Time and context-controlled depletion of proteins in single cells
Final Report Abstract
The traditional approach to deduce the contribution of each protein of a network by comparing key properties of the wild type strain with a mutant carrying the deletion of the respective gene is in many ways not sufficient to fully understand the system properties of the network. We addressed this shortcoming in our DFG supported project by developing an experimental strategy based on Ubiquitin-fusion proteins that were designed in a way that cleavage at the C-terminus of Ubiquitin (Ub) exposes a destabilizing residue at the N-terminus of the investigated protein. We designed and experimentally realized this concept for essential proteins by a strategy named MIND where the fusion proteins will be stable in haploid cells, but highly unstable once two haploid yeast cells of opposing mating type have fused to form a diploid cell. Upon mating these two cells will reconstitute the N-end rule pathway of protein degradation and the diploid cell will thus start its new cell cycle in the (near) absence of the protein of interest. We applied this technique to different essential genes involved at various stages of the cell cycle and could measure the consequences of their deletions online by timelapse fluorescence microscopy of the diploid cells expressing fluorescent variants of suitable marker proteins. The many examples we studied proofed the general applicability of this method to study the functions of essential genes in the model organism Saccharomyces cerevisiae. By putting extra effort into the construction of recombination cassettes and suitable yeast strains we developed the technique into a flexible and efficient tool that complements existing methods to delete genes or deplete proteins to study their cellular functions. Applying MIND to the homologous genes BOI1 and BOI2 uncovered the reason why cells without both genes are not able to grow. Applying MIND to CDC5, IQG1, HOF1 addresses the order and mechanisms by which the proteins involved in cytokinesis assemble at the bud neck.
