Implementation of a screening strategy to decipher Gdt1p degradation in yeast

Details

Supervisors

Morsomme ; Deschamps

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

The Uncharacterized Protein Family 0016 (UPF0016) is the name given to a group of poorly studied membrane proteins well-conserved during evolution. Recent research demonstrated that members of this family are involved in calcium, manganese and, potentially, pH homeostasis. The Golgi-localized human protein of the family, TMEM165, was recently discovered to be involved in a type of Congenital Disorders of Glycosylation (CDG). Indeed, TMEM165 deficiency causes Golgi glycosylation failings in human cells leading to a wide variety of clinical phenotypes. The Saccharomyces cerevisiae ortholog is called Gdt1p. Also located in the Golgi apparatus membrane, it has been widely studied to decipher molecular causes of TMEM165-related CDG. Both orthologs were found to be highly sensitive to manganese excess and to be rapidly degraded in these conditions. However, regulation and degradation mechanisms of these calcium and manganese transporters remain unknown to date. As the classical paths of Gdt1p transport from the Golgi apparatus to the vacuole do not seem to be involved, it is likely that a new pathway, unknown or uncharacterized to date, could be implicated in the degradation of Gdt1p. In this project, a screening strategy was designed, developed and tested with the aim of shedding light on the actors involved in Gdt1p degradation. It consists in the fusion of the pH-sensitive GFP, pHluorin, to Gdt1p. Since the pHluorin emits a different signal depending on the pH environment, the purpose is to determine through pHluorin fluorescence whether the chimeric protein is retained in the Golgi apparatus or located in the vacuole for degradation, and therefore identifying mutants in which Gdt1p degradation no longer occurs after application of manganese stress. In this master’s thesis, the different steps of this method development are presented, from the transgene construction to the characterization of the recombinant protein (function, localization, degradation and fluorescent signal).