[Show abstract][Hide abstract] ABSTRACT: Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency.
[Show abstract][Hide abstract] ABSTRACT: Little is known about how metalloproteins in the secretory pathway obtain their metal ion cofactors. We used the Pho8 alkaline phosphatase of the yeast Saccharomyces cerevisiae to probe this process in vivo. We found that both Pho8 activity and protein accumulation are zinc-dependent and decrease in zinc-limited cells. Low Pho8 accumulation was the result of degradation by vacuolar proteases. Surprisingly, the protective effect of zinc on Pho8 stability was not solely due to Zn(2+) binding to the active-site ligands suggesting that the Pho8 protein is targeted for degradation in zinc-limited cells by another mechanism. Pho8 appears to be a rare example of a metalloprotein whose stability is regulated by its metal cofactor independently of active-site binding. We also assessed which zinc transporters are responsible for supplying zinc to Pho8. We found that the Zrc1 and Cot1 vacuolar zinc transporters play the major role while the Msc2/Zrg17 zinc transporter complex active in the endoplasmic reticulum is not involved. These results demonstrate that the vacuolar zinc transporters, previously implicated in metal detoxification, also deliver zinc to certain metalloproteins within intracellular compartments. These data suggest that Pho8 receives its metal cofactor in the vacuole rather than in earlier compartments of the secretory pathway.
[Show abstract][Hide abstract] ABSTRACT: Zinc deficiency causes oxidative stress in many organisms including the yeast Saccharomyces cerevisiae. Previous studies of this yeast indicated that the Tsa1 peroxiredoxin is required for optimal growth in low zinc because of its role in degrading H(2)O(2). In this report, we assessed the importance of other antioxidant genes to zinc-limited growth. Our results indicated that the cytosolic superoxide dismutase Sod1 is also critical for growth under zinc-limiting conditions. We also found that Ccs1, the copper-delivering chaperone required for Sod1 activity is essential for optimal zinc-limited growth. To our knowledge, this is the first demonstration of the important roles these proteins play under this condition. It has been proposed previously that a loss of Sod1 activity due to inefficient metallation is one source of reactive oxygen species (ROS) under zinc-limiting conditions. Consistent with this hypothesis, we found that both the level and activity of Sod1 is diminished in zinc-deficient cells. However, under conditions in which Sod1 was overexpressed in zinc-limited cells and activity was restored, we observed no decrease in ROS levels. Thus, these data indicate that while Sod1 activity is critical for low zinc growth, diminished Sod1 activity is not a major source of the elevated ROS observed under these conditions.
PLoS ONE 01/2009; 4(9):e7061. · 3.53 Impact Factor