Fig. 4. YGR168C (PEX35) is a new PEX gene.

(A) Ygr168c colocalizes with a peroxisomal marker. Overexpressed (OE) N-terminally GFP-tagged Ygr168c colocalizes with Pex3-mCherry, with a notable phenotype of about one large peroxisome per cell (top). Overexpression of Ygr168c with a C-terminal mCherry tag displays colocalization with Pex3-GFP (bottom) with wild-type (WT) peroxisome abundance. Scale bars: 5 μm. (B) Predicted transmembrane topology of Ygr168c (Pex35). Transmembrane helices were predicted by the TMHMM algorithm (v2.0). The schematic representation demonstrates the five transmembrane domains, as well as a long cytoplasmic C-terminus for the protein. (C) Deletion of PEX19 leads to the cytosolic localization and ER accumulation of Ygr168c. sfGFP, superfolder GFP. Scale bar: 5 μm. (D) Identification of GFP-Ygr168c on purified wild-type peroxisomes isolated from oleate-induced cells. Western blot analysis of the post-nuclear supernatant (PNS), and cytosol (Cyt), peroxisomal (Px) and mitochondrial (Mito) fractions from the Nycodenz step gradient. Anti-Por1 antibody is used as a mitochondrial marker. (E) Overexpression of amino acids 1-107 of Ygr168c (Ygr168c_1-107; including the N-terminal region and the first two predicted transmembrane domains) tagged with mCherry displays colocalization with Pex3-GFP, and also shows localization to the ER. Scale bar: 5 μm. (F) YGR168C mutants grow more slowly in medium containing oleic acid as the sole carbon source. WT, Δygr168c, OE-YGR168C and Δpex3 strains were grown in oleic acid for 128 h, and the OD₆₀₀ was measured to assess growth. YGR168C mutants display a significant growth defect, at an intermediate level between WT and the Δpex3. Error bars indicate s.d., n=4. (G) YGR168C mutants consume less oleic acid. Media from the end-point of the experiment described in D (128 h) were used to assess the relative oleic acid consumption of each strain. Error bars indicate s.d., n=4. ** P<0.05 (t-test). (H) Induction of GFP-Ygr168c leads to accumulation of the protein in peroxisomes, and to aberrant peroxisome content. A strain expressing both Pex3-RFP and GFP-Ygr168c under the control of the inducible GAL1 promoterwas transferred from glucose to galactose medium. Time-lapse imaging was carried out starting from 90 min (t=0). GFP-Ygr168c becomes highly induced under these conditions and colocalizes with Pex3-RFP over time. Notably, cells in which GFP-Ygr168c is not expressed (arrows) display normal peroxisome content, while cells in which expression is induced display one large peroxisome in each cell. Scale bar: 5 μm. Dashed lines in images highlight cell outlines.