Abstract
Nutrient metals such as zinc are both essential to life and potentially toxic if overaccumulated by cells. Non-essential toxic metals like cadmium can enter cells through the uptake transporters responsible for nutrient metal acquisition. Therefore, in the face of ever changing extracellular metal levels, organisms tightly control their intracellular levels of nutrient metals and prevent accumulation of toxic metals. We show here that post-translational inactivation of the yeast Zrt1 zinc uptake transporter is important for zinc homeostasis. During the transition from zinc-limiting to zinc-replete growth conditions (i.e. zinc shock), the Zrt1 transporter is ubiquitinated, endocytosed, and subsequently degraded in the vacuole. To further understand this process at a molecular level, we mapped a region of Zrt1 required for ubiquitination and endocytosis in response to zinc to a domain located on the intracellular surface of the plasma membrane. This domain is a critical cis-acting component of the metal signaling pathway that controls Zrt1 protein trafficking. Using mutant alleles defective for metal-responsive inactivation, we also show that Zrt1 inactivation may be an important mechanism for preventing cadmium uptake and toxicity in zinc-limited cells.
Highlights
The transition from limiting to replete conditions is very stressful to microbial cells because of the insult this transition imposes on cellular homeostasis
We show here that post-translational inactivation of the yeast Zrt1 zinc uptake transporter is important for zinc homeostasis
We show here that Zrt1 inactivation is an important part of zinc shock tolerance
Summary
Yeast Strains and Growth Conditions—Strains used were DY1457 (␣ ade can his leu trp ura3), ZHY3 (DY1457 zrt1::LEU2 zrt2::HIS3), DEY1531 (DY1457 end4::LEU2), MSYS1 (␣ ade can his leu trp ura3), MSYS2 (MSYS1 ZRT1K195R), MSYS3 (MSYS1 zrc1⌬), MSYS4 (MSYS1 ZRT1K195R zrc1⌬), and MKY1 (MSYS1 ZRT1⌬205Ϫ211). Yeast were grown in SD1 medium (0.67% yeast nitrogen base without amino acids) [18] supplemented with auxotrophic requirements and 2% galactose to induce expression of ZRT1 from the GAL1 promoter or 2% glucose to repress this expression This medium was made zinc limiting by adding 1 mM EDTA. Cells grown to exponential phase (OD600 ϭ 0.5–1) in SD galactose medium were harvested by centrifugation (5 min at 1000 ϫ g) and resuspended in SD glucose medium to shut off expression of the GAL1 promoter. Cells were harvested and assayed for 65Zn uptake rates to assess plasma membrane Zrt activity These assays were performed as described previously for iron uptake [21] except that 65ZnCl2 (Amersham Biosciences) and LZM prepared without EDTA (LZM-EDTA) were substituted for 59FeCl3 and LIMEDTA, respectively. Indirect immunofluorescence microscopy was performed as described previously [7]
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