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BleretAndréa_00891700_2021-2022.pdf
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- Gdt1p is a protein localized in the membrane of the cis- and medial Golgi in Saccharomyces cerevisiae. It is believed to act as a Ca2+/H+ antiporter and has also recently been linked to Mn2+ homeostasis. In addition, Gdt1p belongs to the uncharacterized protein family 0016 (UPF0016) and has a human ortholog, TMEM165, the dysfunction of which was linked to a metabolic disease called “Congenital Disorders of Glycosylation”. Recently, an excess of extracellular Mn2+ was discovered to induce Gdt1p degradation in the vacuole. Using different strains that were deficient for the classical degradation pathway genes, only Pep12p and the ESCRT complex were identified as proteins potentially involved in Gdt1p degradation. Despite numerous attempts, no explanation for Gdt1p capture at the Golgi level can be proposed. Therefore, focusing on Gdt1p degradation might lead us to a new pathway which is yet to be characterized. Since the degradation process is a key factor of protein homeostasis, this aspect of Gdt1p research studies is particularly relevant. In order to provide the mediators of Gdt1p degradation, a genetic screen strategy has been implemented. More precisely, my work aimed at designing and validating a genetic screen tool, corresponding to recombinant proteins constituted of Gdt1p and one auxotrophic marker (His3p or Ura3p). Indeed, the latter allows the selective growth of degradation mutants in case of Mn2+ stress, as they are unable to degrade Gdt1p and the fused marker. Eventually, the genes deleted or mutated in these cells can be identified as important genes involved in Gdt1p degradation. To fulfill their final function, the chimeric proteins have been characterized in terms of expression, activity and degradation. Results led to the conclusion that those bearing Ura3p were promising genetic screen tools.