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Membrane promotes tBID interaction with BCLXL

Abstract

Two important questions on the molecular mechanism of the B cell CLL/lymphoma 2 (BCL2) proteins involve the interaction network between pro- and antiapoptotic members and the role of their translocation to the mitochondrial membrane during apoptosis. We used fluorescence correlation spectroscopy to quantify the molecular interactions of BH3-interacting domain death agonist (BID) and its truncated form tBID with the B cell lymphoma extra-large protein truncated at the C terminus (BCLXLΔCt) in solution and in membranes, and we found that (i) only the active form tBID binds to BCLXLΔCt and (ii) that the membrane strongly promotes binding between them. Particularly, a BH3 peptide from BID disrupts the tBID–BCLXL complex in solution, but only partially in lipid bilayers. These data indicate that tBID–BCLXL interactions in solution and lipid membranes are distinct, and they support a model in which BCLXL inhibition of tBID takes place predominantly at the membrane. Our findings imply an active role of the membrane in modulating the interactions between BCL2 proteins that has so far been underestimated.

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Figure 1: Fluorescently labeled tBID and BCLXLΔCt retain their apoptotic activity.
Figure 2: Binding of tBIDred and BCLXLΔCtgreen to giant unilamellar vesicles.
Figure 3: tBIDred and BCLXLΔCtgreen cooperate in membrane permeabilization.
Figure 4: Measurement of tBID–BCLXLΔCt interactions by fluorescence cross-correlation (CC) spectroscopy.
Figure 5: Inhibition of tBID–BCLXLΔCt interactions in solution and in the membrane.
Figure 6: Proposed model for the interactions between tBID and BCLXL.

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Acknowledgements

We thank J. Salgado, E. Petrov and S. Chiantia for useful discussions and J. Suckale for careful reading of the manuscript. We are grateful to F. Pan-Montojo (Max Planck Institute for Cell Biology and Genetics and TU Dresden) for supplying the mouse livers and S. Rubio and A. Borrmann for technical assistance in protein mutagenesis and purification. This work was supported by a Marie Curie Intra-European Fellowship (A.J.G.-S.) within the 6th European Framework Program. Work in the E.P.-P. laboratory is supported by the Spanish Ministry of Science and Innovation grants BIO2007-60066 and CSD2008-00005.

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Contributions

A.J.G.-S. proposed the project, prepared materials, performed the experiments and analyzed the data; J.R. developed the method for FCS data analysis and helped with data analysis; M.O. and E.P.-P. prepared materials; P.S. provided the infrastructure and expertise in FCS and model membranes; A.J.G.-S. wrote the manuscript with the help of all other authors.

Corresponding authors

Correspondence to Ana J García-Sáez or Petra Schwille.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Methods (PDF 278 kb)

Supplementary Movie 1

Kinetics of GUV permeabilization in the presence of tBIDred and BCLXLΔCtgreen. We monitored vesicle permeabilization by the internalization of free DiI added to the external medium. Total duration of the movie is 2 h. Scale bar, 50 μm. (MOV 768 kb)

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García-Sáez, A., Ries, J., Orzáez, M. et al. Membrane promotes tBID interaction with BCLXL. Nat Struct Mol Biol 16, 1178–1185 (2009). https://doi.org/10.1038/nsmb.1671

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