Rot1, an essential yeast protein, is degraded through the ER-associated protein degradation system (ERAD)

S. cerevisiae ROT1 is an essential gene which has been related to cell wall biosynthesis, the actin cytoskeleton and protein folding. Rot1 protein is primarily located at the endoplasmic reticulumnuclear membrane facing the lumen where it is translocated through two internal topogenic elements by an SRP-independent posttranslational mechanism which depends on Sec62 to then be N-glycosylated at the lumen of the ER. Despite Rot1 protein levels are critical for yeast survival, proper cell cycle progression and morphogenesis, Rot1 protein levels has not been investigated so far. Here we carry out an in vivo study to analyse the Rot1 protein levels and show that Rot1 is a short-lived protein and its turnover is mediated by the ubiquitin proteasome system (UPS), dependent on the main degradation pathway located at the ER, the ER-associated degradation system (ERAD). This finding correlates with genetic interactions we previously found and that suggested a link between Rot1 and the ubiquitin-proteasome system. In addition, by using mutant cells components of the ERAD pathway, we demonstrated that Rot1 is degraded through the ubiquitin conjugating enzymes (E2) components of ERAD, Ubc6 and Ubc7, and it seems to require only one of the E3 ubiquitin ligases involved in ERAD, Hrd1 but not Doa1