Purification and reconstitution of Gdt1p, a secondary cation transporter

Details

Supervisors

Morsomme, Pierre ; Gaiffe, Alexandra

Faculty

Faculté des bioingénieurs

Degree label

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

Abstract

The Uncharacterized Protein Family 0016 was first identified in 2012 when a link was highlighted between mutations in the gene coding for the human member TMEM165 and a disease called Congenital Disorder of Glycosylation. UPF0016 is a well conserved family and has members in nearly all organisms. Recent studies showed that proteins belonging to this family are involved in calcium, manganese and probably proton homeostasis. Nowadays, UPF0016 is not considered as uncharacterized anymore thanks to the S. cerevisiae ortholog Gdt1p and has been renamed “GDT1 protein family”. Several experiments performed in vivo using Gdt1p suggested that these proteins may act as secondary cation transporters. However, information concerning transport mechanism and direction of transport are still unknown and need to be further investigated. In this project, Gdt1p was purified and reconstituted in liposomes to measure in vitro transport activity. First, a His-tagged version of Gdt1p was overexpressed in L. lactis bacteria and purified by Immobilized Metal Affinity Chromatography (IMAC). Early results showed presence of contaminants and slight degradation of the protein in eluted fractions. Purification conditions were therefore optimized and showed that column washes with increasing imidazole gradients removed most contaminants. Then, asolectin liposomes were produced and characterized by Dynamic Light Scattering (DLS). This technique confirmed the expected size distribution of liposomes supporting the reproducibility of experiments. Fluorescence assays on liposomes with internal calcium indicator Fura-2 were performed and a strategy for transport activity measurements was determined. Reconstitution of purified Gdt1p was realized by a detergent-mediated method with DDM and different detergent-lipid-protein ratios were assessed. Flotation assays confirmed an efficient protein incorporation for most tested ratios. Finally, in vitro transport assays were done but no significant results could be concluded due the low concentration of proteoliposomes. Optimization of transport assays is still required but these early results are promising and represent the first steps towards in vitro characterization of the GDT1 family.