Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation

Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. It have been recently reported in our lab that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and form a new group of Golgi-localized cation/Ca2+ exchangers. Both share topological characteristics reminiscent of the cation/calcium exchanger superfamily. They are composed of two hydrophobic regions containing a highly conserved motifs separated by a large cytosolic loop. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. The Ca2+ transport activity of Gdt1p was demonstrated using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis. The Ca2+ uptake activity in cell expressing GDT1 was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. This work focuses on the functional and structural study of Gdt1p in the yeast Saccharomyces cerevisiae. We firstly found that Gdt1p controls cellular calcium content and plays a major role in the calcium response following an osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external Ca2+, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Additionally, we highlighted that Gdt1p levels are controlled by external Ca2+ via a mechanism involving the calcineurin signaling pathway along with vacuolar proteolysis. Finally, by site-directed mutagenesis and characterization of the mutants, we found that the cations binding pocket of Gdt1p is most likely composed of glutamate, aspartate, serine and threonine residues located in the repeated motifs.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 201