The community dynamics of protein phosphatase 2A subunits in Saccharomyces cerevisiae
Date of Award
Doctor of Philosophy (PhD)
Richard L. Hallberg
Protein phosphatase 2A, Holoenzymes, Cell cycle
Biochemistry, Biophysics, and Structural Biology | Cell and Developmental Biology | Cell Biology | Genetics and Genomics | Life Sciences | Molecular Biology
Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This holoenzyme is a collection of varied heterotrimeric complexes, composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit.
To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of PP2A subunits in Saccharomyces cerevisiae . We found: the level of each subunit remained constant throughout the cell-cycle; there is at least ten times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding; the two regulatory subunits display distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Additionally, we identified some cis- and trans-acting machinery involved in PP2A Cdc55p and PP2A Rts1p localizations.
In vitro binding assays demonstrated that C subunit post-translational modification(s) affects PP2A heterotrimer stabilities. However, the degree to which different types of heterotrimers are affected in vivo unknown. Using subunit localization to assess in vivo binding stabilities, we found that Pph21p C-terminal mutants that are not efficiently modified and cells lacking the C subunit's methyltransferase, PPM1 , both show reduced PP2A localization, presumably because the formation of stable PP2A heterotrimers is decreased. Furthermore, Cdc55p-directed PP2A localizations are reduced to a greater degree than Rts1p-directed localizations. Thus, the formation of stable PP2A heterotrimers is dependent on C subunit modification and PP2A Cdc55p stability is more dependent on this than is PP2A Rtrs1p .
In vitro data also suggest that specific regions of the A subunit are necessary for heterotrimer formation. However, there is little to no in vivo data to support this finding. Thus, we determined the role of various A subunit regions in in vivo heterotrimeric makeup. Overall, we found that PP2A heterotrimers are more stable in vivo than they are in vitro . Using PP2A mutant phenotypes, we found that PP2A Rts1p is more resistant to A subunit mutations than is PP2A Cdc55p .
Gentry, Matthew Shawn, "The community dynamics of protein phosphatase 2A subunits in Saccharomyces cerevisiae" (2003). Biology: Dissertations. 35.