The MAL proteolipid restricts detergent-mediated membrane pore expansion and percolation.

Differential solubilization of membrane components by cold 1% Triton X-100 extraction is common practice in cell biology and membrane research, used to define components of, or localization within membrane domains called lipid rafts. In this study, extraction of biological membranes was continuously monitored in single cells by confocal microscopy. The distributions of fluorescently-tagged proteins that label raft and non-raft membranes, cytosolic and cytoskeletal proteins were continuously monitored upon addition of the detergent. Membranes containing the non-raft membrane protein VSVG-GFP were immediately extracted from the plasma membrane, whereas raft-membrane proteins were predominantly resistant to the detergent. The morphological characteristics of differential membrane solubilization consisted of the formation of pores that expand and percolate as the detergent-mediated solubilization proceeds. Pore expansion and percolation was much slower and more restricted in non-polarized MDCK cells than in COS-7 cells. Heterologous overexpression in COS-7 cells of the fluorescently-tagged human MAL, a tetra-spanning, lipid-raft-associated protein, significantly slowed and limited membrane pore expansion and percolation. Extensive percolation resulting in large holes in the membrane was observed for the raft-associated, GPI-GFP-labeled membranes in COS-7 cells. Quantitative analysis carried out using pixel intensity variance as an indicator of membrane pore expansion demonstrated that the MAL protein is capable of modifying the plasma membrane, thereby increasing its resistance to detergent-induced pore formation.

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.