Mechanisms and signals inducing ubiquitylation and endocytosis of amino acid transporters
One of the main objectives of the lab is to further characterize the role of ubiquitin (Ub) and Ub-binding proteins in endocytosis and sorting into the multivesicular body (MVB) pathway of plasma membrane proteins. For this work we mainly use the yeast Gap1 permease as a model system but we recently initiated a novel research axis extending this study to amino acid transporters in human cells.
The ubiquitylation of Gap1 is mediated by a HECT-type Ub ligase, Rsp5, well conserved in mammals. This Rsp5 enzyme is recruited to Gap1 by two redundant adaptor proteins, Bul1 and Bul2, which are members of the arrestin family. In a recent study, we found that Gap1 ubiquitylation is stimulated by a regulatory cascade stimulated in response to an increase of the internal pool of amino acids. This cascade involves the TORC1 kinase complex and ultimately controls the phosphorylation and ubiquitylation states of the Bul proteins. For instance, when the Bul proteins are phosphorylated, they are bound to and inhibited by the 14-3-3 proteins, but when TORC1 is stimulated, the Buls are dephosphorylated and dissociate from the 14-3-3 proteins, thus allowing them to promote Gap1 ubiquitylation (Merhi and André, 2012).
Our recent work unravelled novel conditions inducing Gap1 ubiquitylation and endocytosis. For instance, the process of substrate transport catalyzed by Gap1 itself stimulates the ubiquitylation of the permease (thus avoiding excess accumulation of substrates) and this modification is also dependent on Rsp5 and the Bul proteins (Ghaddar et al. 2014). Furthermore, stress conditions or inhibition of the TORC1 kinase complex also triggers Gap1 ubiquitylation and endocytosis (Crapeau et al. 2014).
Our previous work showed that Gap1 is modified with a short K63-type Ub chain and that this Ub chain provides a specific signal for sorting into the MVB pathway. When Gap1 is only modified with a single Ub moiety, it is normally internalized by endocytosis but fails to sort into intra-endosomal vesicles and the protein recycles from endosomes to the cell surface. One of current objectives of the lab is to further investigate the exact mechanisms allowing K63-type Ub chains to serve as a signal for sorting proteins into the multivesicular body pathway.
Finally, our lab recently initiated a novel study on the intracellular traffic of a human amino acid transport, LAT-1. This protein belongs to the same superfamily of transporters (named APC) that also includes yeast amino acid permeases. LAT-1 is overproduced in cancer cells where it catalyzes uptake of important amino acids like leucine, which activates the mTORC1 kinase complex promoting cell growth. If the activity of LAT-1 is inhibited, cell growth is thus impaired. Furthermore, LAT-1 is involved in immunosuppression as it also catalyzes uptake of tryptophan (thus starving surrounding T cells for this essential amino acid).