The function of Hok1 is conserved. (A) Phylogenetic tree of Hook and closest non-Hook proteins and the correlation with the presence of kinesin-3 motors. No Kif1A-like sequence was found at NCBI for A. fumingatus, and no Hook was listed for A. nidulans. The tree is based on Hook domains, identified in Pfam, and calculated in MEGA 5.10 (Tamura et al., 2011). (B) Domain organization of human Hook3 and the chimeric proteins Hok1^HsH3_1–600, Hok1^{HsH3_166–436}, and Hok1^{HsH3_293–345}. (C) Colocalization of Hok1^HsH3_1–600 and Hok1^{HsH3_166–436} with mCherry-Rab5a on apical EEs. (D) Morphology of Δhok1 expressing Hok1, Hok1^HsH3_1–600, Hok1^{HsH3_166–436}, and Hok1^{HsH3_293–345}. The conserved region of human Hook3 restores the mutant phenotype in U. maydis. (E) Co-motility of Hok1^{HsH3_293–345} and mCherry-Rab5a–labeled EEs. See also Video 8. (F) Bidirectional flux of EEs in wild type and Δhok1 expressing Hok1^{HsH3_293–345}. Bars represent two experiments and are means ± SE; sample size is indicated. No significant difference was found (P = 0.285, Kruskal–Wallis test). (G) Endocytic sorting defect in Δhok1 and dynein (Dyn2^ts) mutants treated with the dye FM4-64. In wild type, the dye appears in the vacuoles (stained with Cell Tracker Blue CMAC), whereas in the mutants, it accumulates next to GFP-Rab5a–carrying EEs and does not reach the vacuoles (asterisks). The arrowhead indicates the FM4-64–stained “cloud.” (H) The late endocytic compartment in Δhok1. In wild-type cells, the late endosomes marker Rab7 localizes to vacuoles and motile structures (arrowhead). In Δhok1, Rab7 colocalizes with the apical FM4-64–positive cloud, whereas the localization on vacuoles is not affected. Images in C, D, E, G, and H were adjusted in brightness, contrast, and γ settings. Horizontal bars are in micrometers, and the vertical bar is in seconds.