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Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells

Nature Methods volume 2, pages 261–268 (2005) Cite this article

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Abstract

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-cross-linking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

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Figure 1: New photoreactive amino acids are incorporated into proteins.
Figure 2: Photo-cross-linking induced by photo–amino acids.
Figure 3: Specificity of incorporation and cross-linking by photo-Met and photo-Leu.
Figure 4: Photo-cross-linking shows specific interactions of proteasome subunits in vivo.
Figure 5: Direct interaction in vivo between Insig-1 and PGRMC1.

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Acknowledgements

We are grateful to J. Rink for help with Rab experiments, M. Zerial for the GFP-Rab expression constructs and antibody against EEA1, R. Prohaska for antibody against Stomatin, F. Buchholz for the β-galactosidase expression vector, A. Rudolph and M. Gruner for recording of NMR spectra, G. Wiebe for DNA sequencing and K. Simons and T. Kurzchalia for critical reading of the manuscript.

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  1. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden, D-01307, Germany

    Monika Suchanek, Anna Radzikowska & Christoph Thiele

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  1. Monika Suchanek
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  2. Anna Radzikowska
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  3. Christoph Thiele
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Corresponding author

Correspondence to Christoph Thiele.

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The author's employer, the Max Planck Society, is preparing to file a patent application covering the photo–amino acids and their application to detect protein-protein interactions in living cells.

Supplementary information

Supplementary Fig. 1 (download PDF )

Localization and fluorescence of EGFP-Rab5, EGFP-Rab5QL and EGFP-Rab5SN are not affected by the presence of photo-amino acids in the growth medium. (PDF 1099 kb)

Supplementary Table 1 (download PDF )

Effects of photo-amino acids on cellular protein synthesis and cell viability. (PDF 21 kb)

Supplementary Table 2 (download PDF )

Effects of UV irradiation in the absence or presence of photo-amino acids on cell viability. (PDF 22 kb)

Supplementary Table 3 (download PDF )

Activity of beta-galactosidase expressed in cells grown in the absence or presence of photo-amino acids. (PDF 32 kb)

Supplementary Note (PDF 823 kb) (download PDF )

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Suchanek, M., Radzikowska, A. & Thiele, C. Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nat Methods 2, 261–268 (2005). https://doi.org/10.1038/nmeth752

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