Characterization of sequence requirements for translational readthrough in <i>U. maydis</i>.

<p>(A) 3′ sequence of the <i>U. maydis tpi1</i> gene and its translation. PTS1 is highlighted. (B) Tpi1 with indicated C-terminal extensions was fused to GFP and analyzed by Western blot. (C) A reporter construct consisting of <i>mCherry</i> and <i>gfp</i> interrupted by a stop codon was expressed. Stop codon readthrough was analyzed by detection of GFP by Western blot. The termination codon TGA was replaced by TAA or TAG and tested in combination with the downstream sequence CTA for ribosomal readthrough. (D) Nucleotides downstream of TGA were exchanged as indicated and tested as described in (C) for stop codon readthrough. (E) Tpi1 including TGA CTA was fused to GFP. Cells were incubated with different amounts of G418 and analyzed for readthrough by Western blotting. (F) TGA CTA was inserted between <i>mCherry</i> and <i>gfp</i> of the reporter construct. Cells were incubated with different amounts of G418 and analyzed for readthrough by Western blotting. Filled arrows indicate readthrough products while blank arrows indicate products terminated at the first stop codon.</p

Analysis of peroxisomal targeting of LDHB via translational readthrough in HeLa cells.

<p>(A) Western blot analysis of LDHB derivatives fused to HA and Myc. (B) The stop codon of the LDHB construct described in (A) was exchanged by TAA and TAG. (C) Western Blot analysis of LDHB fused to HA and Myc in dependence on Gentamicin (+). (D) GFP-LDHB and GFP-LDHBΔPTS1 were co-expressed with the peroxisomal marker mCherry-SKL. (E) LDHB*-GFP-PTS1 was co-expressed with mCherry-SKL. (F) MDH1*-GFP-PTS1 was co-expressed with mCherry-SKL. Asterisks mark the positions of stop codons. PTS1 indicates the peroxisomal targeting signal downstream of the conventional stop codon of LDHB and MDH1, respectively. Intracellular localization was followed by fluorescence microscopy before and after repeated photobleaching. Magnified areas are indicated and shown below the micrographs. Scale bars represent 10 µm.</p

Translational readthrough derived PTS1 motifs of MDH1 and LDHB are conserved in animals.

<p>(A) 3′ sequences of the human <i>MDH1</i> and its translation. The readthrough element and the predicted PTS1 motifs are highlighted. Enzymatic reactions catalyzed by MDH1 is indicated. NCBI Gene IDs is shown. (B) Human <i>LDHB</i> and its translation as in (A). (C) Phylogenetic conservation of readthrough derived MDH1 and LDHB peroxisomal isoforms is indicated by colouring. PTS1 predictions were carried out with PTS1 predictor <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004685#pgen.1004685-Neuberger1" target="_blank">[66]</a>. PTS1 motifs of MDH1 homologs encoded by the original open reading frame are indicated by no readthr. and …NNN*.</p

Characterization of sequence requirements for translational readthrough in human cells.

<p>(A) A reporter construct consisting of <i>gfp</i> and <i>myc</i> interrupted by a stop codon was expressed in HeLa cells. Stop codon readthrough was analyzed by detection of the myc-epitope by Western blot. The termination codon TGA was replaced by TAA or TAG and tested in combination with the downstream sequence CTA for ribosomal readthrough. (B) Nucleotides downstream of TGA were exchanged as indicated and tested as described in (A) for stop codon readthrough.</p

Peroxisomal targeting of NADH-dependent aldehyde reductase Art1 and D-ribulose-5-phosphate-3-epimerase Rpe1 is conserved in fungi.

<p>(A) 3′ sequences of <i>U. maydis</i> genes <i>art1</i> and <i>rpe1</i> together with their derived polypeptide sequence. PTS1 and readthrough elements are highlighted. All accession numbers are from MIPS <i>Ustilago maydis</i> DataBase (MUMDB). (B) The PTS1 motifs of Art1 and Rpe1 were fused to GFP and co-expressed with the peroxisomal marker mCherry-SKL in <i>U. maydis</i> cells. BF indicates bright field. Scale bars represent 10 µm. (C) Conservation of readthrough derived peroxisomal isoforms of Art1 and Rpe1 orthologs in fungi. Predictions were carried out with a PTS1 predictor <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004685#pgen.1004685-Neuberger1" target="_blank">[66]</a>. In some organisms the PTS1 is part of the original open reading frame indicated by no readthr. and …NNN*.</p

Inorganic pyrophosphatase is targeted to peroxisomes in different species by various mechanisms.

<p>(A) 3′ sequence of the <i>C. elegans pyp-1</i> gene encoding inorganic pyrophosphatase (Gene ID: 177856) and its translation. The readthrough element and the PTS1 are highlighted. The reaction catalyzed by inorganic pyrophosphatase is shown. (B) Schematic drawings illustrating different mechanisms leading to peroxisomal isoforms of inorganic pyrophosphatase in a variety of species. Asterisks mark the positions of stop codons. M represents an alternative methionine start codon. (C) The readthrough derived PTS1 motif of human PPA2 was fused to GFP and analyzed for peroxisomal localization in HeLa cells. Scale bar represents 10 µm.</p

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

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

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

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