Title

Functional study of RTS1 and CDC55, two genes encoding regulatory subunits of protein phosphatase 2A in Saccharomyces cerevisiae

Date of Award

2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Richard L. Hallberg

Second Advisor

John Russel

Keywords

RTS1, CDC55, Encoding regulatory subunits, Protein phosphatase 2A, Saccharomyces cerevisiae

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Life Sciences | Molecular Biology

Abstract

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase in eukaryotic cells. Its activity has been implicated in the control of many cellular processes. It is a heterotrimeric complex comprised of a catalytic, a structural and a regulatory subunit.

In Saccharomyces cerevisiae, RTS1 encodes a regulatory subunit of PP2A. rts1Δ cells are temperature sensitive for growth and have a global stress response defect. In order to find out what other cellular functions require RTS1 , a search for multicopy suppressors of the temperature sensitive rts1 -null cells was carried out. The genes CLB2, PIR2, MSB1, PH085 , YLR426W and CAK1 were found to be multicopy suppressors. As indicated by the suppressors' functions, rts1 -null cells were found to have a G2/M delay at 37°C, weakened cell walls at 37°C, and a possible budding defect at 39°C.

The mechanism of ts suppression achieved by overproducing CLB2 was further investigated. We found that Clb2p levels in cells arrested in S phase was decreased relative to that seen in wild type cells, and this decrease was shown to be caused by the anaphase-promoting complex (APC)-dependent ectopic degradation. In investigating the role of two APC regulators, we also found that the ectopic degradation of Clb2p in rts1 -null cells was Cdc20p-dependent but Cdh1p-independent.

Loss of CDC55 , another regulatory subunit of PP2A, causes the cells to exhibit a failure of cytokinesis and the production of abnormally elongated buds at low temperature. As cdc55 -null cells exhibit a hyperphosphorylation of Y19 on the cyclin-dependent kinase Cdc28, we examined wild type and cdc55 -null cells for the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of this phosphorylation. Our results showed that Mih1p contributed little to this hyperphosphorylation. By contrast, Swe1 kinase was significantly elevated in mitosis arrested cdc55 -null cells. This excess buildup of Swe1p in cdc55 -null cells is the result of ectopic stabilization of this protein during G2/M phase. Present evidence indicating that this is a result of a gain-of-function of PP2A complexes lacking Cdc55p. My work shows that different PP2A regulatory subunits are required for the proper function of distinctly different cell cycle related pathways.

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