Figure 2.
Deletion of INP1, but not PEX32, affects peroxisome-PM contact formation, whereas INP1 overexpression increases these contacts. (A) Single focal plane confocal laser scanning microscopy Airyscan images of inp1 cells grown for 8 h on methanol and producing Pex3-GFP under control of the endogenous promoter. Vacuoles are marked with FM4-64. (B) Quantification of peripheral Pex3 patches in WT and inp1 cells. 2 × 45 cells were quantified from two independent experiments. Two-tailed Student’s t test was performed. *, P < 0.05. Error bar represents SD. (C) Tomographic reconstruction of glucose-grown inp1 and pex32 cells. Blue, peroxisomal membrane; orange, ER; cyan, PM. (D) SuperPlots showing the distance between the peroxisomal membrane and the PM or the ER in the indicated strains. 2 × 10 cell sections were quantified from two independent experiments. The duplicate experiments are color-coded. The circles represent individual data points. The squares are the averages from each experiment. The error bars indicate the SD. (E and F) Single focal plane confocal laser scanning microscopy Airyscan images from cells grown for 16 h on a mixture of glycerol and methanol and producing Inp1-GFP controlled by the endogenous promoter (WT) or PADH1 (Inp1++) together with Pex3-mKate2 or DsRed-SKL (peroxisomal matrix marker). The vacuole lumen is marked with CMAC. Note that upon growth in these conditions, the cells contain multiple peroxisomes; therefore, in the WT control cells, more Pex3-mKate2 signal and Inp1-GFP spots are present. Because in glycerol/methanol-grown cells peroxisomes harbor an alcohol oxidase crystalloid, the red fluorescence of DsRed-SKL is only present at the periphery of the organelle. Cells were grown on a mixture of glycerol and methanol because Inp1 overproduction affects peroxisome function. (G) Electron micrographs of cryofixed WT or Inp1++ cells, grown for 16 h on methanol/glycerol-containing medium. The arrows indicate peroxisome–PM contact sites. CW, cell wall; P, peroxisome; V, vacuole; M, mitochondrion. (H) Western blot analysis of Inp1-GFP levels in the indicated strains grown for 16 h on methanol/glycerol. Blots were decorated with α-GFP, α-Pex3, or α-Pyc1 antibodies. Pyc1 serves as a loading control. (I) Quantification of the distance between peroxisome membrane and PM in the indicated strains grown in medium containing methanol/glycerol. Error bars indicate SD. 2 × 34 cell sections from two independent cultures were counted. The data are plotted in a SuperPlot. The duplicate experiments are color-coded. The circles represent the single peroxisomes. Two-tailed Student’s t test was performed. *, P < 0.05.

Deletion of INP1, but not PEX32, affects peroxisome-PM contact formation, whereas INP1 overexpression increases these contacts. (A) Single focal plane confocal laser scanning microscopy Airyscan images of inp1 cells grown for 8 h on methanol and producing Pex3-GFP under control of the endogenous promoter. Vacuoles are marked with FM4-64. (B) Quantification of peripheral Pex3 patches in WT and inp1 cells. 2 × 45 cells were quantified from two independent experiments. Two-tailed Student’s t test was performed. *, P < 0.05. Error bar represents SD. (C) Tomographic reconstruction of glucose-grown inp1 and pex32 cells. Blue, peroxisomal membrane; orange, ER; cyan, PM. (D) SuperPlots showing the distance between the peroxisomal membrane and the PM or the ER in the indicated strains. 2 × 10 cell sections were quantified from two independent experiments. The duplicate experiments are color-coded. The circles represent individual data points. The squares are the averages from each experiment. The error bars indicate the SD. (E and F) Single focal plane confocal laser scanning microscopy Airyscan images from cells grown for 16 h on a mixture of glycerol and methanol and producing Inp1-GFP controlled by the endogenous promoter (WT) or PADH1 (Inp1++) together with Pex3-mKate2 or DsRed-SKL (peroxisomal matrix marker). The vacuole lumen is marked with CMAC. Note that upon growth in these conditions, the cells contain multiple peroxisomes; therefore, in the WT control cells, more Pex3-mKate2 signal and Inp1-GFP spots are present. Because in glycerol/methanol-grown cells peroxisomes harbor an alcohol oxidase crystalloid, the red fluorescence of DsRed-SKL is only present at the periphery of the organelle. Cells were grown on a mixture of glycerol and methanol because Inp1 overproduction affects peroxisome function. (G) Electron micrographs of cryofixed WT or Inp1++ cells, grown for 16 h on methanol/glycerol-containing medium. The arrows indicate peroxisome–PM contact sites. CW, cell wall; P, peroxisome; V, vacuole; M, mitochondrion. (H) Western blot analysis of Inp1-GFP levels in the indicated strains grown for 16 h on methanol/glycerol. Blots were decorated with α-GFP, α-Pex3, or α-Pyc1 antibodies. Pyc1 serves as a loading control. (I) Quantification of the distance between peroxisome membrane and PM in the indicated strains grown in medium containing methanol/glycerol. Error bars indicate SD. 2 × 34 cell sections from two independent cultures were counted. The data are plotted in a SuperPlot. The duplicate experiments are color-coded. The circles represent the single peroxisomes. Two-tailed Student’s t test was performed. *, P < 0.05.

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