title: Extracellular protease digestion to evaluate membrane protein cell surface localization.
abstract: Membrane proteins have crucial roles in signaling and as anchors for cell surface display. Proper secretion of a membrane protein can be evaluated by its susceptibility to digestion by an extracellular protease, but this requires a crucial control to confirm membrane integrity during digestion. This protocol describes how to use this approach to determine how efficiently a protein is secreted to the outer surface of Gram-negative bacteria. Its success relies upon careful selection of an appropriate intracellular reporter protein that will remain undigested if the membrane barrier remains intact, but that is rapidly digested when cells are lysed before evaluation. Reporter proteins that are resistant to proteases (e.g., maltose-binding protein) do not return accurate results; in contrast, proteins that are more readily digested (e.g., SurA) serve as more sensitive reporters of membrane integrity, yielding more accurate measurements of membrane protein localization. Similar considerations apply when evaluating membrane protein localization in other contexts, including eukaryotic cells and organelle membranes. Evaluating membrane protein localization using this approach requires only standard biochemistry laboratory equipment for cell lysis, gel electrophoresis and western blotting. After expression of the protein of interest, this procedure can be completed in 4 h.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
authors.forename: Richard N
authors.initials: RN
authors.lastname: Besingi
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.|Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana, USA.
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Clark
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D058619:Maltose-Binding Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D010447:Peptide Hydrolases