Kinesin-1 autoinhibition is not critical for its own cargo - transport function. (A) Microscopic images showing the hyphal-tip signals of GFP-dynein in the ∆hookA single and the kinA^K895*, ∆hookA double mutant. Hyphal tip is indicated by a yellow arrowhead. Bar, 5 μm. (B) A quantitative analysis on GFP-dynein comet intensity in the ∆hookA single and the ∆hookA, kinA^K895* double mutant. All values are relative to the average value for the ∆hookA single mutant, which is set as 1. Scatter plots with mean and SD values were generated by Prism 9. The Mann–Whitney test (unpaired, two tailed) was used for analyzing the two data sets without assuming normal distribution of the data. (C) Colony phenotypes of the various single and double myosin-V (myoV) and kinesin-1 (kinA) mutants. (D) Quantitative analyses of colony diameters of the alcA-myoV (n = 25) and the kinA^(1–894)-GFP, alcA-myoV (n = 23) strains (unpaired, Mann–Whitney test) and of the ∆myoV (n = 25) and the kinA^(1–894)-GFP, ∆myoV (n = 28) strains (unpaired, Mann–Whitney test). (E) Microscopic images showing the hyphal-tip signals of RabE-GFP in the ∆myoV single mutant and the ∆myoV, kinA^K895* double mutant. Hyphal tip is indicated by a yellow arrowhead. Bar, 5 μm. (F) Line scans of RabE-GFP fluorescence intensity in the ∆myoV single mutant and the ∆myoV, kinA^K895* double mutant (n = 20 for each strain). XY graphs with mean (solid lines) and SEM (shading) were generated by Prism 9. RabE-GFP intensity was significantly higher in the ∆myoV, kinA^K895* double mutant than in the ∆myoV single mutant near the hyphal tip (between 0.65 and 1.43 μm from hyphal tip) (P < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test). (G) Kymographs showing RabE-GFP signals (diagonal lines indicating movements of RabE-GFP-marked vesicles). Green arrows indicate plus-end-directed movements toward the hyphal tip, and a yellow arrow indicates minus-end-directed movement away from the hyphal tip. (H) A quantitative analysis on the frequency of minus-end-directed transport in the ∆myoV single mutant (n = 31 hyphal tips) and the ∆myoV, kinA^K895* double mutant (n = 50 hyphal tips; unpaired, Mann–Whitney test, Prism 9). Scatter plots with mean and SD values were generated by Prism 9. (I) A quantitative analysis on the speed of minus-end-directed transport in the ∆myoV single mutant (n = 140 movements) and the ∆myoV, kinA^K895* double mutant (n = 28 movements; unpaired, Mann–Whitney test, Prism 9). Scatter plots with mean and SD values were generated by Prism 9. (J) A quantitative analysis on the frequency of plus-end-directed transport in the ∆myoV single mutant (n = 31 hyphal tips) and the ∆myoV, kinA^K895* double mutant (n = 50 hyphal tips; unpaired, Mann–Whitney test, Prism 9). Scatter plots with mean and SD values were generated by Prism 9. (K) A quantitative analysis on the speed of plus-end-directed transport in the ∆myoV single mutant (n = 158) and the ∆myoV, kinA^K895* double mutant (n = 144) (unpaired, Mann–Whitney test, Prism 9). Scatter plots with mean and SD values were generated by Prism 9.