Inp1 colocalizes with the peripheral Pex3 patch at peroxisome-PM contacts. (A) Single focal plane confocal laser scanning microscopy Airyscan images of cells producing the indicated proteins under control of their endogenous promoters. Cells were grown for 8 h on methanol medium. Vacuoles were stained with FM4-64. The cell contours are indicated in white. The arrows indicate peripheral Inp1 patches. (B) Quantitative analysis of the position of Inp1-Pex3 spots in budding cells. The budding cell is segmented in four regions as indicated in the scheme. The positions of 2 × 50 spots were determined from two independent experiments. Error bars indicate SD. (C) CLEM analysis of single focal plane Pex3-GFP localization. The upper panel shows an FM image and merged phase contrast/FM image of a 150 nm cryo-section from cells producing Pex3-GFP and grown for 8 h on methanol. The arrowhead indicates the cortical Pex3-GFP patch. The lower panel shows the CLEM image of the Pex3-GFP patch at the PM–peroxisome contact. The dashed square indicates the region shown in the tomographic slice. P, peroxisome. (D) Single focal plane CLEM analysis as described in C of cells producing Inp1-GFP under control of the P_TEF1 together with the peroxisomal membrane marker Pmp47-mKate2. CW, cell wall; N, nucleus; V, vacuole. (E) Tomographic reconstruction of peroxisomes in glucose- or methanol-grown WT cells. (F) Immuno-EM using anti-HA antibodies in WT cells producing Inp1-2HA under control of the P_ADH1. The dashed region indicates the region used for tomography. 3D view of the tomogram. Colors in tomograms indicate the following: cyan, PM; blue, peroxisome; orange, ER.