title: A novel assay for measurement of membrane-protein surface expression using a β-lactamase.
abstract: The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one-step and potentially high-throughput assay, using the β-lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins. In this assay, the βlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell-impermeable substrate of βlac, nitrocefin, is used. We demonstrate the utility of the βlac assay using well-established paradigms of internalization and molecular chaperoning, applied to two G-protein-coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression.
authors.affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Vincent M
authors.initials: VM
authors.lastname: Lam
authors.forename: Pieter
authors.initials: P
authors.lastname: Beerepoot
authors.forename: Stephane
authors.initials: S
authors.lastname: Angers
authors.forename: Ali
authors.initials: A
authors.lastname: Salahpour
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D061505:Cell Surface Display Techniques / Q000379:methods*; D002645:Chickens; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D021381:Protein Transport; D018080:Receptors, GABA-B / Q000235:genetics / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D018080:Receptors, GABA-B; D064569:alpha7 Nicotinic Acetylcholine Receptor; D001618:beta-Lactamases