Deciphering Biochemical and Biophysical Properties of UPF0016 Membrane Proteins – A Link to Human Congenital Disorders of Glycosylation - Poster

Stribny, Jiri;Thines, Louise;Deschamps, Antoine;Morsomme, Pierre
(2017) 28th ICYGMB - International Conference on Yeast Genetics and Molecular Biology — Location: Prague, czech Republic (27.August.2017)

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  • Stribny, JiriUCLouvain
    Author
  • Thines, LouiseUCLouvain
    Author
  • Deschamps, AntoineUCLouvain
    Author
  • Morsomme, Pierreorcid-logoUCLouvain
    Author
Abstract
Congenital Disorders of Glycosylation (CDG) comprise a group of rare inborn human diseases caused by defects in protein glycosylation. Recently, a subtype of CDG has been associated with mutations within the human protein TMEM165. This protein belongs to a family of poorly characterized membrane proteins (UPF0016) which is highly conserved through evolution and widely distributed among kingdoms. Recent results indicate that the UPF0016 proteins play a role in calcium, manganese and pH homeostasis. We have shown the S. cerevisiae UPF0016 member, Gdt1p, to be localized at the Golgi membrane, and to act as calcium and manganese transporter. Bioinformatic analysis of the UPF0016 family predicts three structural states formed through evolution: i) single-domain proteins with 3 transmembrane spans (TMD) which form homodimers, ii) single-domain proteins with 3 TMD (encoded by two adjacent genes on the chromosome) which form heterodimers, or iii) two-domains proteins with 6 TMD. To gain better insight into the conformational topologies of the three evolution states we selected seven genes from different UPF0016 subfamilies: TMEM165 (human), Gdt1 (S. cerevisiae), and five prokaryotic members, Ter1a and Ter2b (Trichodesmium erythraeum), which are predicted to form heterodimers, Dma (Desulfovibrio magneticus), which is predicted to form homodimers, and two (hyper)thermophilic genes from the archea Thermococcus gammatolerans and Pyrococcus furiosus. The codon-optimized synthetic genes were expressed in E. coli and L. lactis. By in vivo transport assays using the fluorescent dye Fura-2 we studied the calcium and manganese transport activity. Furthermore, purification of the proteins is being optimized for reconstitution into liposomes and in vitro transport assays. Altogether, a better understanding of the enzymatic activity, physiological role and structural aspects of the UPF0016 members will enable a better comprehension of the causal link between the development of CDG and the presence of mutations in the human gene TMEM165.
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Stribny, J., Thines, L., Deschamps, A., & Morsomme, P. (2017). Deciphering Biochemical and Biophysical Properties of UPF0016 Membrane Proteins – A Link to Human Congenital Disorders of Glycosylation - Poster. 28th ICYGMB - International Conference on Yeast Genetics and Molecular Biology, Prague, czech Republic. https://hdl.handle.net/2078.5/127788