Characterization of Putative Magnesium Transport systems in yeast

by Franklin, Andrew James

Magnesium (Mg2+) is essential for all life, and is utilized for many important biological processes. All cells must maintain an appropriate concentration of Mg2+ in the cytosol and within organelles in order to maintain key biological processes such as transcription and translation. Despite the fundamental importance of Mg2+ homeostasis, relatively little is known about homeostasis in eukaryotic cells. The goal of this work was to identify membrane transport systems that may be involved in the active transport of Mg2+, using Saccharomyces cerevisiae as a model organism. Although a variety of Mg2+ influx systems have been described, proteins mediating active transport of Mg2+ (which are essential to prevent the overaccumulation of cytosolic Mg2+) have not been identified from any organism. In yeast, a vacuolar Mg2+/H+ exchange activity has been described, but the molecular identify of this protein is not known. To try and identify this activity, a candidate gene approach was used. Four yeast genes of unknown function (PER1, YNL321w, YDL206w, and YJR106w) were screened for phenotypic effects on Mg2+ homeostasis when overexpressed or deleted. PER1 encodes a membrane protein that is essential for growth in high Mg2+ concentrations. YNL321w, YDL206w, and YJR106w are members of the CaCA (calcium/cation antiporter) superfamily, members of which transport a variety of divalent metal cations via a cation/proton exhange mechanism. Experiments to determine the function of Per1 showed that overexpression of this gene did not affect the Mg2+ content of yeast, but the per1 mutation did reduce Mg2+ content. However, information communicated from another research group indicated that this effect was not specific to Mg2+. In addition, Per1 was subsequently identified by another research group as an ER protein mediating a step in the pathway for Franklin, Andrew J. 2007, UMSL, ii glycosylphosphatidylinositol (GPI) anchor synthesis. Of the three remaining candidate proteins, only one (Ynl321w) produced a significant increase in intracellular Mg2+ content when overexpressed. However, the ynl321w deletion mutation did not alter Mg2+ accumulation, Mg2+ tolerance, or tolerance to a range of other potentially toxic cations. Combining the ynl321w mutation with knockout mutations in the other two CaCA proteins also did not affect metal tolerance, indicating that these proteins do not have a redundant function. However, the ynl321w mutant did show a slight sensitivity to a high Ca2+ concentration (700 mM). As a result of this, I screened for other Ca2+ related phenotypes in ynl321w mutants, alone and when combined with other mutations that disrupt Ca2+ homeostasis. I observed that when combined with ynl321w, vcx1 and pmc1 mutations displayed synthetic Ca2+ sensitivity phenotypes. Measurement of cellular Ca2+ content with AAS showed that the ynl321w mutation was associated with an increase in Ca2+ content, and that this effect that was still observed in vcx1 or pmc1 backgrounds, indicating it was independent of vacuolar Ca2+ storage. Thus, these findings suggest a role for Ynl321w in Ca2+ secretion from the cell via the secretory pathway or plasma membrane. Localization studies using fluorescence microscopy and sucrose gradient fractionation showed that Ynl321wp to be localized to the ER membrane. As a consequence of these observations, I propose that Ynl321w may perform a similar function to Pmr1p, a P-type ATPase that transports Ca2+ and Mn2+ into the Golgi (and eventually, releases the ion to the external environment). Therefore, I renamed the Ynl321w protein Ecx1 (for ER calcium exchanger). The identification of Ecx1 is the first described example of a CaCA protein participating in Ca2+ homeostasis within the secretory pathway. Although the work did not provide insight into the molecular mechanisms of Mg2+, it did identify a factor in Ca2+ homeostasis. Franklin, Andrew J. 2007, UMSL, iii Franklin, Andrew J. 2007, UMSL, iv