Identification, characterization and functional study of par1 and par2, two genes encoding B' subunits of protein phosphatase 2A in Schizosaccharomyces pombe
Date of Award
Doctor of Philosophy (PhD)
Richard L. Hallberg
Protein phosphatase 2A, Schizosaccharomyces pombe, par1, par2, Cytokinesis
Biochemistry, Biophysics, and Structural Biology | Cell and Developmental Biology | Cell Biology | Genetics and Genomics | Life Sciences | Molecular Biology
Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase found in all eukaryotic cells. The PP2A holoenzyme is comprised of a catalytic C subunit, a structural A subunit, and a regulatory B subunit.
In my thesis work, I identified and characterized two B ' subunit genes of PP2A in Schizosaccharomyces pombe , namely, par1 and par2 . Both genes share high homology with other known B ' genes. Neither is essential but together they are required for growth at both high and low temperatures, growth under a number of physiologically stressful conditions, and septum positioning and cytokinesis. Cross-organism experiments showed that both par1 and par2 could complement a deletion of Saccharomyces cerevisiae RTS1 , a gene encoding a B ' homolog. Furthermore, both Par1p and Par2p physically associate with the catalytic subunits of PP2A, confirming that both genes encode S. pombe PP2A B ' subunits.
In some par mutant cells, the septum is not centrally located. Fluorescence microscopy revealed that the mitotic actin ring and Mid1p are also eccentrically located in par1Δpar2Δ cells, indicating that par genes are involved in regulating Mid1p and actin ring localization to ensure correct septum positioning. We also found that in par mutants, interphase Mid1p nuclear staining is much weaker than that seen in wild type cells, and Western blots showed that this might be caused by a reduced Mid1p abundance.
par1Δpar2Δ cells also show a multi-septation phenotype, very similar to that seen in hyperactive SIN (septation initiation network) mutants. I examined the genetic interactions between par deletions and SIN mutants, and found that par1Δpar2Δ suppressed Byr4-OP defects, and rescued a loss-of-function allele of spg1 , but could not suppress any mutations in genes downstream of spg1 . I showed further that par deletions suppressed the lethality of the spg1 allele through restoration of the correct localization of Cdc7p to the spindle pole body (SPB). The fact that par mutants themselves exhibited a symmetric localization of Cdc7p to SPBs indicated a hyperactivity of SIN in such cells. I concluded that par genes normally negatively regulate SIN at or upstream of cdc7 insuring that multiple rounds of septation do not occur.
Jiang, Wei, "Identification, characterization and functional study of par1 and par2, two genes encoding B' subunits of protein phosphatase 2A in Schizosaccharomyces pombe" (2001). Biology - Dissertations. Paper 43.