CCC1 Is a Transporter That Mediates Vacuolar Iron Storage in Yeast

Abstract

The budding yeast Saccharomyces cerevisiae can grow for generations in the absence of exogenous iron, indicating a capacity to store intracellular iron. As cells can accumulate iron by endocytosis we studied iron metabolism in yeast that were defective in endocytosis. We demonstrated that endocytosis-defective yeast (⌬end4) can store iron in the vacuole, indicating a transfer of iron from the cytosol to the vacuole. Using several different criteria we demonstrated that CCC1 encodes a transporter that effects the accumulation of iron and Mn 2؉ in vacuoles. Overexpression of CCC1, which is localized to the vacuole, lowers cytosolic iron and increases vacuolar iron content. Conversely, deletion of CCC1 results in decreased vacuolar iron content and decreased iron stores, which affect cytosolic iron levels and cell growth. Furthermore ⌬ccc1 cells show increased sensitivity to external iron. The sensitivity to iron is exacerbated by ectopic expression of the iron transporter FET4. These results indicate that yeast can store iron in the vacuole and that CCC1 is involved in the transfer of iron from the cytosol to the vacuole.While iron is a required element for all eucaryotes, it is also potentially toxic. Organisms tightly regulate the concentration of cytosolic iron through regulation of iron uptake and storage. In the past few years the mechanisms that mediate plasma membrane iron transport in the budding yeast Saccharomyces cerevisiae have been described in molecular detail (1). Many of the genes required for plasma membrane transport have been cloned. Much less is known, however, about iron storage. Yeast is distinguished from most eucaryotes in not having ferritin as an iron storage molecule. In this regard yeast are more analogous to plants than vertebrates. In plants, ferritin is restricted to the chloroplasts, as opposed to the cytosol in animals. Plants and yeast are thought to store iron in the vacuole, although there is little compelling proof of that supposition. Three lines of evidence have been used to support the view that the vacuole is an iron storage organelle: 1) vacuolar mutants show increased metal sensitivity (2, 3); 2) iron can be found in vacuoles (4); and 3) there are transport systems capable of extracting iron from the vacuole (5, 6). The increased metal sensitivity of vacuolar mutants, however, can result from increased metal uptake rather than decreased storage (7). Uptake of extracellular fluid by endocytosis, a steady state process would lead to vacuolar iron accumulation, which could be extracted by vacuolar iron transporters. There are little data that demonstrate that iron accumulated in the cytosol can be stored in the vacuole.To determine whether S. cerevisiae store iron in the vacuole we studied vacuolar iron storage in yeast strains unable to endocytose. Our studies demonstrated that yeast do store iron in the vacuole. We further demonstrated that CCC1 is an iron/Mn 2ϩ transporter responsible for storing these two metals in the vacuole. EXPERIMENTAL PROCEDURESYeast Strains, Plasm...

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions

“…The VIT1 domain is homologous to Arabidopsis VIT1, which is involved in transporting iron into vacuoles (Kim et al, 2006). The domain is also similar to yeast Ca 2+ -sensitive cross-complementer 1 (CCC1) protein, which is localized in vacuolar membranes, and is an iron/manganese transporter that transfers iron from the cytoplasm and into vacuoles (Li et al, 2001). Unlike the ferritins, very little is known about the Er-VIT1 family.…”

A Mitochondrial-Vacuolar Signaling Pathway in Yeast That Affects Iron and Copper Metabolism

“…This could occur through either through a direct reduction in cytosolic iron or by affecting the synthesis of iron-sulfur clusters in mitochondria, as the level of iron-sulfur clusters appears to regulate transcription of the iron-regulon (16). Our previous studies demonstrated that yeast store iron in the vacuole and that Ccc1p is a vacuolar protein that transports iron from the cytosol to the vacuole (15). Vacuoles isolated from ⌬mrs3⌬mrs4 cells showed an increase in iron content compared with vacuoles from wild type cells (Fig.…”

“…Iron Sensitivity of ⌬ccc1 by Reducing Cellular Iron Accumulation-Cells with a deletion in CCC1, which encodes a vacuolar iron and manganese transporter, are more sensitive to growth on high iron medium than wild type cells, presumably because of an inability to remove iron from the cytosol (15). We screened for genes that when overexpressed could suppress the high iron sensitivity of ⌬ccc1 cells.…”

“…A FET3-LacZ construct was constructed and assayed as described previously (15). CTR1-LacZ and CUP1-LacZ constructs were obtained from Dr. Dennis Winge (University of Utah).…”

“…Miscellaneous Procedures-Iron transport activity and iron content assays were performed as described (15). A FET3-LacZ construct was constructed and assayed as described previously (15).…”