Since the concept of ubiquitin-dependent membrane traffic was born in the mid-nineties, substantial progress has been made in understanding the molecular details of how cargo proteins are ubiquitinated by E3 ubiquitin ligases, how sorting components such as GGAs and ESCRT subunits recognize the ubiquitinated cargoes, how ubiquitin recognition controls the intracellular itineraries of membrane proteins, and how DUBs are recruited to remove ubiquitin moieties. Even though sufficient data are available to allow a broad overview of ubiquitin as a tag for protein trafficking, several outstanding questions remain to be addressed before such processes can be modelled in detail.
One of the topics that awaits further clarification is the importance of Lys63-linked polyubiquitin chains as a sorting signal. In mammalian systems there is evidence that Lys63-linked polyubiquitin via the E2s Ube2D1-4 and the E3 Cbl is crucial for endolysosomal sorting of EGFRs (117
), and that Lys63-linked polyubiquitination via the E2 Ubc13 and the E3 K3 is required for Kaposi's sarcoma associated Herpes virus-induced endocytosis and endolysosomal sorting of MHC class I molecules (19
). Likewise, Lys63-linked polyubiquitination appears to be required for vacuolar targeting of the yeast amino acid permease Gap1 (61
). On the other hand, there is convincing evidence that monoubiquitin is a sufficient signal for endocytosis of several yeast membrane proteins such as Gap1, the maltose transporter Mal1, and the mating factor receptor Ste2 (61
). Moreover, Lys63-linked diubiquitination appears sufficient to target Cps1 to the vacuole (61
). It will be important to establish whether the differential requirements for Lys63 polyubiquitination may be related to the types of sorting components that are involved. A few measurements have been made of the affinities and avidities of sorting machinery for ubiquitin chains (59
), but more data are needed, particularly in the context of cargo and membranes.
The cargoes described in this review were selected because they have been so well studied compared to most others. Examples such as the specific recognition of phosphorylated EGFR by the TKB domain of Cbl (79
) beautifully illustrate the specific targeting and regulation of the ubiquitination machinery. Such clear cut examples are still more the exception than the rule. Chain specificity remains just as poorly characterized in most cases, with a few spectacular exceptions such as the mechanism of Lys63-ubiquitin specific cleavage by AMSH (100
). More insights into these mechanisms will be eagerly awaited.
Ubiquitin signals and ubiquitin receptors operate at multiple sequential steps in trafficking. It remains to be understood how ubiquitinated cargoes are handed off at each step. Simple models, such as the possibility of a gradient of sorting complex UBD ubiquitin affinities that follows the directionality of sorting, do not seem sufficient to account for cargo hand-off. In this connection, it is intriguing that at certain steps in the pathway, ubiquitin ligases, DUBs, and ubiquitin-binding proteins appear to form assemblies with one another. For example, the yeast ESCRTs interact, directly or indirectly, with both the ligase Rsp5 (10
) and the DUB Doa4 (3
). This raises a host of possibilities for ubiquitin chain remodeling and compartmentalized, complete ubiquitination/deubiquitination cycles that have barely begun to be explored. The molecular gymnastics of the ubiquitin system in membrane trafficking should continue to offer insights and surprises for years to come.