Investigation of Gdt1 ubiquitination during its Mn2+ -induced degradation, and the link with its transport activity

Details

Supervisors

Morsomme, Pierre ; Alonso Martin, Celia

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

enDespite extensive knowledge regarding protein degradation pathways, the mechanisms controlling the turnover of Golgi membrane proteins remain largely unknown. Elucidating these processes could provide important insights into the general mechanisms of protein quality control and ubiquitin-dependent degradation pathways. To address this question, this work focused on Gdt1, a Golgi membrane protein from Saccharomyces cerevisiae. Gdt1 is involved in the transport of Ca2+ and Mn2+ into the Golgi apparatus in exchange for H+. Consistent with this role, deletion of GDT1 (gdt1delta) causes yeast cells to become sensitive to Ca2+ and impairs their growth compared to WT cells. Interestingly, studies on the transport function of Gdt1 have revealed that the protein undergoes degradation when cells are exposed to high concentrations of Mn2+, suggesting a direct link between its transport activity and its regulated turnover. Preliminary data from our laboratory further indicate that Gdt1 is ubiquitinated before being transported to the vacuole for degradation. Based on these results, the main objectives of my thesis were to identify the cytosolic lysine residues essential for ubiquitination and to determine the relationship between cation transport activity and Mn2+-induced degradation of Gdt1. To this end, HA- and Myc-tagged versions of lysine mutants and transport-deficient mutants were generated with the aim of co-expressing them to assess whether Mn2+-induced degradation depends on the cation pool within the Golgi apparatus. However, experiments involving the single expression of these tagged constructs revealed significant variability between biological replicates, suggesting that C-terminal tagging could influence the stability of Gdt1. These observations highlight both the experimental challenges associated with studying the degradation of Golgi membrane proteins and the complexity of the mechanisms regulating Gdt1 turnover.