Studying protein-reconstituted proteoliposome fusion with content indicators in vitro

Authors

  • Jiajie Diao,

    Corresponding author
    1. Department of Neurology and Neurological Science, Stanford University, Stanford, USA
    2. Department of Structural Biology, Stanford University, Stanford, USA
    3. Department of Photon Science, Stanford University, Stanford, USA
    4. Howard Hughes Medical Institute, Stanford University, Stanford, USA
    5. Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology and Frontier Institute of Life Science, Frontier Institute of Science and Technology (FIST), Xi'an Jiaotong University, Xi'an, China
    • Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA
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  • Minglei Zhao,

    1. Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA
    2. Department of Neurology and Neurological Science, Stanford University, Stanford, USA
    3. Department of Structural Biology, Stanford University, Stanford, USA
    4. Department of Photon Science, Stanford University, Stanford, USA
    5. Howard Hughes Medical Institute, Stanford University, Stanford, USA
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  • Yunxiang Zhang,

    1. Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA
    2. Department of Neurology and Neurological Science, Stanford University, Stanford, USA
    3. Department of Structural Biology, Stanford University, Stanford, USA
    4. Department of Photon Science, Stanford University, Stanford, USA
    5. Howard Hughes Medical Institute, Stanford University, Stanford, USA
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  • Minjoung Kyoung,

    1. Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA
    2. Department of Neurology and Neurological Science, Stanford University, Stanford, USA
    3. Department of Structural Biology, Stanford University, Stanford, USA
    4. Department of Photon Science, Stanford University, Stanford, USA
    5. Howard Hughes Medical Institute, Stanford University, Stanford, USA
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  • Axel T. Brunger

    1. Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA
    2. Department of Neurology and Neurological Science, Stanford University, Stanford, USA
    3. Department of Structural Biology, Stanford University, Stanford, USA
    4. Department of Photon Science, Stanford University, Stanford, USA
    5. Howard Hughes Medical Institute, Stanford University, Stanford, USA
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Corresponding author:

Jiajie Diao

E-mail: diaojj@gmail.com

Axel T. Brunger

E-mail: brunger@stanford.edu

Abstract

In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single-vesicle experiments involving SNARE proteins have been performed only with lipid-mixing, but not content-mixing indicators. Through simultaneous detection of lipid and small content-mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50-seconds observation period, for Ca2+-triggered fusion with SNAREs, full-length synaptotagmin-1, and complexin. Our results illustrate the caveats of commonly used bulk lipid-mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators.

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