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Extracellular protease digestion to evaluate membrane protein cell surface localization

Abstract

Membrane proteins have crucial roles in signaling and as anchors for cell surface display. Proper secretion of a membrane protein can be evaluated by its susceptibility to digestion by an extracellular protease, but this requires a crucial control to confirm membrane integrity during digestion. This protocol describes how to use this approach to determine how efficiently a protein is secreted to the outer surface of Gram-negative bacteria. Its success relies upon careful selection of an appropriate intracellular reporter protein that will remain undigested if the membrane barrier remains intact, but that is rapidly digested when cells are lysed before evaluation. Reporter proteins that are resistant to proteases (e.g., maltose-binding protein) do not return accurate results; in contrast, proteins that are more readily digested (e.g., SurA) serve as more sensitive reporters of membrane integrity, yielding more accurate measurements of membrane protein localization. Similar considerations apply when evaluating membrane protein localization in other contexts, including eukaryotic cells and organelle membranes. Evaluating membrane protein localization using this approach requires only standard biochemistry laboratory equipment for cell lysis, gel electrophoresis and western blotting. After expression of the protein of interest, this procedure can be completed in 4 h.

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Figure 1: Accurate use of protease digestion to confirm protein cell surface display requires monitoring a protease-sensitive intracellular control protein.
Figure 2: Western blots showing the resistance of YapV45, MBP and SurA to digestion by proteinase K (proK) or trypsin (Tryp) in intact cells or sonicated cell lysates.
Figure 3: MBP is resistant to proK digestion even at high (400 μg/ml) proK concentrations.

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Acknowledgements

We thank L.A. Fernandez (Centro Nacional de Biotecnologia, Madrid, Spain), C. Gross (University of California, San Francisco) and J. Beckwith (Harvard Medical School) for providing strains UT5600, MG1655, and KS272 and KS474, respectively; and we thank David Hunstad (Washington University in St. Louis) for the anti-SurA antibody. This project was supported by the US National Institutes of Health (R01 GM097573).

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Authors

Contributions

R.N.B. and P.L.C. developed the concept, designed the experiments and wrote the manuscript. R.N.B. carried out the experimental work, analyzed the data and prepared Figures 2 and 3. P.L.C. prepared Figure 1.

Corresponding author

Correspondence to Patricia L Clark.

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The authors declare no competing financial interests.

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Besingi, R., Clark, P. Extracellular protease digestion to evaluate membrane protein cell surface localization. Nat Protoc 10, 2074–2080 (2015). https://doi.org/10.1038/nprot.2015.131

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