Protein synthesis inhibitors and the chemical chaperone TMAO reverse endoplasmic reticulum perturbation induced by overexpression of the iodide transporter pendrin.
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.
Shepshelovich
Jeanne
J
Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.
Goldstein-Magal
Lee
L
Globerson
Anat
A
Yen
Paul M
PM
Rotman-Pikielny
Pnina
P
Hirschberg
Koret
K
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
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