Abstract
Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni2+, Mn2+, Zn2+ and Co2+. Using Ni2+ uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms.
Highlights
Magnesium (Mg) is the fourth most abundant cation in the body, and the second most abundant within cells [1]
To determine the effect of Mg supply and the mnr2 mutation on the regulation of the Alr systems, we initially attempted to measure the rate of Mg uptake by cells grown over a range of Mg concentrations, using atomic absorption spectroscopy to measure the change in Mg content of cells when subsequently supplied with a dose of excess Mg (AAS) [24,41,42]
The CorA family of Mg channels is widespread in biology, and its members play an important role in the regulation of cytosolic Mg concentration
Summary
Magnesium (Mg) is the fourth most abundant cation in the body, and the second most abundant within cells (after potassium) [1]. In environments with abundant Mg, its tendency to over-accumulate in cells can challenge homeostatic mechanisms [5]. Cytosolic Mg is distributed between a large pool bound to proteins, nucleic acids and small molecules, and a smaller, regulated pool of free-ionized Mg [13,14]. Regulation of the cytosolic free-ionized Mg concentration is likely achieved by three major mechanisms: control of uptake systems, efflux from the cell, and sequestration within organelles. Despite the importance of this cation we are only beginning to understand the molecular basis of Mg homeostasis in eukaryotic cells
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