Lipid flippases are protein complexes composed of two integral membrane proteins: a 10 transmembrane spanning catalytic subunit (P4-ATPase or ALA) and a 2 transmembrane spanning beta subunit (CDC50 protein or ALIS). The P4-ATPase belongs to a family of well-known primary transporters (P-type ATPases) that move cations across membranes (H+, Ca2+, Zn2+ …). However, the preferred substrate of lipid flippases are phospholipids, which are at least 45 times bigger than the biggest cation transported by any other P-type ATPase. How is such a huge substrate transported? This enigma is called the “giant substrate problem” and the scientists at the Biomembranes and Lipid Flippases group are decided to solve it.
In order to solve the mechanism of lipid transport we are trying to resolve the structure of the complex. For this, we need to produce the proteins in big amounts with tags that can help purification and visualization.
Your tasks will be to (modifications of the project are possible if you have new ideas):
- Generate a construct containing a SNAP-tagged flippase complex in a plasmid that can be used in
Saccharomyces cerevisiae with your plasmid and other plasmids already available in the lab that contain characterized flippase complexes as controls.
- Test lipid transport by use of fluorescent lipids and flow cytometry assays, as well as functional complementation of mutants on toxic peptides.
- Isolate membrane protein fractions from Saccharomyces and test expression of the protein by SDS-PAGE and western blot.
- Generate yeast extracts and label the SNAP tag with fluorescent substrates that can be visualized by in-Gel fluorescence.
- Medium-scale protein expression and purification of the flippase in complex with its beta subunit (affinity chromatography and size exclusion)
- Carry out biochemical and biophysical studies (activity assays, protein reconstitution in liposomes, cryo-EM structural determination)
The project will be embedded within a project financed by the Independent Research Fund Denmark. The work will be carried out in the Section for Transport Biology, Department for Plant and Environmental Sciences, Faculty of Science at Copenhagen University, under the supervision of Associate Professor Rosa L. Lopez-Marques (firstname.lastname@example.org).