Applying Protein-Peptide Interactions to Fluorescently Label Proteins in S. Cerevisia

Cells are highly complex, organized and dynamic machines, driven by intricate protein-based machinery. Understanding how this protein machinery produces the cellular processes we observe requires more than simply characterizing the individual parts in isolation. Complete understanding requires knowledge of a protein's spatial distribution in the cell, how this distribution relates to that of other proteins in the cell, and how these distributions change over time. Fluorescent labeling of proteins is an unrivaled technique for visualizing protein localizations and dynamics in living cells and can provide great insight into many of the mechanisms that underlie cellular processes.;While a number of methods can be used to fluorescently label a protein of interest, there is no one method that is suitable for all imaging applications. Oftentimes, picking the appropriate labeling method requires balancing a variety of competing factors such as label size, fluorophore properties, and labeling specificity. Label size in particular can be a cause for concern, as many labeling methods require modifications that are nearly as big as the protein itself and can affect the protein's native localization and/or function. I sought to develop new methods for fluorescently labeling proteins in vivo that do not require such large modifications to the protein of interest, but that can still be readily applied to fluorescently label any protein of interest. Rather than make direct fusions to a fluorescent protein, our approach is to use protein/peptide interactions to mediate protein labeling. Proteins of interest are tagged at the C-terminus with a short peptide tag, and the corresponding binding protein that recognizes the peptide tag is fused to a fluorescent protein and expressed in the same cell. Interaction between the binding protein and the peptide drives association of the fluorescent protein to the protein of interest, fluorescently labeling the target protein. I used two different protein/peptide interaction pairs to mediate protein labeling; the irreversible SpyCatcher/SpyTag pair, and the reversible Tetratricopeptide Repeat Affinity Protein (TRAP) binding domain.;SpyCatcher/SpyTag are a protein/peptide pair that bind one another and form an irreversible isopeptide bond in solution. I harnessed this interaction pair to fluorescently label proteins in vivo by fusing SpyTag (13 amino acids) to the protein of interest, and separately expressing SpyoIPD, a more stable derivative of SpyCatcher, fused to a fluorescent protein (FP). 1 have developed the molecular biology tools and labeling protocol required to fluorescently label proteins in S. Lerevisiae, and successfully used our strategy to fluorescently label a range of proteins in live yeast. I have also shown that labeling proteins in this manner can be less disruptive to protein function than direct fusion to a fluorescent protein, and that pulsed labeling of SpyTagged proteins with SpyolPD-FP can be used to track the turnover rate of a plasma membrane protein in living cells. 1 have also investigated the potential of different SpyoIPD-FP sequestration strategies to reduce fluorescent signal from unbound SpyoIPD-EGFP, demonstrated that SpyCatcher can be used in combination with a variety of fluorescent proteins, and that SnoopCatcher/SnoopTag, another permanent protein/peptide interaction pair, can also be used to fluorescently label proteins in vivo.;Additionally. I have also investigated the use of a reversible protein/peptide interaction to fluorescently label proteins in S. cerevisiae. TRAPs are artificial binding domains developed in the Regan lab that bind 5 amino acid long, C-terminal peptide tags with affinities as low as 300 nM. Previous work in the Regan lab has demonstrated that TRAP domains can be used to fluorescently label a protein in E. Coli. Here I extend upon this work, showing that TRAPs can also be used to fluorescently label a collection of proteins in S. cerevisiae.;Thus, I have shown that a variety of protein/peptide interactions can be used to fluorescently label proteins in vivo and have developed the tools and protocols necessary to fluorescently label proteins of interest in yeast. Our SpyCatcher/SpyTag based strategy in particular offers a useful tool for fluorescently labeling target proteins that are sensitive to large modifications, or for experiments that require studying protein dynamics over time.