Document Type

Thesis

Degree

Master of Science

Major

Biology, Molecular and Cellular Biology

Date of Defense

11-16-2007

Graduate Advisor

Colin MacDiarmid, PhD.

Committee

Colin MacDiarmid

Wendy Olivas

Marc Spingola

Abstract

Magnesium (Mg2+) is essential for all life, and is utilized for many important biological processes. Despite the fundamental importance of Mg2+ homeostasis to life, relatively little is known about Mg2+ homeostasis in eukaryotes. The goal of this work was to identify proteins required for the active transport of Mg2+, which are essential to prevent the overaccumulation of cytosolic Mg2+, but 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 unknown. 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 effects on Mg2+ homeostasis. The per1 mutation reduced Mg2+ content, but overexpression of PER1 had no effect. Subsequently, Per1 was identified as component in the pathway of 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 mutations in the other two CaCA proteins had no effect on Mg tolerance, indicating that these proteins do not have a redundant function in homeostasis. However, the ynl321w mutant did show sensitivity to high Ca2+ (700 mM). As a result of this, ynl321w mutant were screened for other Ca2+ related phenotypes and synergistic interactions. 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. Thus, these findings suggest a role for Ynl321w in Ca2+ secretion from the cell. Localization studies using fluorescence microscopy and sucrose gradient fractionation showed that Ynl321wp is localized to the ER membrane. Ecx1 is the first described example of a CaCA protein participating in Ca2+ homeostasis within the secretory pathway.

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