A conserved region in the first CC is essential for Hok1 function. (A) Organization of truncated Hok1^Δ225–624, Hok1^1–224PX, which carries an EE-targeting PX domain, and Hok1^225–930. (B) Colocalization of truncated Hok1 proteins and mCherrry-Rab5a–labeled EEs in Δhok1 and colocalization of Hok1-GFP and EEs in cells expressing the peptide pHk^361–385. Cell edges are indicated in blue. All constructs localize to EEs without rescuing Δhok1. (C) Morphology of Δhok1 mutants expressing Hok1^Δ225–624, Hok1^1–224PX, Hok1^225–930, and Hok1^Δ361–385. Neither rescues the morphology defect. (D) Localization of a highly conserved region within the first CC of Hok1 and human Hook3. Identical amino acids are shown in light blue, and similar amino acids are shown in pink. The alignment was generated in ClustalW. H.s., Homo sapiens; U.m., U. maydis. (E) Organization of the truncated proteins Hok1^Δ361–385 and Hok1^Δ333–355. (F) Anterograde and retrograde flux of mCherry-Rab5a–labeled EEs in control cells and Hok1^Δ361–385 and Hok1^Δ333–355 (Δ333–355 and Δ361–385) at ∼10 µm behind the cell tip. Bars represent data from two experiments and are means ± SE; sample size is indicated. (G) Anterograde and retrograde flux of mCherry-Rab5a–labeled EEs in control cells and cells expressing the peptide pHk^361–385 (see D for sequence). Bars represent data from two experiments and are means ± SE; sample size is indicated. Images in B and C were adjusted in brightness, contrast, and γ settings. Significant difference is indicated: ***, P < 0.0001. Bars are in micrometers.