Dissecting cell cycle protein complexes using the pptimized yeast cytosine deaminase protein-fragment complementation assay
“You too can play with an edge”
Proteins are the end-products of gene interpretative machinery.  Proteins serve essential roles in defining the structure, integrity and dynamics of the cell and mediate most chemical transformations needed for everything from metabolic catalysis to signal transduction.  We know that the central dogma of molecular biology, one gene = one mRNA = one protein is a gross simplification and that a protein may do different things depending on the form in which its mRNA was spliced, how and where it is post-translationally modified, what conformational state it may be in or finally, which other proteins it may interact with.  Formation of protein complexes may, themselves, be governed by the states in which proteins are expressed in specific cells, cellular compartments or under specific conditions or dynamic phases such has growth or division.  Protein complexes involved in mitotic cell cycle regulation are particularly challenging to dissect since they could only form during specific phases of the cell cycle, are highly regulated by post-translational modifications and can be found in any subcellular compartments.  To date, no general methods have been developed to allow fine dissection of these protein complexes.  The goal of this thesis was to establish and demonstrate a novel strategy for dissecting protein complexes regulating the budding yeast Saccharomyces cerevisiae (S. cerevisiae) mitotic cell cycle.    In this thesis, I describe my development and optimization of a simple survival-selection Protein-fragment Complementation Assay using the prodrug-converting enzyme, yeast cytosine deaminase as reporter (OyCD PCA).  I further describe, in a series of proof of principle studies, applications of the OyCD PCA to dissect the mechanism of transcriptional activation by key mitotic transcription factors and to dissect protein-protein interactions (PPIs) among regulators of the mitotic cell cycle.  A key feature of the OyCD PCA is that it can be used to detect both formation and disruption of PPIs by virtue of having positive readouts for both assays.    I applied the OyCD PCA in a strategy to dissect interactions between the key transcription factors of the G1/S phase: SBF and MBF.  These two heterodimeric transcription factors are composed of, respectively, two distinct DNA-binding subunits named Swi4 and Mbp1 and a common transcription activation subunit called Swi6. I took advantage of the dual selection by OyCD PCA to engineer a specific mutant of Swi6 in order to demonstrate the rewiring of a transcriptional network.  We isolated Swi6 with mutations found in its C-terminal domain previously identified for binding Swi4 and Mbp1 and important for SBF and MBF activities.  Our results support a model where Swi6 undergoes a conformational change upon binding to Swi4 or Mbp1.  In addition, this Swi6 mutant was used to dissect the regulatory mechanism that governs the entry of S. cerevisiae to a new round of cell division also known as START. We found that the SBF and MBF repressor Whi5 directly binds to the C-terminal domain of Swi6.  Finally, I applied the OyCD PCA to dissect the yeast cyclin dependent kinase Cdk1-protein complexes.  Cdk1 is the essential kinase that regulates cell cycle progression and can phosphorylate a large number of different substrates by teaming up with one of nine cyclin regulatory proteins (Cln1-3, Clb1-6). I describe a strategy to identify interaction partners of Cdk1 that can easily be scaled up for a genome-wide screen and associate the complexes with the appropriate cyclin(s) required for mediating the interaction using the OyCD PCA and deletion of the cyclin genes.  My results allow us to postulate which phase(s) of the mitotic cell cycle Cdk1 may phosphorylate proteins and what function potential or known substrates of Cdk1 may take on during that phase(s). For example, we identified the interaction between Cdk1 and the γ-tubulin (Tub4) to be dependent upon Clb3, consistent with its role in mediating nucleation and growth of mitotic microtubule bundles on the spindle pole body.  This strategy can also be applied to study other PPIs that are contingent upon accessory subunits.
Saccharomyces cerevisiae 5-fluorocytosine Swi6 positive and negative selection Protein-fragment Complementation Assay yeast cytosine deaminase 5-fluorocytosine cell cycle transcription factors rewiring transcriptional activities mechanism of regulation, G1/S phase cyclin dependent kinase Cdk1 cyclin protein-protein interactions substrates and protein complexes