Zap1p, a Metalloregulatory Protein Involved in Zinc-Responsive Transcriptional Regulation in Saccharomyces cerevisiae

Abstract

Zinc ion homeostasis in Saccharomyces cerevisiae is controlled primarily through the transcriptional regulation of zinc uptake systems in response to intracellular zinc levels. A high-affinity uptake system is encoded by the ZRT1 gene, and its expression is induced more than 30-fold in zinc-limited cells. A low-affinity transporter is encoded by the ZRT2 gene, and this system is also regulated by zinc. We used a genetic approach to isolate mutants whose ZRT1 expression is no longer repressed in zinc-replete cells, and a new gene, ZAP1, was identified. ZAP1 encodes a 93-kDa protein with sequence similarity to transcriptional activators; the C-terminal 174 amino acids contains five C 2 H 2 zinc finger domains, and the N terminus (residues 1 to 706) has two potential acidic activation domains. The N-terminal region also contains 12% histidine and cysteine residues. The mutant allele isolated, ZAP1-1 up , is semidominant and caused high-level expression of ZRT1 and ZRT2 in both zinc-limited and zinc-replete cells. This phenotype is the result of a mutation that substitutes a serine for a cysteine residue in the N-terminal region. A zap1 deletion mutant grew well on zinc-replete media but poorly on zinc-limiting media. This mutant had low-level ZRT1 and ZRT2 expression in zinc-limited as well as zinc-replete cells. These data indicate that Zap1p plays a central role in zinc ion homeostasis by regulating transcription of the zinc uptake system genes in response to zinc. Finally, we present evidence that Zap1p regulates transcription of its own promoter in response to zinc through a positive autoregulatory mechanism.Zinc is essential for all organisms. This metal is a catalytic component of over 300 enzymes, including alcohol dehydrogenase, carbonic anhydrase, and carboxypeptidases (33). Zinc also plays a structural role in many proteins. For example, several motifs found in transcriptional regulatory proteins are stabilized by zinc, including the zinc finger, zinc cluster, and RING finger domains (28). Proteins containing these domains are very common; as many as 1% of all human gene products contain the C 2 H 2 zinc finger motif first identified in transcription factor IIIA (TFIIIA) (18).Although it is essential for many different cellular functions, zinc can also be toxic. When the intracellular zinc level rises to some critical level, the metal can interfere with vital processes, perhaps by competing with other metal ions for enzyme active sites, transporter proteins, and other biologically important ligands. The delicate balance of intracellular zinc is accomplished through precise homeostatic regulation mediated by a number of mechanisms. These include binding of the metal by cytoplasmic macromolecules such as metallothioneins (14) and phytochelatins (27), zinc storage in intracellular compartments (3,20,25), and transport of the metal out of the cell (26).The primary control point for zinc ion homeostasis is the regulation of zinc uptake across the plasma membrane. Little is known about the mechanism and regulat...

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