Integration of a mitochondrial marker at a URA3 safe harbor. (A) Schematic showing the integration strategy to replace ura3::HYGR with URA3:CIT1-tdTomato. (B and C) Number of transformants (uracil prototrophs) and (C) % of hygromycin-sensitive URA3:CIT1-tdTomato integrants from three independent transformations. (D) Inverted greyscale image of A. pullulans strains growing on YPD. Colonies 1–6 are URA3:CIT1-tdTomato integrants from three independent transformations. (E) Quantification of colony diameter from the images shown in D. (F) Maximum intensity projection of confocal z-stacks of wild-type (WT) cells and example transformants expressing Cit1-tdTomato. Scale bar, 5 μm. (G) Quantification of the average tdTomato signal in confocal images of WT cells and cells expressing Cit1-tdTomato from three separate imaging experiments. Dark magenta symbols represent the mean for each experiment and light pink symbols indicate values for individual cells (n = 247 WT cells and 352, 376, 181, 386, 408, and 263 cells from colonies 1–6). (H) Maximum intensity projection of confocal images showing colocalization of Cit1-tdTomato (magenta) and MitoTracker dye (cyan). Scale bar, 5 μm. (I and J) Number of transformants (uracil prototrophs) and (J) % of hygromycin-sensitive URA3:CIT1-tdTomato integrants from three independent transformations using competent cells frozen at −80°C for 1 wk before transformation. Statistical significance calculated by two-tailed Student’s t test in B and I and by one-way ANOVA in E and G; P values indicated (not shown, P > 0.05; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001).