ANOVA with a Sidak’s multiple comparison test.

Fig 4: vps35 does not genetically interact with pink1. (A–F) Climbing ability was assessed at different ages in animals single and double heterozygous for null alleles of pink1 (pink1B9) and two independent null alleles of vps35, vps35MH20 (A–C) and vps35E42 (D–F). n = 77–94 (A), 53–87 (B), 27–42 (C), 23–39 (D), 50–85 (E) and 42–73 (F). (G) Overexpression of vps35 does not suppress climbing deficits in pink1 mutants (n = 60–100 animals) but slightly enhances the flight defect (H) (n = 35–60 animals). Histograms show mean ± SEM. Statistical analysis: ns = not significant, **P < 0.01, ***P < 0.001 and ****P < 0.0001; one-way ANOVA with Bonferroni's correction. Comparisons are with pink1-mutant control (pink1B9; da(GAL4)/+) unless otherwise indicated.

Fig 5: Overexpression of vps35 rescues some parkin-mutant phenotypes. Ubiquitous overexpression of all variants of Drosophila or human vps35 rescues parkin-mutant climbing defect (A), but not the flight defect (B). n = 50–150 animals. Sensitivity of parkin mutants to 5 mm paraquat (PQ) exposure was completely rescued by transgenic expression of all variants of Drosophila (C) or human (D) vps35. n = 40–100 animals. Control genotype is heterozygous da-GAL4 driver line alone. Histograms show mean ± SEM. Statistical analysis: ns = not significant, **P < 0.01, ***P < 0.001 and ****P < 0.0001; one-way ANOVA with Bonferroni's correction, or Log-rank test for survival. All comparisons are with park25.