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The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology

Nature Protocols volume 1, pages 276–279 (2006)Cite this article

Abstract

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.

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Figure 1: Cartoon of the FPP assay illustrating a single cell before and after digitonin and trypsin treatment.
Figure 2: The FPP assay indicates the topology of the ER membrane protein CD3δ.

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Acknowledgements

We thank the members of the Lippincott-Schwartz lab for their support and comments on the protocol. This work was supported by the Intramural Research Program at the US National Institutes of Health in the National Institutes of Child Health and Human Development.

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  1. Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 18T Library Drive, Bethesda, 20892, Maryland, USA

    Holger Lorenz, Dale W Hailey, Christian Wunder & Jennifer Lippincott-Schwartz

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  1. Holger Lorenz
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  2. Dale W Hailey
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  3. Christian Wunder
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  4. Jennifer Lippincott-Schwartz
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Correspondence to Holger Lorenz.

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Lorenz, H., Hailey, D., Wunder, C. et al. The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology. Nat Protoc 1, 276–279 (2006). https://doi.org/10.1038/nprot.2006.42

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