Molecular analysis of lipid transporters at the single vesicle level

• Applicant: Professor Dr. Thomas Günther-Pomorski
• Subject Area: Biochemistry ; Biophysics
• Term: from 2017 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 373191975

Lipid transporters are integral membrane proteins that play a central role in moving lipids across cellular membranes, thereby controlling numerous cellular processes including vesicle biogenesis, cell signaling, morphogenesis and migration. They can be classified into two categories: (i) energy-independent transporters, termed scramblases, which randomize the distribution of lipids across the bilayer and (ii) ATP-driven, vectorial transporters, including members of a conserved subfamily of P-type ATPases, that actively translocate specific lipids from one membrane leaflet to the other. Current concepts suggests that the activity of lipid transporters is regulated by the lipid bilayer composition and that the dynamic process of lipid translocation may help driving membrane bending and vesicle budding. However, direct experimental evidence for these concepts is lacking. On one hand, this can be attributed to difficulties in handling of integral membrane proteins, their delicacy in production, purification, and characterization of their assembly with membrane lipids. On the other hand, sophisticated techniques are required for studying membrane proteins on the molecular level. Our approach will tackle this problem from both these sides by establishing defined reconstituted systems based on giant unilamellar vesicles and state-of-the-art imaging techniques to enable the molecular analysis of lipid transporters at different levels of compositional complexity (from simple lipid mixtures to cell-like lipid mixtures). Reconstitution of the purified transporters into giant unilamellar vesicles will allow us to study their activity in defined lipid environments by time-resolved fluorescence microscopy. We expect that these studies at the single vesicle level should reveal (i) the impact of lipid composition on the activity of the lipid transporters, and (ii) the role of ATP-dependent lipid translocation in membrane bending and vesiculation. In conclusion, the powerful combination of membrane protein chemistry and imaging techniques as proposed here should provide important new insight into the molecular characteristics, regulation and working mechanism of lipid transporters as vital components of cells.

• DFG Programme: Research Grants