The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology.
Correct localization and topology are crucial for the cellular function of a protein. To determine the topology of membrane proteins, a new technique, called the fluorescence protease protection (FPP) assay, can be applied. This assay uses the restricted proteolytic digestibility of GFP-tagged transmembrane proteins to indicate their intramembrane orientation. The sole requirements for FPP are the expression of GFP fusion proteins and the selective permeabilization of the plasma membrane, which permits a wide range of cell types and organelles to be investigated. The FPP assay can be carried out in a straightforward manner to obtain reliable results within minutes. Here we provide a step-by-step protocol for the assay. As an example, we use FPP to determine which terminus of an endoplasmic reticulum (ER) transmembrane protein is lumenal and which one is facing the cytosol.
Lorenz
Holger
H
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 18T Library Drive, Bethesda, Maryland 20892, USA. holgerl@mail.nih.gov
Hailey
Dale W
DW
Wunder
Christian
C
Lippincott-Schwartz
Jennifer
J
D002463:Cell Membrane Permeability; D049452:Green Fluorescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010447:Peptide Hydrolases; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases