Jcb_200810114 493..502

Ubiquitin (Ub) is a highly conserved protein composed of 76 aa that can be covalently linked to a lysine residue of a target protein. Attachment of a single Ub monomer to one or several lysines of a protein (monoor multimonoubiquitylation, respectively) is notably involved in endocytosis and the regulation of histones (Hicke, 2001). Ub itself possesses several lysines that can be used for the attachment of another Ub molecule, allowing substrates to be modified with different types of Ub chains (Peng et al., 2003; Pickart and Fushman, 2004). The consequences of this polyubiquitylation appear to depend on the length of the Ub chain and on the type of linkage used. The most abundant Ub chains in living cells are K48linked chains, which constitute a signal for protein degradation by the 26-S proteasome (Thrower et al., 2000), and K63-linked chains, which function in a variety of cellular processes, including DNA repair (Spence et al., 1995), stress responses (Arnason and Ellison, 1994), signal transduction (Sun and Chen, 2004; Mukhopadhyay and Riezman, 2007), and intracellular trafficking of membrane proteins (Hicke, 1999; Mukhopadhyay and Riezman, 2007). Among the protein cargoes of Saccharomyces cerevisiae used to investigate the roles of Ub and Ub chains in membrane trafficking is the general amino acid permease Gap1 (Haguenauer-Tsapis and André, 2004). Gap1 is highly active and stable at the plasma membrane in cells growing on poor nitrogen sources like proline. Upon addition of a favored nitrogen source like ammonium, it is ubiquitylated on its amino-terminal lysines 9 and 16 by the HECT (homologous to E6-associated protein carboxy terminus)-type Rsp5/Npi1 Ub ligase (Soetens et al., 2001), thus causing its internalization (Springael and André, 1998). After reaching the late endosome, Gap1 is sorted into vesicles that bud into the lumen of this compartment as it matures into a multivesicular body (MVB). Upon fusion of the MVB with the vacuole, internal vesicles are released in the vacuolar lumen where the permease is ultimately degraded (Nikko et al., 2003). Both K9 and K16 of Gap1 are modified with K63linked Ub chains (Springael et al., 1999). Although monoubiquitylation is sufficient to trigger endocytic internalization of Gap1, K63-linked ubiquitylation appears necessary for this process to occur at a maximal rate (Springael et al., 1999). The control by nitrogen of Gap1 trafficking also affects the newly synthesized protein in the secretory pathway. On poor nitrogen 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 monoubiquitylation 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 Tom1) domain of Gga (Golgi localizing, gammaear 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. K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway