Development of an in vitro transport assay to measure the activity of the yeast cation transporter Gdt1p

Gdt1p is a transporter localized in the membrane of the Golgi apparatus of Saccharomyces cerevisiae which is involved in calcium, manganese, and pH homeostasis. Over the past decade, research on this protein and its family has intensified, as the human ortholog of Gdt1p, named TMEM165, has been linked to a subclass of congenital disorders of glycosylation. From these studies, it is known that the GDT1 family presents one or two copies of a highly conserved motif (E-x-G-D-(K/R)-(S/T)) and in vivo transport assay showed that Gdt1p may act as an antiporter transporting Ca2+, Mn2+ and H+. However, more information is still needed to fully understand the functioning of Gdt1p, such as the mechanism and the direction of transport. In this project, Gdt1p was produced, purified, and reconstituted in liposomes in order to develop in vitro transport experiments. First, a His-tagged Gdt1p was overexpressed in Lactococcus lactis and purified using affinity chromatography. Results showed that production and purification were reproducible although some contaminants were still present at the end of the process. Liposomes of different lipids composition were produced and their solubilization by two different detergents (OG and DDM) was investigated. Flotation assays showed efficient reconstitution of Gdt1p into liposomes. Two fluorescent probes were used to follow the in vitro transport of cations: Fura-2 for Ca2+ and Mn2+, and ACMA for protons. While the ACMA assays gave promising information on the sealing of DOPC/DOPE liposomes, Fura-2 assays were not conclusive. The residual probe concentration inside liposomes at the end of the whole reconstitution procedure was indeed too low to monitor cations transport across membranes. Overall, this project has advanced the realization of functional in vitro transport assays and provided tools that will be useful for future reconstitutions of Gdt1p in liposomes. Ways of improvement and diversification are possible, such as the use of other fluorescent probes, or the reconstitution of Gdt1p in other types of liposomes (e.g., larger liposomes or other lipid composition).