Optogenetics is a developing field of biotechnology in which an organism is genetically engineered so that a concrete biological process can be specifically activated by light. This technique is heavily used to study neuron signal transmission in the brain, because it allows the precise activation and inactivation of neurons by using light of different colours. In this project, we will try to generate a yeast-based optogenetics system to study the function of proteins called lipid flippases. Such system should allow the lipid flippase under study to be activated only in the presence of light of specific colours. Lipid flippases are integral membrane proteins that transport lipids towards the cytosolic side of biological membranes. In humans, lipid flippases are involved in diseases like Parkinson’s and Alzheimer’s, while in plants they have a role in the response to different types of biotic and abiotic stress (heat, cold, pathogens,...). We expect that our optogenetics system could be adaptable to lipid flippases from different organisms and would be a tool for future characterization of flippase regulation.
In yeast, two lipid flippases at the plasma membrane are activated by kinases called Fpk proteins. Recently, a study demonstrated that Fpk proteins can be substituted for a light-activated kinase from a green algae. The green algae kinase (CrPHOT1) can be expressed in yeast and activated by illuminating the yeast with blue light. CrPHOT1 can then phosphorylate and activate endogenous lipid flippases. Our group has recently generated a yeast strain that lacks the Fpk proteins and several lipid flippases. This strain can be used as a chassis for the generation of our optogenetics system. The idea is to express PHOT proteins from the plant Arabidopsis thaliana together with lipid flippases from the same plant, and test whether lipid transport activity can be induced by illuminating the yeast with blue light, followed by characterization and validation of the system. This will generate a proof-of-concept that a yeast-based optogenetics system can be developed to study lipid transporters, not only from plants, but also from other organisms, including humans.
Techniques involved: traditional and homologous recombination-based strategies, yeast culture and transformation, single-point mutant generation, lipid transport assays based on fluorescent lipids and flow cytometry, functional complementation assays, heterologous protein-expression and purification in yeast and E. coli, MS of phosphorylated residues, kinase activity assays.
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).