Addressing membrane protein topology using the fluorescence protease protection (FPP) assay.
Determining a protein's correct topological distribution within the cell is essential for understanding the proper functioning of many proteins. Here, we describe a fluorescence-based technique, termed FPP for fluorescence protease protection, to determine protein topology in living cells. The FPP assay uses the restricted proteolytic digestibility of green fluorescent protein-tagged membrane proteins to reveal their intramembrane orientation. Membrane protein topology can be assessed using this technique for proteins residing in organelles as diverse as the Golgi apparatus, the endoplasmic reticulum (ER), peroxisomes, mitochondria, and autophagosomes. To illustrate the technique, we describe its use for deciphering the topology of a membrane protein in the ER.
Lorenz
Holger
H
Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, MD, USA.
Hailey
Dale W
DW
Lippincott-Schwartz
Jennifer
J
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases; D004072:Digitonin