![Figure 4. Coiled-coil region of PxdA is necessary and sufficient for peroxisome motility and endosomal hitchhiking. (A) Schematic of PxdA protein constructs used in this analysis relative to FL PxdA. UR1 (white) and UR2 (yellow) are on the N and C terminus, respectively. Predicted regions of coiled-coil (CC1, 2, and 3) are indicated in orange. All strains contain a C-terminal fluorescent protein tag (either mKate or TagGFP). (B) Normalized line scans of peroxisome distribution from the hyphal tip for FL, C-terminal deletion (ΔCT), N-terminal deletion (ΔNT), and ΔUR2 PxdA strains (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 0.51 and 3.71 µm for FL vs. ΔCT; n = 51–54 hyphae per genotype). (C) Bar graph of the flux of peroxisome movements in FL, ΔCT, and ΔUR2 hyphae. Peroxisome movements were 3.69 ± 0.47 (SEM)/min for FL, 0.44 ± 0.14/min for ΔCT, and 3.10 ± 0.50/min for ΔUR2 (P < 0.0001, one-way analysis of variance; *, Bonferroni post hoc test significant for FL vs. ΔCT; FL vs. ΔUR2 was not significantly different; n = 29–35 hyphae per genotype). (D) Normalized line scans of peroxisome distribution along hyphae expressing either FL or CC1–3 PxdA (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 1.19 and 3.03 µm from hyphal tip; n = 78 [FL] and n = 68 [CC1–3]). (E) Normalized line scans of peroxisome distribution along hyphae expressing either FL or ΔCC2/3 PxdA (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 0.87 and 4.11 µm from hyphal tip; n = 28 [FL] and n = 32 [ΔCC2/3]). (F) Bar graph of the flux of peroxisome movements in FL and ΔCC2/3 hyphae. Peroxisome movements were 3.69 ± 0.47 (SEM) per min for FL (this is the same FL data depicted in C) and 0.51 ± 0.13 per min for ΔCC2/3 (P < 0.0001, one-way analysis of variance; *, Bonferroni post hoc test significant for FL vs. ΔCC2/3; n = 29–35 hyphae per genotype). (G) Representative kymographs generated from simultaneous time-lapse movies of EEs with FL or ΔCC2/3 PxdA. Colocalization represents the number of EEs that colocalize with PxdA. FL is 54.8% ± 3.8% (Fig. 3 B) and ΔCC2/3 3.2% ± 1.3% (n = 10 kymographs). (H) Model for peroxisome (P) hitchhiking on EEs mediated by PxdA. HookA recruits the transport machinery to EEs. PxdA is required to tether EEs to peroxisomes and may have distinct binding partners (x) on each organelle. The arrow depicts the direction of movement.](https://cdn.rupress.org/rup/content_public/journal/jcb/212/3/10.1083_jcb.201512020/6/jcb_201512020_fig4.jpeg?Expires=1773484376&Signature=xvANOzI2Fp~~lgDq~VMLnhsjweUzEaG4~i8e-Nd4M~QfKJQNUiIlRuhe027vtP~mzoaoKgfpuYQxmwga-9eM2oOb2bti2ELG-aOfGVC2m3N3lBfulYNfcvn9uMSCpyf2So51gbUn5OU1sXqq~JqP0MUCfDr0h4KPrQolKfhsM6JAnxg9gpCklsaXgSZaLybJHfcObsyxhTE1JzsvsnmFqm8wsauSEKkhH-5hTjYd03Q8~hnWtaM88qsrDnFpbaoZ5vYNbwJDbVVuTdcSTDcDVfa02jmTXDm1erb9vnFGLqK0HFh6h8aI3R2z-4CudUiLxYJs2MZpoEEpTpw2n0wwig__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Coiled-coil region of PxdA is necessary and sufficient for peroxisome motility and endosomal hitchhiking. (A) Schematic of PxdA protein constructs used in this analysis relative to FL PxdA. UR1 (white) and UR2 (yellow) are on the N and C terminus, respectively. Predicted regions of coiled-coil (CC1, 2, and 3) are indicated in orange. All strains contain a C-terminal fluorescent protein tag (either mKate or TagGFP). (B) Normalized line scans of peroxisome distribution from the hyphal tip for FL, C-terminal deletion (ΔCT), N-terminal deletion (ΔNT), and ΔUR2 PxdA strains (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 0.51 and 3.71 µm for FL vs. ΔCT; n = 51–54 hyphae per genotype). (C) Bar graph of the flux of peroxisome movements in FL, ΔCT, and ΔUR2 hyphae. Peroxisome movements were 3.69 ± 0.47 (SEM)/min for FL, 0.44 ± 0.14/min for ΔCT, and 3.10 ± 0.50/min for ΔUR2 (P < 0.0001, one-way analysis of variance; *, Bonferroni post hoc test significant for FL vs. ΔCT; FL vs. ΔUR2 was not significantly different; n = 29–35 hyphae per genotype). (D) Normalized line scans of peroxisome distribution along hyphae expressing either FL or CC1–3 PxdA (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 1.19 and 3.03 µm from hyphal tip; n = 78 [FL] and n = 68 [CC1–3]). (E) Normalized line scans of peroxisome distribution along hyphae expressing either FL or ΔCC2/3 PxdA (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 0.87 and 4.11 µm from hyphal tip; n = 28 [FL] and n = 32 [ΔCC2/3]). (F) Bar graph of the flux of peroxisome movements in FL and ΔCC2/3 hyphae. Peroxisome movements were 3.69 ± 0.47 (SEM) per min for FL (this is the same FL data depicted in C) and 0.51 ± 0.13 per min for ΔCC2/3 (P < 0.0001, one-way analysis of variance; *, Bonferroni post hoc test significant for FL vs. ΔCC2/3; n = 29–35 hyphae per genotype). (G) Representative kymographs generated from simultaneous time-lapse movies of EEs with FL or ΔCC2/3 PxdA. Colocalization represents the number of EEs that colocalize with PxdA. FL is 54.8% ± 3.8% (Fig. 3 B) and ΔCC2/3 3.2% ± 1.3% (n = 10 kymographs). (H) Model for peroxisome (P) hitchhiking on EEs mediated by PxdA. HookA recruits the transport machinery to EEs. PxdA is required to tether EEs to peroxisomes and may have distinct binding partners (x) on each organelle. The arrow depicts the direction of movement.
