K48-ubiquitin-dependent proteases cut-up post-ER proteins.

Abstract

Polyubiquitin chains, linked via K48 or K63 of ubiquitin, direct membrane proteins in the secretory system to distinct degradative fates. However, it's unclear whether these linkage isomers are functionally interchangeable. Here we show that for post-endoplasmic reticulum (ER) proteins, K63-linked polyubiquitination induces sorting into multivesicular bodies (MVBs) and lysosomal degradation. In contrast, K48-linked polyubiquitination induces shearing from the membrane and proteasomal degradation. This process involves two ubiquitin-dependent proteases: Ddi1, a conserved cytosolic ubiquilin that generates fragments from soluble and membrane proteins, and Rbd2, an intramembrane rhomboid protease that produces lumenal fragments from membrane proteins at Golgi/endosomes and the vacuolar membrane. Ddi1's catalytic core, the HDD-RVP domain, is sufficient for ubiquitin-dependent proteolysis. It binds ubiquitin directly and its activity is enhanced by 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 reveal two proteases that target ubiquitinated integral membrane proteins in a process we call CUT-UP (Cleavage of Ubiquitinated Targets by Ubiquitin-dependent Proteases).