MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces cerevisiae

Magnesium (Mg) is an essential enzyme cofactor and a key structural component of biological molecules, but relatively little is known about the molecular components required for Mg homeostasis in eukaryotic cells. The yeast genome encodes four characterized members of the CorA Mg transporter superfamily located in the plasma membrane (Alr1 and Alr2) or the mitochondrial inner membrane (Mrs2 and Lpe10). We describe a fifth yeast CorA homolog (Mnr2) required for Mg homeostasis. MNR2 gene inactivation was associated with an increase in both the Mg requirement and the Mg content of yeast cells. In Mg-replete conditions, wild-type cells accumulated an intracellular store of Mg that supported growth under deficient conditions. An mnr2 mutant was unable to access this store, suggesting that Mg was trapped in an intracellular compartment. Mnr2 was localized to the vacuole membrane, implicating this organelle in Mg storage. The mnr2 mutant growth and Mg-content phenotypes were dependent on vacuolar proton-ATPase activity, but were unaffected by the loss of mitochondrial Mg uptake, indicating a specific dependence on vacuole function. Overexpression of Mnr2 suppressed the growth defect of an alr1 alr2 mutant, indicating that Mnr2 could function independently of the ALR genes. Together, our results implicate a novel eukaryotic CorA homolog in the regulation of intracellular Mg storage.MAGNESIUM (Mg) is a critical factor in a wide variety of biological processes (Elin 1994), and there are at least 300 Mg-dependent enzymes (Williams 1993; Cowan 1995). Given its diverse roles in biology, understanding how cells maintain Mg homeostasis is of fundamental importance. Maintaining a consistent Mg concentration in the cytosol and organelles is likely to require tight regulation of passive influx, active efflux, and sequestration mechanisms. Despite the importance of these mechanisms, relatively little is known about the molecular identity, function, and regulation of Mg transporters in eukaryotic cells.The yeast Alr1 and Alr2 proteins were the first eukaryotic Mg transporters identified. ALR1 inactivation conferred Mg-dependent growth and blocked Mg uptake (MacDiarmid and Gardner 1998; Graschopf et al. 2001). The closely related ortholog Alr2 was not essential for growth, but could compensate for the loss of Alr1 when overexpressed and was found to physically associate with Alr1 in vivo (MacDiarmid and Gardner 1998; Wachek et al. 2006). Further studies identified two related proteins in the mitochondrial inner membrane (Mrs2 and Lpe10). Both proteins were required for the entry of Mg into the mitochondrial matrix, and loss-of-function mutations in either gene caused similar reductions in mitochondrial function and Mg content (Bui et al. 1999; Gregan et al. 2001a,b). All four of these proteins are members of the metal ion transporter (MIT) superfamily, the founder member of which is CorA from Salmonella typhimurium (Gardner 2003; Knoop et al. 2005). In general, MIT proteins mediate rapid, membrane-potential-dependent transport, suggesting that they form Mg-selective channels (MacDiarmid and Gardner 1998; Liu et al. 2002; Kolisek et al. 2003; Froschauer et al. 2004; Schindl et al. 2007). Although divergent in primary sequence, typical MIT proteins possess two conserved structural features: a pair of transmembrane domains close to the C terminus and a triad of conserved residues (glycine–methionine–asparagine) that are essential for Mg transport (Knoop et al. 2005). The low number of transmembrane domains predicted to be present in MIT proteins suggested that oligomerization was required for ion transport (Kolisek et al. 2003; Warren et al. 2004), and independent crystallographic studies of a CorA homolog from Thermotoga maritima support this model (Eshaghi et al. 2006; Lunin et al. 2006; Payandeh and Pai 2006). T. maritima CorA formed a homopentamer of subunits in which the C-terminal transmembrane domains clustered together to form a membrane-spanning pore. The N-terminal regions of the subunits formed a cytosolic “funnel” domain that incorporated several apparent Mg-binding sites, suggesting a regulatory role for this domain. Genetic studies provided evidence that the binding of Mg ions to these sites altered the conformation of the complex and decreased channel activity (Payandeh and Pai 2006; Payandeh et al. 2008). The activity of the Mrs2 protein was also shown to be dependent on Mg concentration (Schindl et al. 2007), suggesting that both prokaryotic and eukaryotic MIT proteins can respond directly to the cytosolic or matrix Mg concentration to promote homeostasis.A fifth CorA homolog (YKL064w) is present in the yeast genome, but has not been characterized (MacDiarmid and Gardner 1998). The Ykl064w protein shares the two predicted transmembrane domains and conserved GMN motif characteristic of Mg-transporting members of the MIT family (MacDiarmid and Gardner 1998; Knoop et al. 2005). Phylogenetic analysis revealed that Ykl064w belongs to a subgroup of fungal MIT proteins in which a tryptophan residue replaces the conserved phenylalanine preceding the GMN motif (Knoop et al. 2005). Most sequenced yeast and fungal genomes include at least one Alr1 and one Ykl064w-type ortholog (Knoop et al. 2005). The presence of two discrete groups of fungal CorA proteins suggested that the Ykl064w-related proteins perform a novel function. For this reason, we decided to investigate the role of Ykl064w in ion homeostasis. Here we report that Ykl064w is a vacuolar membrane protein that is required for Mg homeostasis and present evidence implicating this protein in the regulation of intracellular Mg storage.