Protein degradation under butanol stress. The gene deletion that resulted in strains with a reduced butanol tolerance are shown in red. After their ubiquitination, membrane proteins are internalized via endocytosis and endocytic vesicles fuse with the membrane of Multivesicular Bodies (MBVs). In the MBVs, proteins are deubiquitinated and sorted into vesicules. When the membrane of the MBVs fuses with the vacuole, releases the vesicles that are degraded by vacuolar hydrolases. Ubiquitinated cytosolic proteins are degraded in the proteasome, producing small peptides and free ubiquitin. Rpn4p induces the expression of proteasome genes including PRE9, and was found to be relevant for butanol tolerance in the evolutionary engineering approach.