Figure 1.
Characterization of sequence requirements for translational readthrough in U. maydis.
(A) 3′ sequence of the U. maydis tpi1 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 mCherry and gfp 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 mCherry and gfp 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.
Figure 2.
Peroxisomal targeting of NADH-dependent aldehyde reductase Art1 and D-ribulose-5-phosphate-3-epimerase Rpe1 is conserved in fungi.
(A) 3′ sequences of U. maydis genes art1 and rpe1 together with their derived polypeptide sequence. PTS1 and readthrough elements are highlighted. All accession numbers are from MIPS Ustilago maydis DataBase (MUMDB). (B) The PTS1 motifs of Art1 and Rpe1 were fused to GFP and co-expressed with the peroxisomal marker mCherry-SKL in U. maydis 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 [66]. In some organisms the PTS1 is part of the original open reading frame indicated by no readthr. and …NNN*.
Figure 3.
Characterization of sequence requirements for translational readthrough in human cells.
(A) A reporter construct consisting of gfp and myc 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.
Figure 4.
Translational readthrough derived PTS1 motifs of MDH1 and LDHB are conserved in animals.
(A) 3′ sequences of the human MDH1 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 LDHB 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 [66]. PTS1 motifs of MDH1 homologs encoded by the original open reading frame are indicated by no readthr. and …NNN*.
Figure 5.
Analysis of peroxisomal targeting of LDHB via translational readthrough in HeLa cells.
(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.
Figure 6.
Inorganic pyrophosphatase is targeted to peroxisomes in different species by various mechanisms.
(A) 3′ sequence of the C. elegans pyp-1 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.