With our recent studies we have demonstrated the essential function of protein Alr1 for the Mg 2+ homeostasis in
the yeast Saccharomyces cerevisiae. The alr1 knock out mutation causes a conditional lethal phenotype, which can
be suppressed by high external Mg 2+ concentrations (100mM Mg 2+) in the growth media. Standard growth
conditions (~1mM Mg 2+) result in a 60% decrease in intracellular Mg 2+ concentrations, likely to be responsible for
several not clearly defined cellular malfunctions, which consequently triggers cell death. We have investigated the
Mg 2+ dependent expression and stability of the Alr1 protein and observed a specific control of both processes by
Mg 2+ concentrations. Mg 2+ dependent Alr1p turnover involves polyphosphorylation followed by endocytosis in an
End4/Rps5-dependent manner and ends degradation in the vacuole.
Here we want to focus our efforts on the control which the Mg 2+ ion exerts on synthesis and degradation of Alr1p.
This system has the potential to serve as a paradigm for ion-controlled processes. Emphasis will be on the Mg 2+
dependent initiation of Alr1p endocytosis. By random and site-specific mutation of the ALR1 gene we want to
define sequences in cis which are important for this process. With a genetic screen we will then search for factors
interacting with Alr1p to mediate endocytosis. In order to enable us to properly interpret phenotypes of mutants
with changes in Alr1p itself and in proteins affecting its synthesis and stability it will be important to better
characterize (Mg2+-dependent) regulation of Alr1p synthesis and assembly as well as its topology and structure in
a proposed Mg 2+ transport system. The proposed studies aim at unravelling essential facets of the control of small
ions on cellular processes, particularly on the initiation of endocytosis.