Discovery of Ubiquitin Based Sorting

Ubiquitin is a 76-amino acid polypeptide virtually invariant in sequence throughout eukaryotes. The carboxy-terminal residue, 76Gly, can be conjugated by an isopeptide linkage to lysine side chains on target proteins. Conjugation is the result of a sequential series of reactions involving the generation and transfer of a thioester-bound ubiquitin intermediate by the E1, E2, and E3 conjugation machinery proteins (165). Several endogenous lysines (e.g., 29Lys, 48Lys, 63Lys) within the protein-linked ubiquitin molecule can, subsequently, be self-conjugated to additional ubiquitin molecules, generating polyubiquitin chains. Post-translational ubiquitination is at the core of regulated intracellular protein turnover, and 48Lys-linked polyubiquitin chains of tetraubiquitin or greater serve as a signal for protein degradation via the 26S proteasome (166). Appreciation that ubiquitin also functions as an authentic trafficking signal came from the discovery that, in S. cerevisiae, internalization of the G protein-coupled a-factor

(Ste2p) and a-factor (Ste3p) pheromone receptors is accompanied by ubiquiti-nation of these proteins (167, 168). Genetic background mutations that prevent endocytosis cause ubiquitinated species to accumulate, whereas compromising the cellular ubiquitination machinery retards surface uptake and prolongs receptor half-life (167, 168). It was quickly established that a multitude of yeast plasma membrane receptors and permeases/transporters (e.g., Ste6p, Gal2p, Gap1p, Fur4p, Pdr5p, Zrt1p,) use ubiquitination as an endocytic signal (169 -171).

Apparently the major endocytic signal in budding yeast, in higher organisms ubiquitin addition also regulates the internalization of certain transmembrane proteins. One of the first indications came from studies on the growth hormone (GH) receptor in the CHO-ts20 cell line that exhibits a temperature-sensitive E1 ubiquitin-activating enzyme. At the nonpermissive temperature, no uptake or lysosomal degradation of transfected GH receptor occurs (172). Mutation of 327Phe in the cytosolic domain of the receptor blocks endocytosis and also inhibits receptor ubiquitination (173). Specific inhibitors of the proteasome, which cause depletion of free ubiquitin within the cell, abrogate ligand-stimu-lated destruction of the GH receptor, but transferrin receptor endocytosis is unaffected (174, 175). Also, blocking endocytosis by cyclodextran-mediated cholesterol depletion, or with a dominant-negative dynamin mutant, causes ubiquitinated GH receptor to accumulate on the plasma membrane (176). These experiments firmly establish that internalization of the GH receptor requires the ubiquitin-conjugation machinery and that the receptor is ubiquitinated directly.

There is now good evidence for ubiquitination regulating the internalization of the EGF (177-179), MET (180), and CSF-1 (181, 182) receptors, the epithelial sodium channel (ENaC) (171), the aggregated IgG-bound Fc receptor FcRyIIA (183), and the transmembrane Notch ligand Delta (184-186). The a1 subunit of the glycine receptor is modified with one to three ubiquitin molecules at the plasma membrane as a prelude to uptake (187), and ubiquitination of E-cadherin precedes internalization (188). There is also evidence that endocytosis of the pre-T cell receptor in thymocytes is dependent upon ubiquitination (189). In C. elegans, targeted neural overexpression of ubiquitin reduces the surface density of glutamate receptors (190) in a manner that is dependent on clathrin-mediated endocytosis, as mutant unc-11 (AP180) counteracts the effect of the excess ubiquitin. Direct ubiquitination of the glutamate receptor can be demonstrated biochemically (190). Final corroboration of the role of ubiquitination in endocytic mobilization comes from the demonstration that the Karposi's sarcoma-associated herpes virus protein K3 can route surface MHC class I for lysosomal degradation by possessing inherent E3 ubiquitin ligase activity that ubiquitinates a single MHC class I lysine (191, 192).

Multiple Sorting Steps Involving Ubiquitin

Ubiquitination has now been found to effect protein sorting at other intracellular stations as well. Ubiquitin conjugation is a cyclical process; because ubiquitin is a long-lived protein, the bulk of activated ubiquitin generated by the E1 enzyme comes from ubiquitin recovered by deubiquitinating enzymes (193). Inactivation of one deubiquitinating enzyme in yeast, Doa4p, results in pleiomorphic effects, including defects in surface uptake (194 -197). Bypass suppressors of a doa4 mutation turn out to be six class E vps (vacuolar protein sorting) mutants (194). Class E vps mutants are defective in multivesicular body formation, and as these suppressors essentially negate the role of Doa4p, the data indicate that the late endosome/prevacuolar compartment is a major sink for ubiquitin (198). However, even though overexpression of ubiquitin overcomes the doa4 phenotype (195, 199), this does not indicate that Doa4p simply retrieves and recycles ubiquitin from tagged proteins only originating from the cell surface (198). Biosynthetic delivery of newly synthesized vacuolar proteins carboxypeptidase S (CPS) and the polyphosphatase Phm5p into the vacuolar lumen is also ubiquitin dependent (196, 200). These proteins are ubiquitinated directly; a single major lysine acceptor has been mapped in both Phm5p (196) and CPS (200), but other potential acceptors are also utilized in Phm5p (196). Biosynthetic ubiquitination of CPS occurs post Golgi but prior to delivery to late endosomes (200). If ubiquitination of CPS is blocked, the unprocessed form accumulates on the limiting membrane of the vacuole instead of inside (200). The same is true for Phm5p (196). In-frame addition of either a single unextendable ubiquitin (196, 201) or a ubiquitination-directing sequence (200) routes into the vacuole lumen a variety of proteins that normally traffic through the late endosome/prevacuolar compartment without entering intralumenal structures. This does not involve passage through the plasma membrane, showing that ubiquitin-directed sorting of proteins not originating from cell surface certainly occurs (Figure 6).

In mammalian cells, signaling receptors, like the EGF receptor, move into intralumenal membranes of multivesicular endosomes following ligand stimulation and internalization (202). TGFa, an alternate ligand for the EGF receptor, does not induce this translocation, nor substantial receptor degradation, because the ligand dissociates from the receptor within early endosomes (203, 204). TGFa also does not cause protracted ubiquitination of the EGF receptor (204). Proteasome inhibitors prevent both the sequestration of EGF-activated receptors into the interior of multivesicular bodies (204) and degradation of internalized receptors (204, 205). Also, lysosomal turnover of the HIV coreceptor CXCR4 requires a degradation motif (206). Mutation of the three lysine residues within this motif abolishes degradation of the G protein- coupled receptor while monou-biquitination of the receptor correlates with lysosomal delivery (206). Interestingly, proteasome inhibitors selectively disrupt delivery of membrane proteins into the lysosome; soluble protein traffic is normal (175, 204). Together, these studies reveal that transmembrane protein relocation into the interior elements of the multivesicular late endosome is a regulated step in lysosomal delivery that is under the control of ubiquitin.

Sorting decisions are, of course, made before reaching the late endosomal compartment. Minutes after endocytosis, EGF and GH receptors are actively

Animal

Plasma membrane

Ste2p Ste3p Ste6p Gal2p

Rsp5p

Prevacuolar compartment

Late Golgi

Vps27p Hselp

Entlp Ent2p Edelp

Vps23p Vps28p Vps37p (ESCRT-1

Rsp5p

Plasma membrane

Vps23p Vps28p Vps37p (ESCRT-1

Prevacuolar compartment

Late Golgi

Vps27p Hselp

Bul1/2p

Gaplp Fur4p Tat2p Put4p

Entlp Ent2p Edelp

Ste2p Ste3p Ste6p Gal2p

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