Single-channel images related to Fig 8E and 8G.

(A) Fluorescence microscopic images of indicated strains expressing mNeonGreen-Pex15 (green), mCherry-PTS (magenta), and Sec63-CFP (blue). (B) Fluorescence microscopic images indicated strains expressing mNeonGreen-Pex15 (green), mCherry-PTS (magenta), and Tim50-CFP (blue). (C) Fluorescence microscopic images of control and indicated mutant cells expressing mNeonGreen-Pex15 (green) and mCherry-PTS (magenta). Scale bar represents 5 μm. (TIF)</p

Expression of the synthetic PerMit tether in control cells.

Fluorescence microscopic images of cells co-expressing the peroxisomal marker RFP-PTS (red, A) or Pxp2-RFP-PTS (red, B), the peroxisomal membrane protein Ant1-YFP (green), and the mitochondrial protein Tim50-CFP. Scale bars represent 5 μm. (TIFF)</p

Movement of peroxisomes in indicated strains.

Frames were captured with the following excitation times and gain settings, control, 75 ms, gain 23; Δpex30, 80 ms, gain 79; Δget2, 100 ms, gain 63; Δmdm10; 100 ms, gain 48. (MP4)</p

Localization of RFP-PTS and Ptc5 derivatives in Δ<i>mdm10</i> cells.

(A) Localization of Ptc5-RFP-PTS (magenta, upper panel) and RFP-PTS (magenta, lower panel) in mdm10 mutants expressing a synthetic ER–mitochondria tether (green) [43] and quantification of peroxisome number per cell in strains of the lower panel. (B) Fluorescence microscopic images showing expression of the peroxisomal marker RFP-PTS (magenta) in indicated strains. (C) Fluorescence microscopic images showing expression of Ptc5 derivatives in Δmdm10 cells (magenta). Quantifications show the ratio of peroxisomal versus total Ptc5-RFP-PTS signal. The peroxisomal membrane protein Ant1-YFP (green, B and C) and the mitochondrial protein Tim50-CFP (blue, C) served as marker proteins. Scale bar represents 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent standard error of the mean. P-values were calculated using a two-sided unpaired Student’s t test. Underlying data for quantifications can be found in S1 Data. (TIF)</p

Analysis of peroxisomes in ERMES mutants upon expression of a synthetic tether.

Images of strains deleted for mdm10 or mdm34 co-expressing RFP-PTS (A, red) or Pxp2-RFP-PTS (B, red), the peroxisomal membrane protein Ant1-YFP (green), and the mitochondrial protein Tim50 (blue) either in the presence of Tom70-ProteinA-TAPex15 or a control protein. Scale bars represent 5 μm. (TIFF)</p

Cat2 and Pxp2 localize to mitochondria and peroxisomes and induce interorganellar contacts.

(A) Scheme showing Cat2 and Pxp2. Cat2 contains an N-terminal MTS that is likely processed sequentially by 2 different mitochondrial proteases (MPP, Oct1) [109] and contains an acetyl-carnitine transferase (CAT) domain and a PTS1 [110]. Pxp2 contains an N-terminal MTS, a putative carboxymuconolactone decarboxylase domain (CMD) and a PTS1 [11]. Prediction of protein domains was performed with the HMMER web server. (B) Fluorescence microscopic images of yeast cells expressing either Cat2-RFP-HA or Cat2-RFP-HA-PTS (red) together with the peroxisomal membrane protein Ant1-YFP (green) and the mitochondrial inner membrane protein Tim50-CFP (blue). White arrows denote peroxisomes overlapping mitochondria. (C) Quantification of the fraction of peroxisomes in contact with mitochondria (PxM) relative to the total peroxisome number (PxT) (upper plot) and quantification of the number of peroxisomes per cell (lower plot) of cells shown in (B). (D) Fluorescence microscopic pictures of control and Δpex5 cells co-expressing Pxp2-RFP-HA-PTS (red) together with Ant1-YFP (green) and Tim50-CFP (blue) (left). White arrows denote peroxisomes overlapping mitochondria. (E) Quantification of the number of peroxisomes per cell (upper plot) and the correlation between the Pxp2-RFP-HA-PTS signal and Ant1-YFP signal (lower plot) of cells shown in (D). PCC refers to Pearson’s correlation coefficient. (F) Subcellular localization of endogenously tagged Pxp2-3xMyc-PTS and Pxp2-3xMyc was determined using density gradient centrifugation. Twelve fractions, collected from the top of the gradient, were analyzed by SDS–PAGE and immunoblot. Ant1-YFP is a peroxisomal membrane protein and Por1 is localized in the mitochondrial outer membrane. (G) Quantification of the fraction of peroxisomes contacting mitochondria (PxM) in relation to the total peroxisome count (PxT) of control cells and cells overexpressing the indicated fusion proteins. (H) Yeast cells expressing N-terminally tagged Pxp2 (red) together with Ant1-YFP (green) and Tim50-CFP (blue) were analyzed by fluorescence microscopy. The progenitor strain without RFP-HA-Pxp2 served as control. White arrows denote peroxisomes overlapping mitochondria. The fraction of peroxisomes in contact with mitochondria (PxM) relative to the total peroxisome number of peroxisomes was plotted (right). Single-channel pictures are shown in S2D Fig. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent SEM. P-values were calculated using a two-sided unpaired Student’s t test. For plots showing multiple comparison, a one-way ANOVA combined with a Tukey test was performed. Scale bars represent 5 μm. Underlying data for quantifications can be found in S1 Data. MTS, mitochondrial targeting signal; PTS, peroxisome targeting signal.</p

Ultrastructural characterization of Um_Ptc5 induced contacts.

(A) Scheme of U. maydis Ptc5 highlighting the targeting signals (MTS, PTS), a TMD and the phosphatase domain (PP2C). Prediction of the PP2C domain was performed with the HMMER web server. Note that this sequence contains a putative cleavage site between TMD and PP2C, which has been experimentally validated only in yeast so far [11,12]. (B) Cells expressing Um_Ptc5-GFP or Um_Ptc5-GFP-PTS (green) under control of the constitutive Otef-promoter [33] and together with the peroxisomal marker mCherry-PTS (magenta) were analyzed by fluorescence microscopy. (C) Pictures were obtained by TEM. mCherry-PTS was stained via immunogold labeling. White arrows indicate PerMit contacts. Scale bar: 0.2 μm. (D) and (E) Quantifications are based on n = 3 experiments. Each color represents 1 experiment. At least 25 peroxisomes per experiment were analyzed. Error bars represent SEM. P-values were calculated using a two-sided unpaired Student’s t test. (F) Cells expressing an internally GFP-tagged derivative of Um_Ptc5 (green) under control of the endogenous promoter together with the peroxisomal marker mCherry-PTS (magenta) were analyzed by fluorescence microscopy following incubation with indicated carbon sources. Insets show single channels and merged channels. Scale bar: 5 μm. (G) Cells expressing a C-terminally GFP-tagged derivative of Um_Ptc5 preserving the PTS (green) under the control of the endogenous promotor and the peroxisomal marker mCherry-PTS (magenta) were analyzed by fluorescence microscopy following incubation with indicated carbon sources. Insets show single channels and merged channels. Scale bars in fluorescence microscopic images: 5 μm. Underlying data for quantifications can be found in S1 Data. MTS, mitochondrial targeting signal; PTS, peroxisome targeting signal; TEM, transmission electron microscopy; TMD, transmembrane domain.</p

Depletion of components of the peroxisomal import machinery reduces PerMit contacts.

(A) Fluorescence microscopic picture of control and Δpex5 cells expressing endogenously tagged Pex3-GFP (green) and Tim50-RFP (magenta). White arrows denote peroxisomal signal overlapping with mitochondrial signal. (B) Quantification of the fraction of peroxisomes contacting mitochondria (PxM) in relation to the total peroxisome count (PxT) of control cells and Δpex5 cells. (C) Quantification of the fraction of peroxisomes contacting mitochondria (PxM) in relation to the total peroxisome count (PxT) of control cells and Δpex5 cells expressing Pxp2-RFP-PTS, Ant1-YFP, and Tim50-CFP. (D) The number of peroxisomes per cell was quantified in the indicated strains expressing Pex3-GFP. (E) Scheme of the genetic modifications used for auxin-dependent depletion of Pex13 in (F)–(I). The endogenous PEX13 locus was genetically engineered to encode a translational fusion of Pex13 with a C-terminal AID and 6 hemagglutinin (HA) tags. Pex13 degradation is mediated by the F-box protein AFB2 from Arabidopsis thaliana, which was expressed from the ADH1 promotor. (F) Auxin-dependent depletion of Pex13-AID-HA at indicated time points was analyzed by SDS-PAGE and immunoblot. Por1 served as a loading control. (G) Fluorescence microscopic images of indicated strains expressing the peroxisomal membrane protein Ant1-YFP (green) and RFP-PTS (magenta) in the absence (-Auxin) or presence (+Auxin; 4 h) of 2 mM indole-3-acetic acid. (H) Subcellular localization of Ant1-YFP (magenta) and Tim50-CFP (green) of indicated strains was analyzed in the presence of 2 mM indole-3-acetic acid at indicated time points (left). White arrows indicate peroxisomes in proximity to mitochondria. The fraction of peroxisomes in contact with mitochondria (PxM) relative to the total peroxisome count (PxT) of the indicated strain was quantified (right). (I) Identical to (H), except that the cells also expressed Cat2-RFP-HA-PTS (red) to increase PerMit contacts. Scale bars represent 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent SEM. A one-way ANOVA combined with a Tukey test was performed to assess statistical significance for multiple comparisons. Otherwise, a two-sided unpaired Student’s t test was performed. Underlying data for quantifications can be found in S1 Data. AID, auxin-inducible degron; PTS, peroxisome targeting signal; RFP, red fluorescent protein.</p

Lack of ERMES components changes the distribution of dual affinity proteins.

(A and B) Subcellular localization of indicated dual affinity proteins (red), the peroxisomal membrane protein Ant1-YFP (green), and the mitochondrial inner membrane protein Tim50-CFP (blue) in indicated strains was analyzed using fluorescence microscopy. Scale bars represent 5 μm. (TIF)</p

Dual targeting of Pex15 supports ER-peroxisome contacts.

(A) Fluorescence microscopic images of control and Δget3 cells expressing PA-GFP-Pex15g (green) and RFP-PTS (magenta). (B) Localization of PA-GFP-Pex15g (green) in the ER in cells lacking the chaperone and targeting factor Pex19. RFP-HDEL (magenta) is a marker protein for the ER; RFP-PTS (magenta) is a marker for peroxisomes. (C) Quantification of peroxisome number of indicated strains expressing the peroxisomal marker mCherry-PTS. (D) Proteins extracted from indicated strains were subjected to high-resolution SDS-PAGE and immunoblot to visualize glycosylation of PA-GFP-Pex15g. To confirm glycosylation, extracts were treated with endoglycosidase H (EndoHF). (E) Fluorescence microscopic images of control and Δspf1 cells expressing mNeonGreen-Pex15 (green), mCherry-PTS (magenta), and Sec63-CFP (blue). (F) Quantification of the mNeonGreen-Pex15 signal distribution in strains shown in (D). (G) Fluorescence microscopic images of control and indicated mutant cells expressing mNeonGreen-Pex15 (green) and mCherry-PTS (magenta). (H) Quantification of peroxisome number of indicated strains either grown in glucose medium or galactose medium for 3 h to induce expression of GFP-Pex15g. (I) Induced expression of the chimeric protein RFP-Pex15-TATom22 (magenta) in cells expressing Ant1-YFP (green) and Tim50-CFP (blue) (left). Quantification of the number of peroxisomes proximal to mitochondria (right). White arrows specify peroxisomes close to mitochondria. Scale bars represent 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent standard error of the mean. P-values were calculated using a two-sided unpaired Student’s t test. For multiple comparisons, a one-way ANOVA combined with a Tukey test was performed. Underlying data for quantifications can be found in S1 Data. ER, endoplasmic reticulum; PTS, peroxisome targeting signal; RFP, red fluorescent protein.</p