Systems analysis of metabolic control in saccharomyces cerevisiae [electronic resource]

How cells respond to intrinsic and extrinsic perturbations engages the exquisite regulation of metabolic pathways. In this study, we developed an approach based on targeted metabolomics, gene disruption, and modeling to profile novel regulatory interactions and identify key regulatory nodes in the yeast ergosterol biosynthesis pathway. Ergosterol pathway metabolites were quantified in yeast heterozygous strains with individual ergosterol biosynthetic genes deleted, and enrichment results were analyzed by modeling to discover regulatory nodes. Numerous known and new regulatory interactions were identified in this study. Common regulatory nodes in almost all deletion strains were MVD1, ERG6, and ERG27, whereas ERG12 and ERG20 were uniquely regulated in mvd1Δ, highlighting the importance of MVD1 and its metabolite product isopentenyl pyrophosphate. Regulation was suggested to occur post-transcriptionally rather than at the level of gene expression. The deletion of ERG genes also led to the decrease of a variety of metabolites within the pathway, suggesting a regulatory mechanism to conserve materials. This study provides insights on how cells rewire their metabolism as a response to genetic perturbations and showcases a new technique to analyze the regulatory interactions underlying the response. Natural small metabolites comprise the majority of cellular molecules and participate in a wide variety of biochemical and regulatory processes. However, a global analysis of their interactions with and regulation of proteins in vivo is lacking. A method to systematically investigate in vivo protein-metabolite interactions was developed in an initial study (Li et al. Cell, 2010), and widespread interactions of hydrophobic metabolites with different groups of proteins were found by mass spectrometry. We extended this untargeted and systematic approach to investigate the interactions of both hydrophilic and hydrophobic metabolites with proteins produced by S. cerevisiae. In particular, 200 yeast proteins (3% of the proteome) annotated to be involved in regulating enzyme activities were analyzed. The methods of metabolite extraction and LC-MS analysis were tailored to be suitable for analyzing both hydrophilic and hydrophobic molecules. More than one hundred physical interactions were found in this study, some of which were in accordance with previously reported metabolite-protein interactions and/or function. For instance, Bcy1, a regulatory subunit of the cyclic AMP-dependent protein kinase, showed significant binding to cyclic AMP. Vhs3, a regulatory subunit of protein phosphatase 1 Ppz1 and a homolog of an FMN-binding protein, bound to FMN and its degradation product. Intriguingly, we also discovered numerous novel interactions. Pai3, a proteinase A inhibitor, bound to AMP, and the binding may regulate responses to osmotic stress. Gip2 and seven other proteins bound to sucrose, and four of them have been reported to be implicated in carbohydrate metabolism. In particular, Gip2 is a putative regulatory subunit of type-1 protein phosphatase Glc7. Our analysis of invertase activity and glycogen levels provided evidence that upon binding to sucrose, Gip2 may promote glucose derepression and glycogen synthesis, respectively. These results revealed widespread metabolite-protein interactions in yeast. Systematic investigation of such interactions provides new insights about the regulation of protein functions. Such knowledge may have important implications in promoting human health by altering metabolite levels or use of drugs that control metabolic and/or protein activities.

Keywords: Chemistry.
Advisor: Snyder Michael
Cegelski Lynette advisor. Thesis advisor
Stearns Tim advisor. Thesis advisor