Zinc transporters belonging to the Cation Diffusion Facilitator (CDF) family have complementary roles in transporting zinc out of the cytosol

Abstract

Zinc is an essential trace element that is required for the function of a large number of proteins. As these zinc-binding proteins are found within the cytosol and organelles, all eukaryotes require mechanisms to ensure that zinc is delivered to organelles, even under conditions of zinc deficiency. Although many zinc transporters belonging to the Cation Diffusion Facilitator (CDF) families have well characterized roles in transporting zinc into the lumens of intracellular compartments, relatively little is known about the mechanisms that maintain organelle zinc homeostasis. The fission yeast Schizosaccharomyces pombe is a useful model system to study organelle zinc homeostasis as it expresses three CDF family members that transport zinc out of the cytosol into intracellular compartments: Zhf1, Cis4, and Zrg17. Zhf1 transports zinc into the endoplasmic reticulum, and Cis4 and Zrg17 form a heterodimeric complex that transports zinc into the cis-Golgi. Here we have used the high and low affinity ZapCY zinc-responsive FRET sensors to examine cytosolic zinc levels in yeast mutants that lack each of these CDF proteins. We find that deletion of cis4 or zrg17 leads to higher levels of zinc accumulating in the cytosol under conditions of zinc deficiency, whereas deletion of zhf1 results in zinc accumulating in the cytosol when zinc is not limiting. We also show that the expression of cis4, zrg17, and zhf1 is independent of cellular zinc status. Taken together our results suggest that the Cis4/Zrg17 complex is necessary for zinc transport out of the cytosol under conditions of zinc-deficiency, while Zhf1 plays the dominant role in removing zinc from the cytosol when labile zinc is present. We propose that the properties and/or activities of individual CDF family members are fine-tuned to enable cells to control the flux of zinc out of the cytosol over a broad range of environmental zinc stress.

The Loz1 transcription factor from Schizosaccharomyces pombe binds to Loz1 response elements and represses gene expression when zinc is in excess

“…To overcome this limitation, we generated a new construct to express Loz1GFP from a weaker derivative of the pgk1 promoter that contained a deletion within the canonical TATA box ( pgk1 ΔTATA ). To compare the strength of the pgk1 ΔTATA promoter to the loz1 promoter ( loz1 p ), strains expressing Loz1GFP from these promoters were grown overnight in a zinc‐limited minimal medium (ZL‐EMM) (Choi et al ., ) supplemented with 0 (−Zn) or 100 μM Zn (+Zn) and crude protein extracts prepped for immunoblot analysis. The immunoblot analysis revealed that the pgk1 ΔTATA ‐driven Loz1GFP accumulated in both low and high zinc conditions (Fig.…”

“…Examples of these second‐response genes include TSA1 , which encodes a peroxiredoxin that has a dual role as a peroxidase and a holdase, and ZRG17 , which encodes a CDF family member that is required for zinc transport from the cytoplasm into the endoplasmic reticulum (Wu et al ., ; ). Further analyses of these genes and their protein products have shown that the holdase function of Tsa1 is critical for survival during severe zinc starvation, and that in the absence of the Zap1‐dependent increase in ZRG17 expression, cells experience increased protein unfolding in the endoplasmic reticulum (MacDiarmid et al ., ; Choi et al ., ). Studies of these Zap1 target genes therefore suggest that cells have programmed responses to counter stresses that arise from zinc starvation.…”

Epidermodysplasia Verruciformis: Functional Role of E6 and E7 Oncoproteins as the Link between Viral Infection to Carcinogenesis

“…ZIP and SLC30 designated as cation diffusion facilitator (CDF) or as the zinc transporter (Zn-T). 1,26 ZIP and Zn-T counter-balance each other's work and protect the cell from zinc toxicity. The former is responsible for translocating zinc towards the cytoplasm whereas the latter translocates zinc from the nucleus, outside the cytoplasm.…”

FRET Microscopy in Yeast

“…The design, calibration, and application of this type of FRET sensors have already been carefully discussed [49], so we provide here an update of more recently implemented sensors (see Table 2). These sensors not only assess metabolite/ion concentrations in yeast, but also help to delineate functions and regulatory networks of involved enzymes (e.g., in respective mutant strains) [50][51][52]. Remarkably, some metabolite/ion FRET biosensors were directly developed from specific yeast proteins or systematically optimized in yeast.…”