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Proc Natl Acad Sci U S A. 2019 Jan 22;116(4):1309-1318. doi: 10.1073/pnas.1817498116. Epub 2019 Jan 8.

Chemically induced vesiculation as a platform for studying TMEM16F activity.

Han TW1,2,3, Ye W1,2,3, Bethel NP4, Zubia M1,2,3, Kim A1,2,3, Li KH5, Burlingame AL5, Grabe M4, Jan YN1,2,3, Jan LY6,2,3.

Author information

1
Howard Hughes Medical Institute, University of California, San Francisco, CA 94143.
2
Department of Physiology, University of California, San Francisco, CA 94143.
3
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.
4
Department of Pharmaceutical Chemistry, Cardiovascular Research Institute, University of California, San Francisco, CA 94143.
5
Mass Spectrometry Facility, University of California, San Francisco, CA 94143.
6
Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; Lily.Jan@ucsf.edu.

Abstract

Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.

KEYWORDS:

GPMV; TMEM16F; calcium influx; extracellular vesicles; phospholipid scrambling

PMID:
30622179
PMCID:
PMC6347726
DOI:
10.1073/pnas.1817498116
[Indexed for MEDLINE]
Free PMC Article

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