Rsp5 Ubiquitin ligase-mediated quality control system clears membrane proteins mistargeted to the vacuole membrane.

Maintenance of organelle identity is profoundly dependent on the coordination between correct targeting of proteins and removal of mistargeted and damaged proteins. This task is mediated by organelle-specific protein quality control (QC) systems. In yeast, the endocytosis and QC of most plasma membrane (PM) proteins requires the Rsp5 ubiquitin ligase and ART adaptor network. We show that intracellular adaptors of Rsp5, Ear1, and Ssh4 mediate recognition and vacuolar degradation of PM proteins that escape or bypass PM QC systems. This second tier of surveillance helps to maintain cell integrity upon heat stress and protects from proteotoxicity. To understand the mechanism of the recognition of aberrant PM cargos by Ssh4-Rsp5, we mistarget multiple PM proteins de novo to the vacuolar membrane. We found that Ssh4-Rsp5 can target and ubiquitinate multiple lysines within a restricted distance from the membrane, providing a fail-safe mechanism for a diverse cargo repertoire. The mistargeting or misfolding of PM proteins likely exposes these lysines or shifts them into the "ubiquitination zone" accessible to the Ssh4-Rsp5 complex.