Manganese and calcium homeostasis in Saccharomyces cerevisiae is regulated by Gdt1p and various transporters in the secretory pathway

Details

Supervisors

Morsomme, Pierre

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enCalcium and manganese homeostasis is an essential process in all living organisms: these two ions are vital for numerous cellular functions but become toxic in excess, which requires precise regulation of their availability. In eukaryotes, an imbalance of these ions in the secretory pathway can cause serious pathologies, such as \acf{CDG} in humans. The yeast \textit{Saccharomyces cerevisiae} is a relevant study model, as it possesses transporters homologous to those of humans, involved in the homeostasis of these two ions. Among them, Gdt1p is the ortholog of TMEM165, a human protein whose gene mutations are associated with \acs{CDG}. Work in the laboratory has mainly focused on Gdt1p, a transporter of both ions, but it is not the only transporter involved in this homeostasis. This master's thesis therefore focuses on the process as a whole, studying four transporters in parallel: Gdt1p, Pmr1p, Smf2p, and Smf1p. Intracellular calcium and manganese concentrations were measured by \acf{ICP-AES} in ten strains (the wild-type and nine deletion mutants) grown in rich medium alone or supplemented with calcium or manganese. Growth assays on solid rich medium, alone or supplemented with calcium, manganese, or both, were used to link the growth phenotypes to the ionic imbalances. This work shows that calcium and manganese doesn't only depend on the amount of an ion in the medium but also on the balance between the two cations, which directly influences growth; the two homeostases, thus, appear closely linked. The \acs{ICP-AES} measurements were broadly consistent with the expected trends but did not reach statistical significance. Several discrepancies with the growth assays suggest that the total ionic concentration doesn't reflect the intracellular distribution of the ions, which is nevertheless biologically decisive. Finally, these results suggest that Pmr1p and Gdt1p are the main importers of calcium and manganese into the secretory pathway, and are consistent with the hypothesis that Smf2p would export manganese from the secretory pathway into the cytosol. Smf1p, by contrast, appears to be an adaptive transporter, mainly required under manganese deficiency, whose deletion only slightly affects growth under the conditions tested in this study.