Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4⌬ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4⌬ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2 E301K ) suppressed the ubi4⌬ growth defect. The rpt2 E301K mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4⌬ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2 E301K mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ϳ5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4⌬ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells.Zinc is an essential element with diverse roles in biology. Unlike transition metals, such as iron and copper, zinc ions (Zn 2ϩ ) are not redox-active under physiological conditions and do not play a direct role in redox reactions. Zn 2ϩ strongly interacts with ligands, such as cysteine, histidine, and acidic amino acids in proteins (1). When bound by three or fewer ligands, Zn 2ϩ can act as a Lewis acid to facilitate catalysis by diverse classes of enzymes, including oxidoreductases, transferases, and hydrolases (2). In contrast, binding of Zn 2ϩ by four ligands produces a relatively inert, structurally rigid tetrahedral complex, which provides stability to many classes of protein domains (1-3). Because of its catalytic and structural roles, Zn 2ϩ has been estimated to be required for the folding and function of ϳ10% of proteins encoded by eukaryotic genomes and ϳ5% of proteins in prokaryotes (4, 5). One abundant example is the enzyme alcohol dehydrogenase, which contains two Zn 2ϩ atoms per subunit, one serving in catalysis and the other playing a structural role (1, 6, 7). Consistent with the importance of zinc to Adh 2 folding, mutants of Adh lacking structural zinc site ligands are unstable and quickly degraded in ...
