Study of the molecular basis of congenital disorders of glycosylation using yeast as a model organism

Treball de fi de grau en Biologia Humana Director: Oriol Gallego Moli Tutor acadèmic: J. Miguel López-Botet Arbona Congenital Disorders of Glycosylation (CDG) are a group of inherited human disorders caused by defects on protein glycosylation. Rft1-CDG is a form of CDG caused by mutations in the gene RFT1. Rft1 is a conserved protein essential for the N-linked glycosylation pathway in the ER. The subcellular localization of Rft1 has not been explored experimentally, although it is crucial for Rft1 function and protein glycosylation. The purpose of this research is to characterize the subcellular distribution of Rft1 and to investigate the molecular defects caused by Rft1-CDG mutations using S. Cerevisiae as a model organism. To answer this question, we investigated the colocalization between GFP tagged Rft1 and several mCherry tagged subcellular structures using fluorescence microscopy. We found that wild-type GFP-Rft1 localizes in the Endoplasmic Reticulum (ER), the Golgi Apparatus, endosomes and mitochondria. The Rft1-CDG mutations introduced to the GFP-Rft1 strain were R179E, M446V, I340K, G320D, I340R and R75C. Results show that in Rft1-R179E mutant yeast strain, the localization of Rft1 within the cell remains similar to the wild-type. However, the intensity distribution of the GFP-Rft1 fluorescent signal in mutants Rft1-R179E and Rft1-I340K is altered and the duplication time of these particular mutants and Rft1-G320D mutant is increased. Our results provide, for the first time, observations of the impact of Rft1-CDG mutations that can contribute identifying the molecular basis of human CDG.