Abstract
The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by five C-terminal C(2)H(2) zinc fingers and each finger is required for high-affinity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc fingers. Furthermore, mutations that disrupt some of these fingers cause constitutive activity of a bifunctional Gal4 DNA-binding domain-Zap1 fusion protein. These results demonstrate a novel function of Zap1 zinc fingers in zinc sensing as well as DNA binding.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Cell Nucleus / chemistry
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Cell Nucleus / drug effects
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Cell Nucleus / metabolism
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DNA, Fungal / genetics
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DNA, Fungal / metabolism
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Fluorescent Antibody Technique
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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Gene Expression Regulation, Fungal / drug effects
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Genes, Reporter / genetics
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Mutation
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Protein Binding
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Protein Biosynthesis / drug effects
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins / chemistry
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / drug effects
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins*
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Signal Transduction / drug effects
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Trans-Activators / chemistry*
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Trans-Activators / genetics
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Trans-Activators / metabolism*
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Zinc / deficiency
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Zinc / metabolism*
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Zinc / pharmacology
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Zinc Fingers / genetics
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Zinc Fingers / physiology*
Substances
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DNA, Fungal
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DNA-Binding Proteins
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Fungal Proteins
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GAL4 protein, S cerevisiae
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Nuclear Proteins
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Recombinant Fusion Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Trans-Activators
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Transcription Factors
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ZAP1 protein, S cerevisiae
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Zinc