Analysis of the three domains of Inp1. (A) Schematic representation of truncated and mutant forms of Inp1. White, N terminus with conserved positive charges; blue, MHD/PH-like domain; purple, C-terminal domain. In Inp_Mut 13 lysines (K) and arginines (R) were substituted by glutamic acids (E). (B) Predicted secondary structures of H. polymorpha Inp1 obtained with Foundation (Bordin et al., 2018) and sequence features. The black horizontal line represents the protein sequence. The predicted β-strands and α-helices are depicted by bars above the line in cyan and magenta, respectively, with the height of the bars representing the confidence of the prediction. The black box represents the stretch of positively charged residues. The green box represents the predicted conserved PEST sequence. White, blue, and purple blocks are as in A. (C) Predicted structure of H. polymorpha Inp1 residues 99–218 (MHD). The alignment of this region with human Ran-binding protein 2 (1XKE_A) was used to seed Modeller to predict the structure. Residues 121–134 between strands 1 and 2 are omitted as they do not align to any known structure, have no conserved residues, and are predicted to form an unstructured loop. (D) Single focal plane FM images of glucose-grown cells producing Pex3-mKate2 and the indicated Inp1 variants C-terminally tagged with GFP and produced under control of the INP1 promoter. The GFP fluorescence images were processed differently in order to visualize the fluorescence optimally. (E) Quantification of peroxisome inheritance in the indicated strains. 2 × 20 cells were quantified from two independent experiments. Error bars indicate SD. (F) Western blot analysis of cells producing the indicated Inp1 truncations. Blots were decorated with α-GFP or α-Pyc1 antibodies. Pyc1 was used as a loading control.