Post-transcriptional role of SNF1 protein kinase in maltose permease synthesis of Saccharomyces cerevisiae.
Item
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Title
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Post-transcriptional role of SNF1 protein kinase in maltose permease synthesis of Saccharomyces cerevisiae.
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Identifier
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AAI3232002
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identifier
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3232002
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Creator
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Cheema, Saima Arshad.
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Contributor
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Adviser: Corinne A. Michels
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Date
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2006
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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Loss of Snf1 kinase blocks MAL gene induction even if MIG1 is also deleted (Hu et al., 2000). Here I show that this defect in MAL gene induction stems from defective maltose permease expression. This dissertation investigates the post-transcriptional requirement of Snf1 kinase in maltose permease synthesis.;Expression of a series of MAL61-LacZ fusions with junction sites in the MAL61 ORF reveals that MAL61 mRNA usage is only modestly affected by loss of SNF1. However, Mal61/HA protein levels are dramatically reduced in a snf1Delta mig1Delta strain even when MAL61/HA is transcribed from the constitutive GPD promoter indicating that maltose permease synthesis requires SNF1 at a post-transcriptional step. GAL2, HXT1 and MAL61, all members of the 12TMD family of sugar transporters, were fused to the GPD promoter and, in contrast to Mal61p, synthesis of Hxt1p and Gal2p and galactose transport activity are unaffected by snf1Delta. Various hybrid MAL61-GAL2 and GAL2-MAL61 genes were constructed and their expression in a snf1Delta mig1Delta strain indicates that the SNF1 requirement for the Mal61p expression is associated with its N-terminal half.;UPR and ERAD are protein quality control pathways involved in the degradation of improperly folded/denatured ER-localized proteins. In the absence of several key components of either UPR or ERAD, particularly Ire1p, Ubc6p, and Doa10p, Mal61p is stabilized suggesting that Mal61p becomes a target of these degradation pathways in the absence of SNF1. Additionally, Mal61p levels increase significantly in a snf1Delta mig1Delta end3Delta strain but this is not associated with an increase in transport activity indicating that permease is rapidly internalized in the snf1Delta strain and that the protein that reaches the plasma membrane is inactive. Consistent with this, Mal61p levels become stable in the absence of PEP4.;Finally, multicopy suppressors of the maltose non-fermenting phenotype of the snf1Delta mig1Delta [pMAL63/43-c] strain were isolated and characterized.;The results show that Snf1 kinase is required post-transcriptionally at several steps in maltose permease expression. In the absence of SNF1, maltose permease is a target of the ER quality control pathways, is rapidly internalized from the plasma membrane, and is functionally inactive.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
