The ubiquitin code of yeast permease trafficking Elsa Lauwers 1 , Zoi Erpapazoglou 2 , Rosine Haguenauer-Tsapis 2 and Bruno Andre ´ 1 1 Physiologie Mole ´ culaire de la Cellule, Institut de Biologie et de Me ´ decine Mole ´ culaires (IBMM), Universite ´ Libre de Bruxelles (ULB), Rue des Pr. Jeener et Brachet 12, 6041 Gosselies, Belgium 2 Institut Jacques Monod, UMR-7592 CNRS, Universite ´ Paris Diderot, 15 rue He ´ le ` ne Brion, 75205 Paris CEDEX 13, France Yeast permeases, that act as transporters for nutrients including amino acids, nucleobases and metals, provide a powerful model system for dissecting the physiologi- cal control of membrane protein trafficking. Modification of these transporters by ubiquitin is known to target them for degradation in the vacuole, the degradation organelle of fungi. Recent studies have uncovered the role of specific adaptors for recruiting the Rsp5 ubiquitin ligase to these proteins. In addition, the role of ubiquitin at different trafficking steps including early endocytosis, sorting into the multivesicular body (MVB) pathway and Golgi-to-endosome transit is now becoming clear. In particular, K63-linked ubiquitin chains now emerge as a specific signal for protein sorting into the MVB path- way. A complete view of the ubiquitin code governing yeast permease trafficking might not be far off. Ubiquitin in permease regulation Transmembrane transport proteins play a crucial role in all cells – from unicellular organisms to mammals – by conferring to plasma and internal membranes selective permeability to a wide range of ions and small molecules. These transport proteins are very often subject to tight regulation allowing cells to adapt to a changing environ- ment. This is particularly well illustrated in the yeast Saccharomyces cerevisiae in which close to a hundred plasma-membrane nutrient-permeases have been ident- ified. The intracellular trafficking of these proteins is typically controlled according to substrate availability, general nutrient supply conditions and/or stresses. Strik- ingly, the mechanisms controlling permease trafficking are essentially conserved from fungi to mammals. In particu- lar, studies on the intracellular trafficking of yeast per- meases have contributed to revealing the central role played by the small protein ubiquitin (Ub, Box 1) as a sorting signal of eukaryotic membrane proteins [1]. Covalent attachment of Ub is the only known signal promoting the internalization of yeast nutrient-permeases from the plasma membrane. These permeases are typically ubiquitylated in response to substrate excess or more global changes of nutrient supply conditions [1]. In all cases, this modification depends on Rsp5, a Ub ligase of the Nedd4 HECT family [2].(Table 1) Human proteins of this Ub ligase family also mediate down-regulation of various channels, transporters and receptors [2]. In yeast, ubiquitylated permeases are proposed to be recognized by epsin/Eps15-like adaptors [3] harboring one or several Ub- binding domains (UBDs) [4] (Figures 1 and 2). This inter- action would mediate sorting of the cargoes into invaginat- ing endocytic vesicles, as demonstrated in mammalian cells [5–7]. When they reach the limiting membrane of the late endosome, ubiquitylated cargoes are sorted into vesicles that bud into the lumen of this compartment as it matures into a multivesicular body (MVB). This MVB sorting is essential to subsequent delivery of the protein into the vacuole/lysosome lumen upon fusion of the MVB with the limiting membrane of this compartment. Per- mease sorting into the MVB pathway (Box 2) requires the presence of Ub attached to the cargo and is mediated by the ESCRT (endosomal sorting complex required for transport) machinery that is composed of five multi-sub- unit complexes named ESCRT-0, -I, -II, -III and Vps4-Vta1 [8,9]. At least one component of each of the ESCRT-0, -I and -II complexes possess a UBD (Figures 1 and 2), and these proteins are proposed to function cooperatively to sort ubiquitylated cargoes into the MVB vesicles [10,11]. Rsp5-mediated ubiquitylation is also involved in the direct sorting of newly synthesized permeases from the trans- Golgi network (TGN) to the vacuolar lumen (without pas- sing by the plasma membrane) via the MVB pathway. This direct sorting typically occurs under the same physiological conditions that trigger downregulation of these cargoes from the plasma membrane [1]. Despite the prevailing role of Rsp5 in ubiquitylation of yeast plasma-membrane transporters, so far there is no evidence for a direct interaction between the Ub ligase and these cargoes. Instead, Rsp5 is recruited to transporters via specific adaptors. The list of specific Rsp5 adaptors for nutrient permeases, some of which are related to mamma- lian arrestins, has recently expanded. Moreover, recent data from several groups has shed light on the nature of the Ub signal required at different trafficking steps. Here we review these latest advances towards elucidating the ‘Ub code’ of permease trafficking in the yeast S. cerevisiae. Ubiquitin as an endocytic signal The first link between Ub and membrane trafficking was made 15 years ago when Ko ¨lling and Hollenberg observed that ubiquitylated forms of the yeast pheromone transporter Ste6 accumulate at the plasma membrane in mutants affecting endocytosis [12]. Furthermore, mutations reducing Ste6-ubiquitylation impaired vacuolar delivery of this protein, suggesting that ubiquitylation of the cargo not only precedes but is also necessary for its endocytic Review Corresponding authors: Haguenauer-Tsapis, R. (haguenauer@ijm.univ-paris-diderot.fr); Andre ´, B. (bran@ulb.ac.be) 196 0962-8924/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2010.01.004