Description |
Protein homeostasis refers to the cellular processes that regulate the folding, unfolding, misfolding, trafficking and degradation of proteins. These cellular processes include the protein expression pathway, adaptive profolding mechanisms and protective degradative pathways. Plasma membrane proteins are located at the interface between the internal and external environment of the cell. They are essential for cellular integrity. Molecules that cannot diffuse across the plasma membrane have to pass through transport proteins that span the length of the plasma membrane. Damage to these transporters can cause unrestricted entry and exit of molecules, an outcome that eventually leads to cellular demise. The levels of these transporters are hence closely regulated by the cell through evolutionarily conserved mechanisms. In the event of an insult to the transporter, the profolding pathways are initially activated. If these fail, the protein is downregulated through the degradative quality control machinery in order to prevent aggregation and persistent defect in function. In Saccharomyces cerevisiae, the downregulation of the uracil transporter Fur4 is mediated through ubiquitin-dependent endocytosis and trafficking to the vacuole for degradation. The ubiquitin ligase Rsp5 is responsible for identifying an unfolded Fur4 and catalyzes its ubiquitination at lysine residues. It remains an open question how the soluble Rsp5 is recruited to the plasma membrane. It is also not clear how lipid microdomains impact this ubiquitination event. Previous studies have described the LID-degron system as a mechanism involved in Fur4 downregulation. Utilizing the LOV2 photosystem and artificial degrons, I have separated conformational changes within the transporter from degron exposure. This has allowed us to independently expose the degron and monitor the dynamics of Rsp5 recruitment. Our results suggest that a transmembrane adaptor is involved in Rsp5 recruitment and that plasma membrane microdomain dynamics ensure that ubiquitination occurs after lateral translocation. |