Hok1 is required for cargo binding of kinesin-3. (A) Localization of an EE cluster, labeled with GFP-Rab5a (arrowhead) in a cell expressing the peptide pHk^361–385 for 2 h. Note that clusters initially appear subapically but later shift to the cell end (tip). (B) Motility of GFP-Rab5a–labeled EEs in a cell expressing the peptide pHk^361–385 for 2 h. A subapical cluster is indicated by an asterisk. Cell end is indicated by tip and the dotted line. See also Video 7. (C) Mean run length of EEs in control cells and cells expressing pHk^361–385 for 2 h. Bars represent data from two experiments and are means ± SE; sample size is indicated. Significant difference is indicated: ***, P < 0.0001. (D) Contrast-inverted kymographs showing anterograde motility of kinesin-3–GFP in control cells, after expressing the peptide pHk^361–385, and in Δhok1 and Δhok1 cells expressing Hok1^Δ361–385. Kinesin-3 signals are strong in the control but weak in all mutants. (E) Kinesin-3-GFP numbers in control cells, after expressing the peptide pHk^361–385, and in Δhok1– and hok1–null cells expressing Hok^Δ361–385. Note that native kinesin-3 levels are shown. All numbers represent two experiments and are given as means ± SD; sample size is indicated. Motor number estimation is based on comparison with an internal calibration standard (Schuster et al., 2011c) and assuming that kinesin-3 is a dimer (Hammond et al., 2009). Red dotted lines indicate medians. Images in A, B, and D were adjusted in brightness, contrast, and γ settings. Horizontal bars are in micrometers, and vertical bars are in seconds.