DUF domain phosphorylation regulates Pex30. (A) Schematic representation of Pex30 domains. Serine 446 (S446) within the DUF is indicated. (B) Localization of Pex30 and the indicated phospho-mutants (Pex30S446D and Pex30S446A) expressed from the endogenous Pex30 locus during the exponential, diauxic shift, and stationary phases. Pex30 and derivatives were expressed as mNG fusions. The concentration of endogenous Nvj1-tdTomato at the NVJ was monitored. Yellow arrowheads highlight colocalization between Pex30 and Nvj1. Bar, 5 µm. (C) Quantification of cells with Nvj1-tdTomato concentrated at the NVJ, during different growth stages as in B. The bars represent the mean and SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were performed to compare the percentage of cells with organized Nvj1 with the WT condition for each time point (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, not significant). (D) Quantification of cells with LDs clustered around the NVJ, during the diauxic shift and stationary phase. The bars represent the mean and SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were performed to compare the percentage of cells with clustered LDs with the WT condition for each time point (*, P < 0.05). (E) Localization of Pex30 and Nvj1-tdTomato in cells with the indicated genotype during the diauxic shift. Yellow arrowheads highlight colocalization between Pex30 and Nvj1. Magenta arrowheads highlight mislocalized Nvj1-tdTomato. Bar, 5 µm. (F) Quantification of cells with Nvj1-tdTomato concentrated at the NVJ, in cells grown as in E. The bars represent the mean and SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were performed to compare the percentage of cells with organized Nvj1 with the WT condition (****, P < 0.0001; *, P < 0.05).