The yeast protein Gdt1 is a newly-identified actor in manganese homeostasis at the Golgi
(2019)
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- Supervisors
- Morsomme, Pierre
- Abstract
- Congenital disorders of glycosylation (CDGs) encompass a broad range of human hereditary diseases characterized by deficiencies in the glycosylation process. In 2012, cases of CDGs were linked to the presence of specific mutations within the human gene TMEM165. The corresponding membrane protein belongs to the uncharacterized protein family 0016 (UPF0016) whose members were, at that time, only poorly characterized and their function, unknown. The project presented in this manuscript aims at studying the Saccharomyces cerevisiae UPF0016 member Gdt1p as a model protein of the family. Previous experiments showed that Gdt1p localizes at the Golgi membrane and is involved both in calcium and pH homeostasis. Transport of calcium cations was further demonstrated by producing Gdt1p in Lactococcus lactis and using the fluorescent Ca2+-sensitive probe Fura-2 in vivo in this bacterial host. More recently, plant and bacterial UPF0016 members were suggested to play a role in Mn2+ homeostasis, but without providing any direct proof of transport. In this context, this work mainly investigated whether the yeast Gdt1p transports manganese cations. To do so, we first took advantage of the fact that manganese quenches the fluorescence emitted by Fura-2 to develop a Mn2+ transport assay in Gdt1p-producing L. lactis cells. The use of this methodology enabled us to highlight Mn2+ transport activity by Gdt1p, in addition to its previously reported Ca2+ transport ability, with an affinity for Ca2+ five-fold higher than for Mn2+. In parallel to these approaches in bacteria, we found in yeast that Gdt1p is involved in the resistance to high Mn2+ concentration, controls the cellular Mn2+ stores, and modulates the activity of the Mn2+-dependent enzyme Sod2p. Similar approaches in yeast and bacteria enabled to highlight additional putative Cd2+ transport by Gdt1p. Besides, the same techniques were also used to demonstrate that the acidic and polar uncharged residues of conserved motifs of the family are essential for proper transport. Investigation of the mechanism and direction of transport further led us to assume that Gdt1p would work as a Ca2+-Mn2+/H+ antiporter, importing both divalent cations in the Golgi lumen in exchange of protons. Taken together, we obtained first evidence that Gdt1p from yeast directly transports manganese, which strongly reinforces the suggested link between the UPF0016 family and Mn2+ homeostasis, and provides new insights into the molecular causes of human TMEM165-associated CDGs. Our results also shed light on how yeast may regulate its Golgi intraluminal concentration of manganese, a key cofactor of many enzymes involved in protein glycosylation.
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Citations
Thines, L. (2019). The yeast protein Gdt1 is a newly-identified actor in manganese homeostasis at the Golgi. https://hdl.handle.net/2078.5/123993