Diffusional Mobility of the Cystic Fibrosis Transmembrane Conductance Regulator Mutant, ΔF508-CFTR, in the Endoplasmic Reticulum Measured by Photobleaching of GFP-CFTR Chimeras
Year: 2002
Type: article
Abstract: Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis. The most common disease-causing mutation, DeltaF508, is retained in the endoplasmic reticulum (ER) and is unable to function as a plasma membrane chloride channel. To investigate whether the ER retention of DeltaF508-CFTR is caused by immobilization and/or aggregation, we have measured the diffusional mobility of green fluorescent protein (GFP) chimeras of wild type (wt)-CFTR and DeltaF508-CFTR by fluorescence recovery after photobleaching. GFP-labeled DeltaF508-CFTR was localized in the ER and wt-CFTR in the plasma membrane and intracellular membranes in transfected COS7 and Chinese hamster ovary K1 cells. Both chimeras localized to the ER after brefeldin A treatment. Spot photobleaching showed that CFTR diffusion (diffusion coefficient approximately 10(-9) cm(2)/s) was not significantly slowed by the DeltaF508 mutation and that nearly all wt-CFTR and DeltaF508-CFTR diffused throughout the ER without restriction. Stabilization of molecular chaperone interactions by ATP depletion produced remarkable DeltaF508-CFTR immobilization ( approximately 50%) and slowed diffusion (6.5 x 10(-10) cm(2)/s) but had little effect on wt-CFTR. Fluorescence depletion experiments revealed that the immobilized DeltaF508-CFTR in ATP-depleted cells remained in an ER pattern. The mobility of wt-CFTR and DeltaF508-CFTR was reduced by maneuvers that alter CFTR processing or interactions with molecular chaperones, including tunicamycin, geldanamycin, and lactacystin. Photobleaching of the fluorescent ER lipid diOC(4)(3) showed that neither ER restructuring nor fragmentation during these maneuvers was responsible for the slowing and immobilization of CFTR. These results suggest that (a) the ER retention of DeltaF508-CFTR is not due to restricted ER mobility, (b) the majority of DeltaF508-CFTR is not aggregated or bound to slowly moving membrane proteins, and (c) DeltaF508-CFTR may interact to a greater extent with molecular chaperones than does wt-CFTR.
Source: Journal of Biological Chemistry
Institutions: University of California, San Francisco, Dartmouth College
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Awards: G1161626946, G2476224966, G3011728751, Physiology and Molecular Biology of Lung Water Transport, Aquaporin-4 in NMO Optic Neuritis: Pathogenesis and New Therapies, G3730553470, Regulation of CFTR Trafficking, APPLICATION OF NOVEL OPTICAL METHODS TO CELL DYNAMICS, Cellular and Molecular Biology of Renal Sodium Channels, ALTERATIONS IN AIRWAY SURFACE LIQUID IN CYSTIC FIBROSIS, G793539597, G951509311, G95487581
OpenAlex ID: W2169890920