Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels

G-protein-coupled receptors and transporters in Saccharomyces cerevisiae are modified with ubiquitin in response to ligand biding. In most cases, the proteasome does not recognize these ubiquitinated proteins. Instead, ubiquitination serves to trigger internalization and degradation of plasma membrane proteins in the lysosome-like vacuole. A number of mammalian receptors and at least one ion channel undergo ubiquitination at the plasma membrane, and this modification is required for their downregulation. Some of these cell-surface proteins appear to be degraded by both the proteasome and lysosomal proteases. Recent evidence indicates that other proteins required for receptor internalization might also be regulated by ubiquitination, suggesting that ubiquitin plays diverse roles in regulating plasma membrane protein activity.     

Growth hormone receptor ubiquitination, endocytosis, and degradation are independent of signal transduction via Janus kinase 2.

The ubiquitin-proteasome system is required in growth hormone receptor (GHR) endocytosis. For cytokine receptors, which lack intrinsic tyrosine kinase activity, signal transduction is initiated by the activation of a member of the Janus kinase (JAK) family. Previously, we have shown that GHR and JAK2 tyrosine (de) phosphorylation are regulated via the ubiquitin system. In this study, we examined the role of JAK2-mediated signal transduction in GHR internalization and down-regulation. Mutation of the attachment site for JAK2, box-1, in the GHR cytoplasmic tail resulted in the complete absence of GHR and JAK2 phosphorylation. This modification did not alter the rate and extent of receptor-bound growth hormone internalization as compared with a functional GHR, nor did it change its turnover and transport to the plasma membrane. In addition, the receptor was still normally ubiquitinated and remained dependent on both an intact ubiquitin system and proteasomal action for its internalization. Thus, GHR ubiquitination, endocytosis, and degradation occur independently of GHR signal transduction via JAK2. We conclude that whereas endocytosis and degradation require the ubiquitin system, they are independent of GHR signal transduction.     

Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis

Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.     

Ubiquitination and Endocytosis of Plasma Membrane Proteins: Role of Nedd4/Rsp5p Family of Ubiquitin-Protein Ligases

In addition to its well-known role in recognition by the proteasome, ubiquitin-conjugation is also involved in downregulation of membrane receptors, transporters and channels. In most cases, ubiquitination of these plasma membrane proteins leads to their internalization followed by targeting to the lysosome/vacuole for degradation. A crucial role in ubiquitination of many plasma membrane proteins appears to be played by ubiquitin-protein ligases of the Nedd4/Rsp5p family. All family members carry an N-terminal Ca²⁺-dependent lipid/protein binding (C2) domain, two to four WW domains and a C-terminal catalytic Hect-domain. Nedd4 is involved in downregulation of the epithelial Na⁺ channel, by binding of its WW domains to specific PY motifs of the channel. Rsp5p, the unique family member in S. cerevisiae, is involved in ubiquitin-dependent endocytosis of a great number of yeast plasma membrane proteins. These proteins lack apparent PY motifs, but carry acidic sequences, and/or phosphorylated-based sequences that might be important, directly or indirectly, for their recognition by Rsp5p. In contrast to polyubiquitination leading to proteasomal recognition, a number of Rsp5p targets carry few ubiquitins per protein, and moreover with a different ubiquitin linkage. Accumulating evidence suggests that, at least in yeast, ubiquitin itself may constitute an internalization signal, recognized by a hypothetical receptor. Recent data also suggest that Nedd4/Rsp5p might play a role in the endocytic process possibly involving its C2 domain, in addition to its role in ubiquitinating endocytosed proteins. Recieved: 19 January 2000/Revised: 6 April 2000     

Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor.

The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome. Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast. By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events. Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system. Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway. Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor.

In addition to its role in selective protein degradation, the conjugation of ubiquitin to proteins has also been implicated in the internalization of plasma membrane proteins, including the alpha-factor receptor Ste2p, uracil permease Fur4p, epithelial sodium channel ENaC and the growth hormone receptor (GHR). Binding of GH to its receptor induces receptor dimerization, resulting in the activation of signal transduction pathways and an increase of GHR ubiquitination. Previously, we have shown that the ubiquitin conjugation system mediates GH-induced GHR internalization. Here, we present evidence that a specific domain of the GHR regulates receptor endocytosis via the ubiquitin conjugation system. This ubiquitin-dependent endocytosis (UbE) motif consists of the amino acid sequence DSWVEFIELD and is homologous to sequences in other proteins, several of which are known to be ubiquitinated. In addition, we show that GH internalization by a truncated GHR is independent of the presence of lysine residues in the cytosolic domain of this receptor, while internalization still depends on an intact ubiquitin conjugation system. Thus, GHR internalization requires the recruitment of the ubiquitin conjugation system to the GHR UbE motif rather than the conjugation of ubiquitin to the GHR itself.     

Rsp5-dependent endocytosis and degradation of the arsenite transporter Acr3 requires its N-terminal acidic tail as an endocytic sorting signal and arrestin-related ubiquitin-ligase adaptors

The yeast plasma membrane transporter Acr3 mediates efflux of toxic arsenite and antimonite. Here, we investigated the mechanisms of Acr3 turnover. We found that after arrival and residence at the plasma membrane, Acr3 is subjected to internalization followed by proteolysis in the vacuole. Endocytic degradation of Acr3 is promoted by the ubiquitin ligase Rsp5 and requires polyubiquitination of Acr3 at multiple lysine residues via lysine 63-linked ubiquitin chains. The turnover of Acr3 also depends on two arrestin-related proteins, Art3/Aly2 and Art4/Rod1, that enable recruitment of Rsp5 to its targets. Finally, we found that a short acidic patch located in the N-terminal tail of Acr3 is needed for its ubiquitination and internalization. We propose that this motif serves as an endocytic signal that facilitates binding of the arrestin-Rsp5 complexes to the Acr3 cargo.     

Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation

Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated - a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.     

Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast.

The Saccharomyces cerevisiae alpha-pheromone receptor, a polytopic, G protein-coupled, membrane protein, is internalized after binding of alpha-factor. Mutational analysis suggested that the first 39 residues of the receptor's cytoplasmic tail carries sufficient information for internalization. A point mutation in one of these 39 residues, K337 to R337, renders the receptor nonfunctional for endocytosis. Other residues, D335 and S338, contribute to the efficiency of internalization. When the sequence DAKSS is added onto a severely truncated receptor, endocytosis of the receptor is restored, showing that this sequence functions to mediate or to signal interaction with the endocytic machinery. Analysis of pheromone response and recovery in strains expressing mutant receptors suggests that receptor internalization is not important for response but contributes to recovery from pheromone.     

p120-Catenin regulates clathrin-dependent endocytosis of VE-cadherin

VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin-p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.     

UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.     

GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.     

The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells.

Lysosomal acid phosphatase (LAP) is synthesized as a type I membrane glycoprotein and targeted to lysosomes via the plasma membrane. Its cytoplasmic tail harbours a tyrosine-containing signal for rapid internalization. Expression in Madine-Darby canine kidney cells results in direct sorting to the basolateral cell surface, rapid endocytosis and delivery to lysosomes. In contrast, a deletion mutant lacking the cytoplasmic tail is delivered to the apical plasma membrane where it accumulates before it is slowly internalized. A chimeric protein, in which the cytoplasmic tail of LAP is fused to the extracytoplasmic and transmembrane domain of the apically sorted haemagglutinin, is sorted to the basolateral plasma membrane. A series of truncation and substitution mutants in the cytoplasmic tail was constructed and comparison of their polarized sorting and internalization revealed that the determinants for basolateral sorting and rapid internalization reside in the same segment of the cytoplasmic tail. The cytoplasmic factors decoding these signals, however, tolerate distinct mutations indicating that different receptors are involved in sorting at the trans-Golgi network and at the plasma membrane.     

Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis

Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast alpha-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on alpha-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.     

Intracellular trafficking and localization of the pseudorabies virus Us9 type II envelope protein to host and viral membranes

The Us9 protein is a phosphorylated membrane protein present in the lipid envelope of pseudorabies virus (PRV) particles in a unique tail-anchored type II membrane topology. In this report, we demonstrate that the steady-state residence of the Us9 protein is in a cellular compartment in or near the trans-Golgi network (TGN). Through internalization assays with an enhanced green fluorescent protein epitope-tagged Us9 protein, we demonstrate that the maintenance of Us9 to the TGN region is a dynamic process involving retrieval of molecules from the cell surface. Deletion analysis of the cytoplasmic tail reveals that an acidic cluster containing putative phosphorylation sites is necessary for the recycling of Us9 from the plasma membrane. The absence of this cluster results in the relocalization of Us9 to the plasma membrane due to a defect in endocytosis. The acidic motif, however, does not contain signals needed to direct the incorporation of Us9 into viral envelopes. In this study, we also investigate the role of a dileucine endocytosis signal in the Us9 cytoplasmic tail in the recycling and retention of Us9 to the TGN region. Site-directed mutagenesis of the dileucine motif results in an increase in Us9 plasma membrane staining and a partial internalization defect.     

A novel endocytosis signal related to the KKXX ER-retrieval signal.

Membrane proteins often contain a sorting signal in their cytoplasmic tail that promotes their clustering into coated vesicles at a specific cellular site. ERGIC-53 contains a cytoplasmic ER-retrieval signal, KKFF. However, overexpressed ERGIC-53 is transported to the cell surface and rapidly endocytosed. Here we report that ERGIC-53 carries a previously undescribed endocytosis signal. Surprisingly, the signal was KKFF and like the ER-retrieval signal required a C-terminal position. In fact, the minimal consensus sequence determined by substitutional mutagenesis (K-K/R-F/Y-F/Y) was related to the ER-retrieval consensus (K-K-X-X). Furthermore, we provide evidence that internalization of VIP36, a protein that cycles between plasma membrane and Golgi, is mediated by a signal at its C-terminus that matches the internalization consensus sequence. The relatedness of the two signals suggests that coatomer-mediated retrieval of proteins may be mechanistically more related to clathrin-dependent sorting than previously anticipated.     

Multiple roles for Rsp5p-dependent ubiquitination at the internalization step of endocytosis

Ubiquitination of integral plasma membrane proteins triggers their rapid internalization into the endocytic pathway. The yeast ubiquitin ligase Rsp5p, a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of multiple plasma membrane proteins, including the alpha-factor receptor (Ste2p). Here we demonstrate that Rsp5p plays multiple roles at the internalization step of endocytosis. Temperature-sensitive rsp5 mutant cells were defective in the internalization of alpha-factor by a Ste2p-ubiquitin chimera, a receptor that does not require post-translational ubiquitination. Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal was not internalized in rsp5 cells. Internalization of these variant receptors was dependent on the catalytic cysteine residue of Rsp5p and on ubiquitin-conjugating enzymes that bind Rsp5p. Thus, a Rsp5p-dependent ubiquitination event is required for internalization mediated by ubiquitin-dependent and -independent endocytosis signals. Constitutive Ste2p-ubiquitin internalization and fluid-phase endocytosis also required active ubiquitination machinery, including Rsp5p. These observations indicate that Rsp5p-dependent ubiquitination of a trans-acting protein component of the endocytosis machinery is required for the internalization step of endocytosis.     

The roles of ubiquitin and lipids in protein sorting along the endocytic pathway

After cell surface receptors are internalized for endocytosis, they are accurately sorted in endosomes. Some are recycled to the plasma membrane and others are downregulated by delivery to lysosomes. Evidence is rapidly accumulating that ubiquitination of cargo proteins acts as a sorting signal during endocytosis. Sorting devices that recognize ubiquitin are distributed to various compartments, probably acting in a concerted manner. Cholesterol is enriched in the plasma membrane and endosomes, and is involved in protein sorting by forming microdomains called lipid rafts. Ubiquitin and cholesterol hold the key to control the endocytic sorting, and they are likely acting cooperatively.     

Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.