We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with a modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. After this step we tested the produced constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosys in S. cerevisiae with a variety of fluorescence and affinity probes. Among the produced modular tags we found several combinations that were optimal for determining sub-cellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and de-phosphorylation. Our results indicate that this approach combining fluorescence microscopy/mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|