Protein Degradation or Regulation: Ub the Judge

The experimental setting for the work of Hicke and Riezman 1996xHicke, L. and Riezman, H. Cell. 1996; 84: 277–287Abstract | Full Text | Full Text PDF | PubMed | Scopus (594)See all ReferencesHicke and Riezman 1996 is very different from that of Chen et al. 1996xChen, Z.I., Parent, L., and Maniatis, T. Cell. 1996; 84See all ReferencesChen et al. 1996, but the inference of a nonproteolytic role for ubiquitin is equally compelling. Many cell surface proteins are rapidly endocytosed and, if not recycled to the plasma membrane, are routed to the lysosome/vacuole, where they are destroyed by vacuolar proteases. Endocytosis of the S. cerevisiae α-factor pheromone receptor, Ste2, takes place constitutively at a slow rate but is stimulated 5-10 fold by α-factor binding. Pheromone binding to the G protein-coupled Ste2 receptor activates the MAP kinase signal transduction pathway, resulting ultimately in alterations in transcription needed by the cell to mate with α-factor secreting cells. Internalized Ste2 follows the endocytic pathway to the vacuole, where it is degraded. For endocytosis, the cytoplasmic C-terminal tail of the polytopic Ste2 protein is critical; within this tail, a nine-residue sequence, SINNDAKSS, is sufficient for receptor internalization (seeHicke and Riezman 1996xHicke, L. and Riezman, H. Cell. 1996; 84: 277–287Abstract | Full Text | Full Text PDF | PubMed | Scopus (594)See all ReferencesHicke and Riezman 1996, for references).In their new work, Hicke and Riezman have examined more closely the requirements for receptor endocytosis. As shown previously, exposure of cells to α-factor led to rapid disappearance of the receptor. Degradation was strongly inhibited in a yeast end4 mutant, which is defective at an early step in receptor endocytosis. Based on SDS-polyacrylamide gel separations, however, Ste2 did not accumulate in its normal form in the mutant cells but rather, as a set of higher molecular mass species, the most prominent of which differed in apparent molecular mass by intervals of ∼9 kD. The higher molecular mass forms of Ste2 are also detectable as transient intermediates in pheromone-stimulated wild-type cells. These results immediately suggested that Ste2 might be modified by ubiquitin, a suspicion confirmed by immunoprecipitation with an antibody to ubiquitin. Furthermore, internalization of ligand-bound receptors was markedly inhibited in yeast cells lacking members of the Ubc1/Ubc4/Ubc5 subfamily of E2 enzymes. Ubiquitination of Ste2 is correspondingly reduced in ubc4 ubc5 mutants. Some of these mutated strains are quite sick, but the defect in Ste2 endocytosis could not be explained by a general defect in membrane trafficking.All this is well and good, but it does not prove ubiquitination of Ste2 is what triggers its endocytosis. For that matter, a very similar story has already been told about yeast Ste6, an ATP-binding cassette transporter protein involved in a-factor export (Kolling and Hollenberg 1994xKolling, R. and Hollenberg, C.P. EMBO J. 1994; 13: 3261–3271PubMedSee all ReferencesKolling and Hollenberg 1994). However, Hicke and Riezman went on to analyze the effect of mutations in the SINNDAKSS internalization signal on Ste2 ubiquitination, and the results were telling. Mutation of the lysine in this element to arginine (in a truncated derivative of Ste2) almost completely blocked endocytosis. The mutation also eliminated Ste2 ubiquitination. There is one other lysine in the cytoplasmic tail of this derivative of Ste2. Changing it to arginine was of no consequence for either receptor internalization or ubiquitination. Finally, mutation to alanine of all three serines in the endocytosis signal eliminated receptor hyperphosphorylation in response to α-factor and inhibited receptor internalization. Correspondingly, ubiquitination of Ste2 was no longer detectable. The simplest interpretation of these data is that endocytosis of Ste2 specifically requires ubiquitination of the lysine in the SINNDAKSS element and that this modification depends on phosphorylation of the flanking serines. Phosphorylation-dependent ubiquitination has been reported for a number of proteins in the last few years, one of the best characterized examples being IκBα, as discussed above.The Ste2 results raise some interesting questions. For one, how does ubiquitination of Ste2 lead to its endocytosis? The ubiquitin (chain?) may provide a binding site for a component of the endocytic machinery or may alter Ste2 structure in a way that facilitates such binding. As an alternative, the authors suggest ubiquitin may promote movement of Ste2 into regions of membrane that actively endocytose. Another question is why ubiquitinated Ste2 is not targeted by the proteasome, as demonstrated convincingly by Hicke and Riezman 1996xHicke, L. and Riezman, H. Cell. 1996; 84: 277–287Abstract | Full Text | Full Text PDF | PubMed | Scopus (594)See all ReferencesHicke and Riezman 1996 with proteasome mutants. It is not simply because Ste2 is a membrane protein. The CFTR transmembrane protein, particularly the mutant form commonly found in cystic fibrosis patients, is multiubiquitinated when in the ER and is apparently degraded by the proteasome (Ward et al. 1995xWard, C.L., Omura, S., and Kopito, R.R. Cell. 1995; 83: 121–127Abstract | Full Text PDF | PubMed | Scopus (976)See all ReferencesWard et al. 1995). Under some conditions, certain receptor tyrosine kinases can also be degraded by a ubiquitin/proteasome-dependent mechanism (Sepp-Lorenzino et al. 1995xSepp-Lorenzino, L., Ma, Z., Lebwohl, D.E., Vinitsky, A., and Rosen, N. J. Biol. Chem. 1995; 270: 16580–16587CrossRef | PubMed | Scopus (174)See all ReferencesSepp-Lorenzino et al. 1995). Structural features of the targeted protein may be important in determining its fate. It is also conceivable that ubiquitin chains on Ste2 rarely reach the lengths thought to be preferred by the 26S proteasome because either chain assembly is slow or chain disassembly by deubiquitinating enzymes is rapid. An intriguing possibility is that the type of ubiquitin chain formed on Ste2 determines its metabolic fate. Several groups have reported that ubiquitin chains with non-lysine 48 isopeptide linkages can form in yeast cells (1xArnason, T. and Ellison, M.J. Mol. Cell Biol. 1994; 14: 7876–7883PubMedSee all References, 13xSpence, J., Sadis, S., Haas, A.L., and Finley, D. Mol. Cell Biol. 1995; 15: 1265–1273CrossRef | PubMedSee all References, 9xJohnson, E.S., Ma, P.C.M., Ota, I.M., and Varshavsky, A. J. Biol. Chem. 1995; 270: 17442–17456CrossRef | PubMed | Scopus (550)See all References, 2xBaboshina, O.V. and Haas, A.L. J. Biol. Chem. 1996; 271PubMedSee all References). Interestingly, lysine 63-linked ubiquitin chains require Ubc4/Ubc5, the same E2 enzymes implicated in receptor endocytosis, and it had previously been argued that such chains serve nonproteolytic functions (Arnason and Ellison 1994xArnason, T. and Ellison, M.J. Mol. Cell Biol. 1994; 14: 7876–7883PubMedSee all ReferencesArnason and Ellison 1994). Such alternative chains could provide distinct binding surfaces that target the ubiquitin conjugate to different binding factors or cellular structures. These considerations are of course relevant not only for membrane protein endocytosis but also for other ubiquitin-dependent, proteasome-independent processes, such as the IκB kinase activation discussed above.A final question is how often ubiquitination is utilized as a signal for endocytosis and vacuolar targeting. Almost certainly, the answer will be that it is extremely common. As mentioned above, the yeast Ste6 protein is very likely to follow a degradation pathway very similar to that which works on Ste2. Other recent studies in yeast suggest the same is true for uracil permease, the general amino acid transporter Gap1, and the multidrug transporter Pdr5 (Egner and Kuchler 1996xEgner, R. and Kuchler, K. FEBS Lett. 1996; 378: 177–181Abstract | Full Text PDF | PubMed | Scopus (97)See all ReferencesEgner and Kuchler 1996references therein). Interestingly, uracil permease appears to use a ubiquitination signal related to the “destruction box” of mitotic cyclins. Internalization and vacuolar degradation of uracil permease and Gap1 require the Npi1/Rsp5 protein, which has characteristics of an E3 ubiquitin-protein ligase. These are not just peculiarities of yeast: a number of mammalian membrane proteins are also known to be multiubiquitinated at the cell surface and degraded in the lysosome (Ciechanover 1994xCiechanover, A. Cell. 1994; 79: 13–21Abstract | Full Text PDF | PubMed | Scopus (1316)See all ReferencesCiechanover 1994).At present, we have little more than a first adumbration of the complexities of the ubiquitin system. Many new physiological roles for this intricate metabolic system are still being discovered. At a very basic mechanistic level, it is still unclear how specific proteins are identified by the ubiquitin conjugation machinery or what the relative importance of the E2 and E3 enzymes is in substrate recognition. We are also just becoming aware of the remarkably large and diverse group of deubiquitinating enzymes that exist (yeast have at least 17 such enzymes); these enzymes are likely to be functionally diverse as well (Papa and Hochstrasser 1993xPapa, F. and Hochstrasser, M. Nature. 1993; 366: 313–319CrossRef | PubMed | Scopus (298)See all ReferencesPapa and Hochstrasser 1993). We now have two clear examples where ubiquitin operates in a way other than as a direct proteolytic signal. With the realization that ubiquitination does more than deliver proteins to the proteasome, questions about the molecular basis of substrate specificity and targeting are given new urgency. Combining mechanistic analysis of the ubiquitin system with the study of its varied biological functions should prove a productive enterprise for some years to come.