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TOR Signalling Regulates Cdk8-Dependent Induction of the GAL Genes

University of British Columbia

The GAL genes in S. cerevisiae are highly regulated at the transcriptional level and make an excellent model for the study of eukaryotic transcription. GAL transcription is activated by Gal4 in response to the availability of galactose. Gal4 activity is positively regulated by Cdk8-dependent phosphorylation, by an uncharacterized mechanism that becomes essential for GAL induction in yeast lacking the galactose inducer protein Gal3. Null mutations of cdk8/ srb10, or mutation of the Cdk8-dependent phosphorylation site on Gal4 at S699, render gal3 yeast incapable of growing on galactose as the sole source of carbon. The work presented in this thesis exploited this phenotype in a genetic screen to identify mutants that prevent Cdk8-dependent GAL expression with the goal of discovering regulators of Cdk8. I isolated 16 complementation groups, termed the gal four throttle (gft) mutants. One mutant, gft1, was found to represent a recessive allele of hom3, which encodes an aspartokinase in the biosynthetic pathway for threonine and methionine. Characterization of gft1 revealed a defect in Tor signalling; strains defective for gft1/ hom3 are hyper-sensitive to rapamycin and cause constitutive Gat1 nuclear localization. Furthermore, null mutations of tor1 or tco89, encoding components of the TORC1 complex, also prevent GAL expression in gal3 yeast. Tetrad analysis revealed that gft7 is allelic to tco89. Further genetic analysis demonstrated that disruption of cdc55, encoding a regulatory subunit of the PP2A protein phosphatase downstream of Tor signalling suppresses the effect of gft1/ hom3, gft7/ tco89, and tor1 mutations on GAL expression. Additionally, a class of gft mutants hyper-sensitive to sublethal concentrations of hygromycin B were identified which led to the discovery that med2 is likely allelic to gft13-2. This group of gft mutants may represent a mechanistic explanation for the gft phenotype that is separate from Tor signalling. Together these results provide novel insight into how induction of transcription by a specific inducer can be modulated by global physiological signals.

Text

2018-08-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/66865

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/