Arrestin-related proteins mediate Plasma Membrane Protein Quality Maintenance in Yeast

Down-regulation of cell surface receptors and transporters is mediated by a series of membrane trafficking steps including ubiquitin-mediated endocytosis, ESCRT-mediated sorting and packaging into vesicles that bud into the lumen of the endosome, and endosome-lysosome fusion that delivers sorted receptors into the lumen of the lysosome where degradation occurs. Failure to attenuate plasma membrane (PM) signaling by growth-factor receptors by endocytic down-regulation can lead to cancer.

In Saccharomyces cerevisiae, ubiquitination mediated by Rsp5, the yeast Nedd4 homolog, is known to be required for the endocytic down-regulation of numerous PM proteins. Properly folded PM proteins, such as signaling receptors, ion channels, and nutrient transporters, exit the ER and traffic through the Golgi to the cell surface where they mediate their specific functions. Maintenance of proper PM proteostasis, particularly with respect to ion channels and nutrient transporters, is crucial to prevent dissipation of chemical gradients and loss of PM integrity. As such, when PM resident proteins become damaged or misfolded, they must be recognized, removed by endocytosis and delivered to the lysosome for degradation. Thus, integral membrane proteins are subject to various layers of quality surveillance, some of which are still poorly understood. Previously, we identified and characterized a family of arrestin-related trafficking adaptors, or ARTs, which recruit the Rsp5 ubiquitin ligase to specific targets at the PM. Research from several groups has demonstrated that ART proteins are key determinants of target selection during ubiquitin-mediated endocytosis. Yet despite the emerging consensus that ARTs function as modular specificity adaptors for Rsp5, we know very little about how the ART proteins recognize cargo or how they are regulated.

Here, we will present evidence that the E3 ubiquitin ligase Rsp5, the yeast homolog of Nedd4, is a key mediator of protein quality control at the PM. In addition, we will show that Art1 undergoes a phosphoregulatory cycle and that dephosphorylation of Art1 triggers its activation and recruitment to PM cargo. Using genetics and biochemistry, we show that this phosphoregulatory cycle is mediated by a TORC1-Npr1 kinase signaling cascade.