Characterization of Gdt1p mutants selectivity

Details

Supervisors

Morsomme, Pierre

Faculty

Faculté des bioingénieurs

Degree label

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

Abstract

The GDT1 family is known for its role in cations homeostasis and the role of its human ortholog, TMEM165, in causing Congenital Disorder of Glycosylation. There is a conserved motif through the family that is hypothesized to form the pore of the transporter. The role of each of the residues composing this motif is not well understood and would be useful for a better comprehension and treatment of the disease. Gdt1p, the yeast ortholog, transports calcium, manganese, and protons through the Golgi membrane. Transport activity is evaluated thanks to a tool developed in the lab, the heterologous expression of the protein in L. lactis cells loaded with Fura-2 fluorescent probe. L. lactis is used as an in-out system, metal cations can reach the probe inside the cell if Gdt1p allows their transport across the bacterium membrane. Thanks to Fura-2 fluorescent features, the transport activity of other cations has been investigated and showed Gdt1p selectivity to calcium, manganese, cadmium, and zinc transport. The transport activity of calcium and manganese by Gdt1p mutants in a L. lactis system has also been studied. Mutations in the motif caused a decreased transport of calcium and no difference in manganese transport compared to Gdt1p activity. This study aimed to characterize the selectivity of Gdt1p and its mutants towards different cations to better understand the role of the residues of this conserved motif. To do so, the project started by studying the mutants E53A and E204A. Transport assays in the L. lactis system were performed with different metal cations. No unexpected transport activity by the mutants was detected, translating no selectivity effect with this approach. To complete those results, growth assays with yeast were led on solid metal-rich media. After a screening of different metal concentration, the strain lacking Gdt1p and Pmr1p, a Golgi ATPase, was chosen for further analysis as a greater it showed a greater sensibility to the medium. A selectivity effect could be observed for both of the mutants on cobalt medium and for the E53A mutant on copper medium. The results of the two different approaches did not converge. Further investigation would be needed to identify if this is due to the different level of metal concentration imposed or to indirect effects.