Fig. 5: A tug-of-war-like mechanism sorts Ptc5 from mitochondria to peroxisomes and prevents detrimental phosphatase activity in the cytosol.
From: Peroxisomal targeting of a protein phosphatase type 2C via mitochondrial transit
a Working model of the mechanism of Ptc5 sorting. Ptc5 (green) either localizes to the mitochondrial intermembrane space or translocates into the peroxisomal matrix after processing by Imp1 (orange). Translocation depends on Pex5 and other components of the peroxisomal protein import machinery (blue). b Fluorescence microscopic pictures of yeast cells expressing either Ptc5∆1–83-RFP or Ptc5∆1–83-RFP-PTS (magenta) together with the peroxisomal membrane protein Ant1-YFP (cyan) in indicated strains. White color indicates colocalization. Scale bar represents 5 µm. c Ptc5∆TM-RFP or Ptc5∆TM-RFP-PTS were co-expressed with Ant1-YFP in indicated strains. Subcellular localization was determined by fluorescence microscopy. White color indicates colocalization. Scale bar represents 5 µm. d Correlation between the signal of Ptc5∆TM-RFP or Ptc5∆TM-RFP-PTS and of Ant1-YFP was quantified. PCC refers to the Pearson’s correlation coefficient. 1: Ptc5∆TM-RFP in WT, 2: Ptc5∆TM-RFP-PTS in WT, and 3: Ptc5∆TM-RFP-PTS in Δpex5. Quantifications are based on n = 3 independent experiments. Each color represents one experiment. Error bars represent standard error of the mean. P-values were calculated with a two-tailed unpaired Student’s t-test. e Quantification of the fraction of peroxisomes contacting mitochondria (PxM) in relation to the total peroxisome count (PxT) of cells co-expressing Ptc5∆TM-RFP or Ptc5∆TM-RFP-PTS with Ant1-YFP, and the mitochondrial membrane protein Tim50 fused to CFP (Supplementary Fig. 5a). Association of Ant1-YFP containing foci with Tim50-CFP was counted in three independent experiments (n = 3). Independent experiments are represented by different colors. Error bars represent standard error of the mean. P-values were calculated with a two-tailed unpaired Student’s t-test. f PhosTag SDS–PAGE of peroxisomal fractions derived from WT and ∆ptc5 cells expressing Gpd1-GFP (Supplementary Fig. 6a) was analyzed by western blot to determine the phosphorylation status of Gpd1-GFP. λ-PP indicates treatment with λ-protein phosphatase prior to PhosTag SDS–PAGE. g Samples shown in f were also analyzed by conventional SDS–PAGE and western blot to assure that migration differences result from altered phosphorylation. h, i Serial dilutions of indicated strains were spotted on SC-HIS medium containing either glucose or ethanol as carbon source. j Western blot showing the expression levels of Ptc5∆1–83-RFP and its phosphatase-dead derivatives D302A and D424A. Por1 served as a loading control. Source data are provided in the Source data file.
