K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway

A growing number of yeast and mammalian plasma membrane proteins are reported to be modified with K63-linked ubiquitin (Ub) chains. However, the relative importance of this modification versus mono-ubiquitylation in endocytosis, Golgi to endosome traffic, and sorting into the multivesicular body (MVB) pathway remains unclear. In this study, we show that K63-linked ubiquitylation of the Gap1 permease is essential for its entry into the MVB pathway. Carboxypeptidase S also requires modification with a K63-Ub chain for correct MVB sorting. In contrast, monoubiquitylation of a single target lysine of Gap1 is a sufficient signal for its internalization from the cell surface, and Golgi to endosome transport of the permease requires neither its ubiquitylation nor the Ub-binding GAT (Gga and Tom 1) domain of Gga (Golgi localizing, gamma-ear containing, ARF binding) adapter proteins, the latter being crucial for subsequent MVB sorting of the permease. Our data reveal that K63-linked Ub chains act as a specific signal for MVB sorting, providing further insight into the Ub code of membrane protein trafficking.