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In vivo characterization of Calcium, Manganese and Proton transport by TMEM165 and its mutants

Hoegener, Victor
(2023)

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Hoegener_51182100_2023.pdf
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Details

Supervisors
Morsomme, Pierre ; Van Raemdonck, Guillemette
Faculty
Faculté des sciences
Degree label
Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie
Abstract
TMEM165 is a human membrane protein belonging to the GDT1 family which is localized in the late Golgi apparatus. Research on the GDT1 family really started in 2012 when TMEM165 was linked to a subtype of congenital disorder of glycosylation (CDG). CDGs are rare genetic diseases that mainly result in growth problems. The study of CDGs highlights the biological importance of glycosylated proteins. TMEM165 is mutated in one type of CDG resulting in impaired Mn2+ transport in the Golgi and a lack of Mn2+ is deleterious to the glycosylation process as it is an essential cofactor for glycosyltransferases. TMEM165 is composed of six transmembrane spans. Two motifs higly conserved among the GDT1 family are localized in the first and fourth span. The presence of charged residues within the motifs suggests that they could be involved in ion coordination and form the pore of the transporter. TMEM165 is able to transport Ca2+ and Mn2+. According to some observations, TMEM165 could also play a role in H+ homeostasis, but there is no experimental data to confirm it. Our current hypotheses is that TMEM165 is either a Ca2+/Mn2+ antiporter or a Ca2+-Mn2+/H+ antiporter. My project aims to provide observations in order to discriminate one of these two hypotheses. As it was shown for the yeast orthologue Gdt1p, the results obtained for TMEM165 point to a proton transport activity. The other part of my project focuses on the transport mechanism. To date, we have no experimental data about the structure but an Alphafold prediction is available. To establish a link between the structure of the protein and its functions, my project focuses on the characterization of the two motifs. The purpose is to show which residues are essential for ion coordination by making an alanine scanning mutagenesis. All the mutants have been obtained and their characterization should be carried out with transport assays for Ca2+, Mn2+ and H+. The mutants E108A and K112A have already been tested for the transport of H+.