Pex30-S446 is highly phosphorylated upon the diauxic shift. (A) Distribution of the posttranslational modifications previously reported for Pex30. Check supplementary data Table S3 for details on the residues and respective studies. (B) Pex30-S446 is more phosphorylated during the stationary phase. Log₂ intensity of peptides phosphorylated in distinct Pex30 residues during exponential and stationary growth phases. White represents no detection of the peptide in the sample. Right: the intensity of peptides that contained phosphorylated S446 was normalized to the amount of total Pex30 protein in the corresponding sample. The bars represent the mean and SD. Three independent experiments were conducted, and Student’s t test (two-tailed) was performed to compare the normalized intensity between conditions (*P < 0.05). (C) Pex29 interacts with Pex30 independently of its phosphorylation status. Crude membrane fractions of cells with the indicated genotypes and expressing endogenous Pex29-V5, or untagged proteins as control, were solubilized with detergent, and extracts were subjected to IP with anti-V5 antibody. Eluted proteins were separated by SDS-PAGE and analyzed by western blotting. Pex29-V5 and Pex30 were detected with anti-V5 and anti-Pex30, respectively. Dpm1, used as a loading control, was detected with anti-Dpm1 antibody. *, IgG light chain. IB, immunoblot; IP, immunoprecipitation. The position of molecular weight markers (in kDa) is indicated. (D) Distribution of peroxisomes in cells with the indicated genotype during exponential growth. Peroxisomes were labeled by the mCherry-PTS1 marker. Please note the increase of cytosolic fluorescence in the mutant cells, corresponding to non-imported mCherry-PTS1. Images correspond to maximum intensity Z-projections. Bar, 5 µm. (E) Quantification of the number of peroxisomes per cell, in cells grown as in D. Three independent experiments were analyzed (>30 cells/genotype/experiment were counted). Each dot corresponds to a cell, and the bars represent the mean and SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were performed to compare the number of peroxisomes between mutants and the WT condition (****, P < 0.0001; ns, not significant). (F) Localization of the NVJ component Tsc13 was analyzed in cells with the indicated genotype during the exponential, diauxic shift, and stationary phases. Endogenous Tsc13 was tagged with GFP (Tsc13-GFP), and endogenously expressed Vph1-tdTomato was used as a vacuole marker. Bar, 5 µm. Source data are available for this figure: SourceData FS5.