Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2018 Mar 20;115(12):3066-3071. doi: 10.1073/pnas.1717956115. Epub 2018 Mar 5.

Single-molecule analysis of phospholipid scrambling by TMEM16F.

Author information

1
Department of Applied Chemistry, The University of Tokyo, Tokyo 113-8656, Japan; wrikiya@nojilab.t.u-tokyo.ac.jp hnoji@appchem.t.u-tokyo.ac.jp snagata@ifrec.osaka-u.ac.jp.
2
Laboratory of Biochemistry & Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.
3
Laboratory of Biochemistry & Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan wrikiya@nojilab.t.u-tokyo.ac.jp hnoji@appchem.t.u-tokyo.ac.jp snagata@ifrec.osaka-u.ac.jp.

Abstract

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.

KEYWORDS:

TMEM16F; membrane protein; microsystem; phospholipid scrambling; single-molecule analysis

PMID:
29507235
PMCID:
PMC5866571
DOI:
10.1073/pnas.1717956115
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center