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Impact of induced Gdt1 degradation on Golgi cationic content in Saccharomyces cerevisiae

Scheen, Arthur
(2025)

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Scheen_56671900_2025.pdf
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Details

Supervisors
Morsomme, Pierre ; Lejeune, Quentin ; Opfergelt, Louis
Faculty
Faculté des sciences
Degree label
Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie
Abstract
The GDT1 family comprises highly conserved transporters found across species, with particular interest in its human orthologue, TMEM165. Mutations in TMEM165 are associated with congenital disorders of glycosylation (CDGs), a group of rare metabolic disorders caused by defects in glycosylation processes. The yeast orthologue, Gdt1, is a Golgi-localised antiporter that mediates Ca2+/Mn2+ and H+ exchange. While Gdt1 is not essential for maintaining steady state Golgi H+ homeostasis, it plays a role during dynamic cellular conditions. This study aimed to investigate the impact of sudden Gdt1 degradation on Golgi cation homeostasis during these conditions, leaving cells no time to adapt. To address this, the auxin-inducible degron 2 (AID2) system was employed to conditionally and selectively degrade Gdt1. Western blot analyses confirmed the efficiency of the AID2 system in achieving rapid and specific Gdt1 degradation, which was subsequently optimised for in vivo applications. Using the Golgi-localised Ca2+-sensitive GoD4cpV probe, time-course fluorometric measurements revealed disruptions in Golgi Ca2+ homeostasis following Gdt1 degradation under calcium-induced stress. In contrast, preliminary measurements using the Mn2+-sensitive GoMnLaMP2 probe during manganese-induced stress showed no detectable disruptions. However, further replicates are required to validate these results. This study demonstrates the utility of the AID2 system as a powerful tool for the rapid and selective degradation of Golgi membrane proteins, enabling the investigation of dynamic cellular processes. Furthermore, it highlights the crucial role of Gdt1 in maintaining Golgi cation homeostasis during dynamic conditions, offering valuable insights into its broader physiological significance.