Impact of TMEM165 depletion on the glycosylation and endocytosis of plasma membrane proteins

Details

Supervisors

Morsomme, Pierre ; Ledoux, Benjamin

Faculty

Faculté des bioingénieurs

Degree label

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

Abstract

TME165 is a transmembrane protein localized in the trans-Golgi membrane of mammalian cells. The protein belongs to the uncharacterized protein family 0016 (UPF0016) and appears to be involved in the transport of Ca2+, Mn2+ and/or H+. The discovery of this protein is linked to the finding of a mutation in its gene causing a type II congenital disorder of glycosylation (CDG) on sibling patients. Then, protein glycosylation defects in these patients were rapidly explained by the crucial role of TMEM165 in allowing the entry of Mn2+ into the Golgi, Mn2+ being a required cofactor for enzymes performing the glycosylation process. Furthermore, TMEM165 depletion was shown to significantly decrease migratory property of mammalian cancer cells. Based on these results and findings, including from our laboratory, this master thesis project was designed to explain the mechanistic pathway linking TMEM165 depletion with modulations in cell migratory property. Our current hypothesis is that TMEM165 depletion causes underglycosylated plasma membrane (PM) glycoproteins that are no longer recognised and subsequently correctly internalised, leading to perturbations in cell adhesion and migration. Using siRNA against TMEM165 in HeLa and HEK293 cell lines, four plasma membrane PM proteins were investigated: CD166, L1CAM, Transferrin-receptor and EGF-receptor. Then, by western blotting and loss of surface assays, we were able to show that only the PM glycoproteins that appeared underglycosylated under TMEM165 depletion (CD166 and L1CAM), show a reduced rate of endocytosis. Therefore, our starting hypothesis is still valid and will be tested in the near future.