TitleRegulation of yeast choline kinase by zinc depletion and the yeast phosphatidate phosphatase by zinc depletion and growth
NameSoto-Cardalda, Anibal (author), Carman, George M (chair), Martin, Charles E (internal member), Kinzy, Terri (internal member), Chikindas, Mikhail (internal member), Han, Gil-Soo (outside member), Rutgers University, Graduate School - New Brunswick,
SubjectMicrobiology and Molecular Genetics,
DescriptionIn the yeast Saccharomyces cerevisiae, the CKI1-encoded choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine via the CDP-choline branch of the Kennedy pathway. Analysis of a PCKI1-lacZ reporter gene revealed that CKI1 expression was regulated by intracellular levels of the essential mineral zinc. Zinc depletion resulted in a concentration-dependent induction of CKI1 expression. This regulation was mediated by the zinc-sensing and zinc-inducible transcriptional activator Zap1p. A purified Zap1p probe interacted with two putative UASZRE sequences (ZRE1 and ZRE2) in the CKI1 promoter. Mutations of ZRE1 and ZRE2 to a nonconsensus UASZRE attenuated the induction of CKI1 expression in response to zinc depletion. A UASINO element in the CKI1 promoter was responsible for stimulating CKI1 expression, but this element was not involved in the regulation by zinc depletion. The induction of CKI1 expression in zinc-depleted cells translated into increased choline kinase activity in vitro and in vivo, and an increase in phosphatidylcholine synthesis via the Kennedy pathway. The yeast PAH1-encoded PA phosphatase (PAP) catalyzes the dephosphorylation of PA to yield DAG and Pi. The DAG produced by the PAP reaction is utilized for the synthesis of TAG and the synthesis of PE and PC via the Kennedy pathway. Analysis of the PPAH1-lacZ reporter gene demonstrated that PAH1 expression was regulated by intracellular levels of zinc. The induction of the PAH1 gene in response to zinc depletion was dependent on the concentration of zinc. PAP regulation by zinc depletion was mediated by the zinc responsive transcription factor Zap1p. Purified Zap1p interacted with three putative UASZRE sequences located in the PAH1 promoter. The Zap1p mediated induction in PAH1 expression in response to zinc depletion resulted in an increase in PAP activity in vitro. The induction in PAP activity in zinc-depleted cells translated into an increase in DAG and TAG levels. The pah1Δ mutation resulted in the decrease of PC synthesis via the Kennedy pathway in response to zinc depletion. The regulation of the yeast PAH1-encoded PAP by growth was examined. The DAG produced in the PAP reaction is converted into the storage lipid TAG throughout growth. As cells progress from the exponential to the stationary phase of growth the PAP activity is induced, resulting in an increased in DAG and TAG levels. The induction in PAP activity coincided with a decrease in PAP protein levels. PAP protein is degraded via the ubiquitin-proteasome pathway as cells progress throughout growth.
NoteIncludes bibliographical references
Noteby Anibal Soto-Cardalda
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.