Lipid Scrambling Pathways in the Sec61 Translocon Complex.

Area of Science:

• Cellular Biology
• Membrane Biophysics
• Protein-Lipid Interactions

Background:

• Cellular homeostasis relies on ATP-independent lipid translocation across the endoplasmic reticulum (ER) membrane.
• Scramblases are membrane proteins facilitating lipid translocation; ER-resident scramblases are recently identified.
• Previous structures suggested the Sec61/translocon-associated protein (TRAP) complex might mediate lipid scrambling due to observed membrane thinning.

Purpose of the Study:

• To investigate the role of the Sec61/TRAP complex in lipid scrambling.
• To identify the specific component and mechanism responsible for lipid translocation within the complex.
• To determine the lipid specificity and regulation of this scrambling activity.

Main Methods:

• Reconstitution of translocon complexes for fluorescence spectroscopy assays.
• Inhibition assays using Sec61 inhibitors.
• Molecular dynamics simulations.
• Kinetic and thermodynamic analyses.

Main Results:

• Translocon complexes exhibited nonselective lipid scrambling activity.
• Scrambling activity persisted even when Sec61's lateral gate was inhibited, suggesting an alternative pathway.
• Molecular dynamics simulations revealed TRAP facilitates lipid translocation via a "credit card" mechanism.
• Membrane thinning enhanced scrambling efficiency; TRAP and Sec61 preferentially scrambled phosphatidylcholine over phosphatidylethanolamine and phosphatidylserine.

Conclusions:

• The trimeric TRAP subunit provides an alternative, Sec61-independent pathway for lipid scrambling.
• This TRAP-mediated lipid translocation is insensitive to the functional state of the Sec61 translocon.
• The findings identify TRAP as a key player in metazoan lipid homeostasis.