title: Protein synthesis inhibitors and the chemical chaperone TMAO reverse endoplasmic reticulum perturbation induced by overexpression of the iodide transporter pendrin.
abstract: An outcome of overloading of the endoplasmic reticulum (ER) folding machinery is a perturbation in ER function and the formation of intracellular aggregates. The latter is a key pathogenic factor in numerous diseases known as ER storage diseases. Here, we report that heterologous overexpression of the green fluorescent protein-tagged iodide transporter pendrin (GFP-PDS) perturbs folding and degradation processes in the ER. Pendrin (PDS) is a chloride-iodide transporter found in thyroid cells. Mutations in PDS can cause its retention in the ER and are associated with Pendred syndrome. Biochemical and live-cell analyses demonstrated that wild-type GFP-PDS is predominantly retained in perinuclear aggregates and in ER membranes, causing their collapse and vesiculation. Inhibition of protein synthesis by cycloheximide (CHX) or puromycin caused dissociation of the GFP-PDS aggregates and returned the ER to its normal reticular morphology. Blocking protein synthesis promoted folding and export of ER-retained GFP-PDS, as demonstrated by surface-biotinylation analysis and by CHX- or puromycin-induced accumulation of YFP-PDS in the Golgi apparatus during a 20 degrees C temperature-block experiment. The chemical chaperone trimethylamine-N-oxide (TMAO) also reversed the GFP-PDS-mediated ER collapse and vesiculation, suggesting that exposed hydrophobic stretches of misfolded or aggregated GFP-PDS may contribute to ER retention. These data suggest that GFP-PDS is a slow-folding protein with a propensity to form aggregates when overexpressed. Thus, we describe a system for the reversible induction of ER stress that is based entirely on the heterologous overexpression of GFP-PDS.
authors.affiliation: Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.
authors.forename: Jeanne
authors.initials: J
authors.lastname: Shepshelovich
authors.forename: Lee
authors.initials: L
authors.lastname: Goldstein-Magal
authors.forename: Anat
authors.initials: A
authors.lastname: Globerson
authors.forename: Paul M
authors.initials: PM
authors.lastname: Yen
authors.forename: Pnina
authors.initials: P
authors.lastname: Rotman-Pikielny
authors.forename: Koret
authors.initials: K
authors.lastname: Hirschberg
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008659:Metabolic Diseases / Q000378:metabolism / Q000503:physiopathology; D008744:Methylamines / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology; D017510:Protein Folding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013312:Stress, Physiological / Q000378:metabolism / Q000503:physiopathology; D000076988:Sulfate Transporters; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D026901:Membrane Transport Proteins; D008744:Methylamines; D016877:Oxidants; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; C005855:trimethyloxamine