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Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating

Nature volume 523, pages 101105 (02 July 2015) | Download Citation

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Abstract

Intramembrane proteases catalyse the signal-generating step of various cell signalling pathways, and continue to be implicated in diseases ranging from malaria infection to Parkinsonian neurodegeneration1,2,3. Despite playing such decisive roles, it remains unclear whether or how these membrane-immersed enzymes might be regulated directly. To address this limitation, here we focus on intramembrane proteases containing domains known to exert regulatory functions in other contexts, and characterize a rhomboid protease that harbours calcium-binding EF-hands. We find calcium potently stimulates proteolysis by endogenous rhomboid-4 in Drosophila cells, and, remarkably, when rhomboid-4 is purified and reconstituted in liposomes. Interestingly, deleting the amino-terminal EF-hands activates proteolysis prematurely, while residues in cytoplasmic loops connecting distal transmembrane segments mediate calcium stimulation. Rhomboid regulation is not orchestrated by either dimerization or substrate interactions. Instead, calcium increases catalytic rate by promoting substrate gating. Substrates with cleavage sites outside the membrane can be cleaved but lose the capacity to be regulated. These observations indicate substrate gating is not an essential step in catalysis, but instead evolved as a mechanism for regulating proteolysis inside the membrane. Moreover, these insights provide new approaches for studying rhomboid functions by investigating upstream inputs that trigger proteolysis.

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Acknowledgements

This work was supported by National Institutes of Health grant 2R01AI066025, the Howard Hughes Medical Institute, and the David and Lucile Packard Foundation. We are grateful to our colleague and friend A. Holland for use of his deconvolution microscope.

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Affiliations

  1. Howard Hughes Medical Institute, Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Room 507 PCTB, 725 North Wolfe Street, Baltimore, Maryland 21205, USA

    • Rosanna P. Baker
    •  & Siniša Urban

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Contributions

S.U. and R.B. designed the research. S.U. performed all Drosophila cell biology experiments, while R.B. performed all biochemistry experiments. S.U. wrote the manuscript and R.B. made the figures.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Siniša Urban.

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https://doi.org/10.1038/nature14357

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