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.