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Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization

Nature Methods volume 3, pages 205–210 (2006)Cite this article

Abstract

Understanding the cell biology of many proteins requires knowledge of their in vivo topological distribution. Here we describe a new fluorescence-based technique, fluorescence protease protection (FPP), for investigating the topology of proteins and for localizing protein subpopulations within the complex environment of the living cell. In the FPP assay, adapted from biochemical protease protection assays, GFP fusion proteins are used as noninvasive tools to obtain details of protein topology and localization within living cells in a rapid and straightforward manner. To demonstrate the broad applicability of FPP, we used the technique to define the topology of proteins localized to a wide range of organelles including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisomes and autophagosomes. The success of the FPP assay in characterizing the topology of the tested proteins within their appropriate compartments suggests this technique has wide applicability in studying protein topology and localization within the cell.

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Figure 1: Extracellular trypsin is unable to enter the interior of intact cells.
Figure 2: Digitonin permeabilization releases the freely diffusible cytosolic content and allows trypsin to access the ER membrane protein CD3δ-CFP.
Figure 3: The FPP assay reveals the topology of the ER membrane protein CD3δ.
Figure 4: FPP assays can be used to determine the topology of membrane proteins localized to the Golgi apparatus and mitochondria.
Figure 5: The FPP assay reveals the localization and orientation of proteins localized to peroxisomes.
Figure 6: Detection of the autophagy substrate GAPDH-RFP in autophagosomes by FPP assay.

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Acknowledgements

We thank E. Wiertz, A.M. Weissman, Y. Ohsumi, M. Raje and P. Kim for providing DNA constructs used in this study. We also thank E.L. Snapp and R.S. Hegde for valuable discussions on cell permeabilization, and the members of the Lippincott-Schwartz laboratory for their support and critical comments on the manuscript. This work was supported by the Intramural Research Program at the US National Institutes of Health in the National Institute 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, Bldg. 18T Library Drive, Bethesda, 20892, Maryland, USA

    Holger Lorenz, Dale W Hailey & Jennifer Lippincott-Schwartz

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  1. Holger Lorenz
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  2. Dale W Hailey
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Correspondence to Jennifer Lippincott-Schwartz.

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Supplementary information

Supplementary Fig. 1

Intracellular organelles remain intact upon incubation with digitonin concentrations routinely used for cell permeabilization. (DOC 3658 kb)

Supplementary Fig. 2

Quantitative analysis of fluorescence intensities of the proteins subjected to FPP assays (DOC 2042 kb)

Supplementary Methods (DOC 59 kb)

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Lorenz, H., Hailey, D. & Lippincott-Schwartz, J. Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization. Nat Methods 3, 205–210 (2006). https://doi.org/10.1038/nmeth857

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