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°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.

The iodide transport defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I− symporter (NIS)

“…None of these treatments [glycerol, DMSO, trimethylamine-N-oxide (TMAO), thapsigargin, the proteasome inhibitor MG101, or low temperature (27˚C)] increased the targeting of R124H NIS to the plasma membrane (data not shown). The inhibition of protein synthesis has been reported to increase plasma membrane targeting of pendrin, a protein retained in the ER in some patients with Pendred syndrome (Shepshelovich et al, 2005). However, treatment of R124H NIS-expressing COS-7 cells with cycloheximide or puromycin did not increase cell surface expression of R124H NIS either (data not shown).…”

Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

“…Single amino acid substitutions may impair the ability of the affected polypeptide to reach the functional conformation; consequently, prolonged interaction with molecular chaperones, enhanced proteolytic degradation and reduced cellular halflife of mutant pro teins are events occurring in a number of genetic diseases (39). A previous report gathered evidence that wildtype pendrinGFP colocalizes with ubiqui tin in the cell, therefore suggesting a role for polyubiquitination in pendrin degradation (40). Accordingly, ubiq uitination prediction programs (http:// www.ubpred.org/) identify pendrin lysine residues at positions 632, 647, 734 and 753 as putative ubiquitination sites ( Supplementary Figure S1).…”

The major facilitator superfamily member Slc37a2 is a novel macrophage- specific gene selectively expressed in obese white adipose tissue

“…Vacuoles of a similar appearance have been noted on ectopic expression of the chloride-iodide transporter protein pendrin in cultured cells. This phenotype was determined to be due to perturbation of the endoplasmic reticulum attributed to the slow-folding nature of the pendrin protein (46). Mutations in pendrin that lead to its retention in the endoplasmic reticulum are associated with Pendred syndrome, an autosomal recessive disorder characterized by congenital deafness and goiter (46).…”