Supplement: Post-ER proteolysis by K48-ubiquitin-activated proteases

Polyubiquitin chains, linked through K48 or K63 of ubiquitin, target membrane proteins in the secretory system to degradative pathways. Whether these linkage isomers are functionally interchangeable is unclear. Here, we show that for post-endoplasmic reticulum (ER) proteins, K63-linked polyubiquitination induces multivesicular bodies (MVBs) sorting and lysosomal degradation. In contrast, K48-linked polyubiquitination induces shearing from the membrane. Substrates are cleaved by the proteasome and by two ubiquitin-activated proteases: Ddi1, a conserved cytosolic ubiquilin that generates cytosolic fragments, and Rbd2, an intramembrane rhomboid protease that produces lumenal fragments. Rbd2 localizes to Golgi/endosomes but also acts on ubiquitinated substrates at the vacuolar membrane. Ddi1’s catalytic core, the HDD-RVP domain, binds ubiquitin directly and is sufficient for ubiquitin-dependent proteolysis. Its activity is amplified by Ddi1’s auxiliary ubiquitin binding domains: an atypical UBL domain and a UBA domain. These findings demonstrate that polyubiquitin chains linked by different residues encode distinct degradative fates for post-ER proteins, and reveals two proteases that target ubiquitinated integral membrane cargo.