Saccharomyces cerevisiae Expresses Three Functionally Distinct Homologues of the Nramp Family of Metal Transporters

Abstract

The baker's yeast Saccharomyces cerevisiae expresses three homologues of the Nramp family of metal transporters: Smf1p, Smf2p, and Smf3p, encoded by SMF1, SMF2, and SMF3, respectively. Here we report a comparative analysis of the yeast Smf proteins at the levels of localization, regulation, and function of the corresponding metal transporters. Smf1p and Smf2p function in cellular accumulation of manganese, and the two proteins are coregulated by manganese ions and the BSD2 gene product. Under manganese-replete conditions, Bsd2p facilitates trafficking of Smf1p and Smf2p to the vacuole, where these transport proteins are degraded. However, Smf1p and Smf2p localize to distinct cellular compartments under metal starvation: Smf1p accumulates at the cell surface, while Smf2p is restricted to intracellular vesicles. The third Nramp homologue, Smf3p, is quite distinctive. Smf3p is not regulated by Bsd2p or by manganese ions and is not degraded in the vacuole. Instead, Smf3p is down-regulated by iron through a mechanism that does not involve transcription or protein stability. Smf3p localizes to the vacuolar membrane independently of metal treatment, and yeast cells lacking Smf3p show symptoms of iron starvation. We propose that Smf3p helps to mobilize vacuolar stores of iron.Nramp (for natural resistance-associated macrophage protein) represents a family of evolutionarily conserved membrane proteins that facilitate the transport of heavy metal ions (5,6,15,17,31). Members of the Nramp family have been found in mammals, birds, insects, plants, fungi, and bacteria (3,5,9,15,16,22,40). Among the best studied are the Nramp1 and Nramp2 transporters of rodents. Although these proteins share 61% homology at the amino acid level, they exhibit distinct functions. Mouse Nramp1 plays an important role in the control of infection against intracellular parasites and is exclusively expressed in monocytes/macrophages and polymorphonuclear leukocytes (2, 15). Nramp2 (also known as DCT1 or DMT1) is more ubiquitously expressed in most tissues (17) and acts as a divalent metal transporter capable of transporting iron, manganese, copper, zinc, cadmium, and lead (17). Mutations in Nramp2 have been associated with defects in duodenal iron uptake and cellular iron utilization in the mk mouse and the Belgrade rat models of anemia (13,14).The baker's yeast Saccharomyces cerevisiae expresses three closely related Nramp homologues, Smf1p, Smf2p, and Smf3p, encoded by SMF1, SMF2, and SMF3, respectively (6,7,29,43). Like mammalian DMT1, the Smf proteins exhibit a somewhat broad substrate specificity. Smf1p was originally defined as a high-affinity manganese transporter (39) and was later shown to contribute to cellular accumulation of cadmium and copper (28). Smf2p can affect cobalt levels in yeast (28) and may also participate in manganese trafficking (43). In more recent studies, Chen and coworkers demonstrated that both Smf1p and Smf2p can stimulate iron uptake into Xenopus oocytes (7). The role of Smf3p in metal homeostasis has not been define...

Manganese transport in eukaryotes: The role of DMT1

“…In animals, the transporter can be located both at the cell surface and in intracellular organelles (Nelson, 1999), whereas in S. cerevisiae, the different isoforms are situated at distinct cellular sites: Smf1p at the cell surface, Smf2p in the intracellular vesicles and Smf3p at the vacuolar membrane (Portnoy et al, 2002;Portnoy et al, 2000;Supek et al, 1996).…”

“…In the Mn depleted state, Smf2p is restricted to intracellular punctuate bodies for mobilizing any Mn that is delivered by Smf1p from the extracellular space into the cytosol and subsequently to the necessary organelles (Portnoy et al, 2000). In contrast, Smf3p is present and stable at the vacuolar membrane independent of the metal availability in the surroundings (Portnoy et al, 2002;Portnoy et al, 2000). The three scenarios are summarized and represented in Figure 4.…”

“…There are three known DMT1 orthologues expressed in yeast cells: Smf1p, Smf2p and Smf3p, which are encoded by SMF1, SMF2 and SMF3 respectively. Overall, the three Smf proteins exhibit ∼26% identity to human NRAMP2 and ∼48% identity to each other at the amino acid level Portnoy et al, 2000;West et al, 1992). In contrast to vertebrate DMT1, no typical IRE is found in any of the SMF genes, and the poor sequence conservation of the C-termini is not predictive of a possible correspondence between the vertebrate −IRE/+IRE and the Smf1p/2p/3p.…”

“…Knockout mutants of the three SMF genes are still able to take up Mn, suggesting the existence of an alternative transporter for Mn uptake from the environment. This figure is modified from Portnoy et al, 2000 and …”

“…SMF3 mutants do not show any impairment in the uptake and homeostasis of Mn; rather, Smfp3 is mainly responsible for trafficking Fe. The SMF3 promoter contains a recognition sequence for the Fe-sensing transcription factor Aft1p which is responsible for its transcriptional regulation by Fe, whereas Smf1p and Smf2p levels are unaffected by Fe concentration (Portnoy et al, 2002;Portnoy et al, 2000). Since no significant staining of Smf2p or Smf3p was observed at the plasma membrane, and SMF1 mutants do not show changes compared to wild type, it has been hypothesized that some extracellular metal transporter(s) other than the three Smfs is responsible for Mn uptake under physiological conditions.…”