In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.
The cation diffusion facilitator (CDF) family of metal ion transporters plays important roles in zinc transport at all phylogenetic levels. In this report, we describe a novel interaction between two members of the CDF family in Saccharomyces cerevisiae. One CDF member in yeast, Msc2p, was shown recently to be involved in zinc transport into the endoplasmic reticulum (ER) and required for ER function. We describe here a newly recognized CDF family member in yeast, Zrg17p. ZRG17 was previously identified as a zinc-regulated gene controlled by the zinc-responsive Zap1p transcription factor. A zrg17 mutant exhibits the same zinc-suppressible phenotypes as an msc2 mutant, including an induction of the unfolded protein response in low zinc. Moreover, a significant fraction of the total Zrg17p protein appears to localize to the ER. Their common phenotypes and localization suggested that these two proteins function together to mediate zinc transport into the ER. Consistent with this hypothesis, Msc2p and Zrg17p physically interact with each other, as determined by co-immunoprecipitation. Therefore, we propose that Msc2p and Zrg17p form a heteromeric zinc transport complex in the ER membrane. We also demonstrate that ZnT5 and ZnT6, mammalian homologues of Msc2p and Zrg17p, functionally interact as well. These results suggest that heteromeric complexes formed by different CDF members may be a common phenomenon for this ubiquitous family of metal ion transporters.
SummaryLittle 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.
Alkaline phosphatase of Saccharomyces cerevisiae (ScALP/Pho8) is a zinc‐dependent vacuolar enzyme. ScALP moves through the secretory pathway as a zymogen before being activated by the Pep4 vacuolar protease. Interestingly, we found that both ScALP activity and protein level are affected by the availability of zinc ions; both decrease dramatically when cells are Zn limited and this effect is very specific to ScALP. Decreased ScALP level in low zinc was dependent on Pep4 suggesting that the apoprotein is degraded. By studying ScALP activity in wild type and various Zn transporter mutants, we found that ALP accumulation and activity is mainly dependent on the major vacuolar Zn transporters Zrc1 and Cot1, rather than zinc transporters acting upstream in the secretory pathway. Our experiments show that this transporter dependency is not due to direct channeling of zinc from these transporters to the enzyme nor is proteolytic activation in the vacuole required for metallation. Our current hypothesis is that the vacuolar environment is specifically favorable for ScALP metallation for two reasons. First, the level of zinc in the vacuole may be higher than in upstream compartments of the secretory pathway. Second, in vitro studies indicate that the low pH of the vacuole (5.5) promotes metal binding relative to more alkaline conditions. Failure to metallate ScALP in the vacuole results in its degradation by vacuolar proteases.
We demonstrate a novel interaction between two members of the cation diffusion facilitator (CDF) family in Saccharomyces cerevisiae: Msc2p and a newly recognized CDF family member, Zrg17p. Both Msc2p and Zrg17p have been previously implicated in zinc homeostasis in yeast. In particular, ZRG17 was previously identified as a zinc regulated gene controlled by the zinc-responsive Zap1p transcription factor. We show that both Msc2p and Zrg17p are localized to the endoplasmic reticulum (ER) when expressed at normal levels. Zinc deficiency in yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. UPR induction in low zinc is exacerbated in msc2 and zrg17 mutants. Genetic and biochemical evidence indicate that this UPR induction is due to genuine ER dysfunction. Notably, ER-associated protein degradation (ERAD) is defective in zinc-limited msc2 mutants. Msc2p and Zrg17p physically interact, as determined by co-immunoprecipitation. Therefore, we propose that Msc2p and Zrg17p form a zinc transport complex in the ER membrane to maintain the function of this compartment. Zinc deficiency also upregulates the mammalian ER stress response, indicating a conserved requirement for zinc in ER function among eukaryotes. Lastly, ZnT5 and ZnT6, the closest mammalian homologues to Msc2p and Zrg17p, may also functionally interact, suggesting that interactions between CDF members may be a common phenomenon.We demonstrate a novel interaction between two members of the cation diffusion.
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