Signals and mechanisms controlling the ubiquitylation and down-regulation of the yeast general amino acid permease

Cell surface transport proteins play a crucial role in all cells, from unicellular organisms to mammals, by conferring to the plasma membrane selective permeability to a wide range of ions and small molecules. The activity of these proteins is very often regulated by controlling their amount at the plasma membrane where they are removed by means of selective endocytosis in response to signals and changes in the environment.One of the membrane proteins of the yeast Saccharomyces cerevisiae whose regulation has been extensively studied is the general amino acid permease. Previous studies on Gap1 and other yeast permeases revealed that ubiquitin plays a key role in the membrane trafficking of these proteins by providing a signal that triggers their internalization in endocytic vesicles and that promote their sorting into intra-endosomal vesicles for subsequent delivery into the lumen of the vacuole, the lysosome of yeast. In the first part of this work, we report the isolation of 64 mutant forms of the Gap1 protein and their exploitation in a systematic functional study of the predicted intracellular regions of the permease. The phenotypic analysis of these mutants revealed an important role of certain amino acid sequences in the (i) transport of the permease through the secretory pathway (ii) intrinsic activity of the permease at the plasma membrane (iii) stability of the protein at the cell surface (iv) sorting of the protein into intra-endosomal vesicles. Further investigation of some of these mutants allowed us to unravel an original mechanism for the degradation of the permease that is independent of its ubiquitylation.In the second part of the work, we used yet other Gap1 mutants to study the signals and pathways inducing the ubiquitylation and endocytosis of the permease. Also, we further investigated the molecular mechanisms inducing Gap1 ubiquitylation. All these results together allow us to better understand the mechanisms controlling the ubiquitin dependent down-regulation of plasma membrane proteins.Doctorat en Sciences