Ubiquitination of the yeast a-factor receptor

The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communication of mating. In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination. Phosphorylation, evident on the unstimulated receptor, increases upon challenge by the receptor's ligand, a-factor. We suggest that this phosphorylation likely functions in the adaptive, negative regulation of receptor activity. Removal of phosphorylation by phosphatase treatment uncovered two phosphatase-resistant modifications identified as ubiquitination using a myc-epitope-tagged ubiquitin construct. Ste3p undergoes rapid, ligand-independent turnover that depends on vacuolar proteases and also on transport of the receptor from surface to vacuole (i.e., endocytosis) (Davis, N.G., J.L.Horecka, and G.F. Sprague, Jr., 1993 J. Cell Biol. 122:53-65). An end4 mutation, isolated for its defect in the endocytic uptake of alpha-factor pheromone (Raths, S., J. Rohrer, F. Crausaz, and H. Riezman. 1993. J. Cell Biol. 120:55-65), blocks constitutive endocytosis of the a-factor receptor, yet fails to block ubiquitination of the receptor. In fact, both phosphorylation and ubiquitination of the surfacebound receptor were found to increase, suggesting that these modifications may occur normally while the receptor is at the cell surface. In a mutant strain constructed to allow for depletion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased. Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally approximately 20 min at 30 degrees C was increased to approximately 2 h under these ubiquitin-depletion conditions. Furthermore, surface residency, normally of short duration in wild-type cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the receptor protein remained surface localized fully 2 h after biosynthesis. Thus, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell. In cells mutant for two E2 activities, i.e., ubc4 delta ubc5 delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5p may play a role in the recognition of the receptor protein as substrate for the ubiquitin system. In addition to ligand-independent uptake, the a-factor receptor also undergoes a ligand-dependent form of endocytosis (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr. 1993. J. Cell. Biol. 122:53-65). Concurrent with ligand-dependent uptake, we now show that the receptor undergoes ligand-induced ubiquitination, suggesting that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive endocytosis. To account for these findings, we propose a model wherein the covalent attachment of ubiquitin to surface receptor triggers endocytic uptake.     

Ubiquitin-independent entry into the yeast recycling pathway

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway links to vacuolar degradation of the receptor, while a ligand-dependent uptake pathway links primarily to recycling and thus, receptor reutilization. Ste3p ubiquitination triggers its uptake into the constitutive pathway. The present work considers the role of the receptor ubiquitination associated with the Ste3p ligand-dependent endocytosis mechanism. The doa4Δ mutation which reduces the cellular availability of ubiquitin blocks the Ste3p constitutive uptake. Uptake into the Ste3p ligand-dependent recycling pathway, however, continues unimpaired. The ubiquitin independence of Ste3p ligand-dependent uptake was further indicated by analysis of receptor mutants having Lys-to-Arg substitutions at all possible ubiquitin acceptor sites. Again, the ligand-induced internalization was unimpaired. Furthermore, no discernible effect was seen on either recycling or on the slow PEP4 -dependent turnover of the receptor (for receptor internalized via the ligand-dependent mechanism, trafficking to the vacuole/lysosome is the minor, alternate fate to recycling). However, one striking effect of the Lys-to-Arg mutations was noted. Following a prolonged exposure of the cells to the a-factor ligand, rather than being delivered to the vacuolar lumen, the Lys-to-Arg receptor was found to localize instead to the limiting membrane of the vacuole. Thus, while receptor ubiquitination clearly is not required for either the a-factor-dependent uptake into recycling pathway or for the recycling itself, it does affect the routing of receptor to the vacuole, likely by affecting the routing through the late endosomal, multivesicular body: ubiquitinated receptor may be selected into the internal, lumenal vesicles, while unmodified receptor may be left to reside at the limiting external membrane.     

A Large PEST-like Sequence Directs the Ubiquitination,Endocytosis, and Vacuolar Degradation of the Yeast a-Factor Receptor

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661–674) and both depend on sequence elements within the receptor''s regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t _1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity—no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences—a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.     

Akr1p and the type I casein kinases act prior to the ubiquitination step of yeast endocytosis: Akr1p is required for kinase localization to the plasma membrane

Ubiquitination of the plasma membrane-localized yeast a-factor receptor (Ste3p) triggers a rapid, ligand-independent endocytosis leading to its vacuolar degradation. This report identifies two mutants that block uptake by blocking ubiquitination, these being mutant either for the ankyrin repeat protein Akr1p or for the redundant type I casein kinases Yck1p and Yck2p. While no obvious defect was seen for wild-type Ste3p phosphorylation in akr1 or yck mutant backgrounds, examination of the Delta320-413 Ste3p deletion mutant phosphorylation did reveal a clear defect in both mutants. The Delta320-413 deletion removes 18 Ser-Thr residues (possible YCK-independent phosphorylation sites) yet retains the 15 Ser-Thr residues of the Ste3p PEST-like ubiquitination-endocytosis signal. Two other phenotypes link akr1 and yck mutants: both are defective in phosphorylation of wild-type alpha-factor receptor, and while both are defective for Ste3p constitutive internalization, both remain partially competent for the Ste3p ligand-dependent uptake mode. Yck1p-Yck2p may be the function responsible in phosphorylation of the PEST-like ubiquitination-endocytosis signal. Akr1p appears to function in localizing Yck1p-Yck2p to the plasma membrane, a localization that depends on prenylation of C-terminal dicysteinyl motifs. In akr1Delta cells, Yck2p is mislocalized, showing a diffuse cytoplasmic localization identical to that seen for a Yck2p mutant that lacks the C-terminal Cys-Cys, indicating a likely Akr1p requirement for the lipid modification of Yck2p, for prenylation, or possibly for palmitoylation.     

The linker region of the ABC-transporter Ste6 mediates ubiquitination and fast turnover of the protein.

Upon block of endocytosis, the a-factor transporter Ste6 accumulates in a ubiquitinated form at the plasma membrane. Here we show that the linker region, which connects the two homologous halves of Ste6, contains a signal which mediates ubiquitination and fast turnover of Ste6. This signal was also functional in the context of another plasma membrane protein. Deletion of an acidic stretch in the linker region ('A-box') strongly stabilized Ste6. The A-box contains a sequence motif ('DAKTI') which resembles the putative endocytosis signal of the alpha-factor receptor Ste2 ('DAKSS'). Deletion of the DAKTI sequence also stabilized Ste6 but, however, not as strongly as the A-box deletion. There was a correlation between the half-life of the mutants and the degree of ubiquitination: while ubiquitination of the deltaDAKTI mutant was reduced compared with wild-type Ste6, no ubiquitination could be detected for the more stable deltaA-box variant. Loss of ubiquitination seemed to affect Ste6 trafficking. In contrast to wild-type Ste6, which was associated mainly with internal membranes, the ubiquitination-deficient mutants accumulated at the plasma membrane, as demonstrated by immunofluorescence and cell fractionation experiments. These findings suggest that ubiquitination is required for efficient endocytosis of Ste6 from the plasma membrane.     

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.     

A PEST-Like Sequence Mediates Phosphorylation and Efficient Ubiquitination of Yeast Uracil Permease

Uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. Uracil permease located at the cell surface is subject to two covalent modifications: phosphorylation and ubiquitination. The ubiquitination step is necessary prior to permease endocytosis and subsequent vacuolar degradation. Here, we demonstrate that a PEST-like sequence located within the cytoplasmic N terminus of the protein is essential for uracil permease turnover. Internalization of the transporter was reduced when some of the serines within the region were converted to alanines and severely impaired when all five serines within the region were mutated or when this region was absent. The phosphorylation and degree of ubiquitination of variant permeases were inversely correlated with the number of serines replaced by alanines. A serine-free version of this sequence was very poorly phosphorylated, and elimination of this sequence prevented ubiquitination. Thus, it appears that the serine residues in the PEST-like sequence are required for phosphorylation and ubiquitination of uracil permease. A PEST-like sequence in which the serines were replaced by glutamic acids allowed efficient permease turnover, suggesting that the PEST serines are phosphoacceptors.     

Deubiquitination Step in the Endocytic Pathway of Yeast Plasma Membrane Proteins: Crucial Role of Doa4p Ubiquitin Isopeptidase

The Fur4p uracil permease, like most yeast plasma membrane proteins, undergoes ubiquitin-dependent endocytosis and is then targeted to the vacuole (equivalent to the mammalian lysosome) for degradation. The cell surface ubiquitination of Fur4p is mediated by the essential Rsp5p ubiquitin ligase. Ubiquitination of Fur4p occurs on two target lysines, which receive two ubiquitin moieties linked through ubiquitin Lys63, a type of linkage (termed UbK63) different from that involved in proteasome recognition. We report that pep4 cells deficient for vacuolar protease activities accumulate vacuolar unubiquitinated Fur4p. In contrast, pep4 cells lacking the Doa4p ubiquitin isopeptidase accumulate ubiquitin-conjugated Fur4p. These data suggest that Fur4p undergoes Doa4p-dependent deubiquitination prior to vacuolar degradation. Compared to pep4 cells, pep4 doa4 cells have huge amounts of membrane-bound ubiquitin conjugates. This indicates that Doa4p plays a general role in the deubiquitination of membrane-bound proteins, as suggested by reports describing the suppression of some doa4 phenotypes in endocytosis and vacuolar protein sorting mutants. Some of the small ubiquitin-linked peptides that are a hallmark of Doa4 deficiency are not present in rsp5 mutant cells or after overproduction of a variant ubiquitin modified at Lys 63 (UbK63R). These data suggest that the corresponding peptides are degradation products of Rsp5p substrates and probably of ubiquitin conjugates carrying UbK63 linkages. Doa4p thus appears to be involved in the deubiquitination of endocytosed plasma membrane proteins, some of them carrying UbK63 linkages.     

Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein.

We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase. Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation. This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains. The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase. Both processes were rescued by overexpression of wild-type ubiquitin. Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination. In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination. Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin. When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate. We have thus identified a natural target of Lys63-linked ubiquitin chains. We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process.     

Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant

Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.     

Site-specific ubiquitination exposes a linear motif to promote interferon-alpha receptor endocytosis

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCF^βTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.     

The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants.

We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life > 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.     

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.     

Cytoplasmic Tail Phosphorylation of the α-Factor Receptor Is Required for Its Ubiquitination and Internalization

G protein–coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The α-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding α-factor. Ubiquitination triggers Ste2p internalization into the endocytic pathway. Here we demonstrate that phosphorylation of Ste2p promotes downregulation by positively regulating ubiquitination and internalization. Serines and a lysine are essential elements of the Ste2p SINNDAKSS internalization signal that can mediate both constitutive and ligand-stimulated endocytosis. The SINNDAKSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence. Mutants defective in yeast casein kinase I homologues are unable to internalize α-factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor. Finally, we provide evidence that the primary function of phosphorylation controlled by the SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.     

Recycling of the yeast a-factor receptor

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.     

The internalization of yeast Ste6p follows an ordered series of events involving phosphorylation, ubiquitination, recognition and endocytosis

A general pathway for the internalization of plasma membrane proteins that involves phosphorylation, ubiquitination, recognition and endocytosis has recently emerged from multiple studies in yeast. We refer to this series of events as the PURE pathway. Here we investigate whether the yeast a-factor transporter Ste6p, an ATP-binding cassette protein, utilizes the PURE pathway. Deletion of a 52-amino acid sequence (the 'A box') within the linker region of Ste6p has previously been shown to block ubiquitination and endocytosis (Kolling R, Losko S. EMBO J 1997; 16:2251-2261). Using wild-type and mutant forms of GFP-tagged Ste6p, we identified two residues (T(613) and S(623)) within the A box as likely sites of Ste6p phosphorylation important for internalization. Mutation of these residues to alanine blocked ubiquitination and endocytosis of Ste6p, similar to the effect of deleting the entire A box, while substitution with glutamic acid (to mimic phosphorylation) suppressed the ubiquitination and endocytic defects. Importantly, a translational fusion of monoubiquitin to the C-terminus of Ste6p-T(613)A, S(623)A or Ste6p-DeltaA restored endocytosis, providing strong evidence that the role of phosphorylation is to direct ubiquitination, which in turn is a critical signal for Ste6p internalization. We also identified multiple (five) lysine residues in the linker that are important for Ste6p ubiquitination. Our results demonstrate that Ste6p follows the PURE pathway and that GFP-tagged Ste6p provides a powerful model protein for studies of endocytosis and post-endocytic events in yeast.     

Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1

In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH(4)(+), a preferential source of nitrogen, is added to cells growing on proline or urea, i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1(K9K16)) remains fully stable at the plasma membrane after NH(4)(+) addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1(K9) and Gap1(K16) mutants, in which a single lysine is mutated, are down-regulated in response to NH(4)(+) although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and André, B. (2001) J. Biol. Chem. 276, 43939-43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Delta mutant, neosynthesized Gap1(K9K16) is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Delta mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking, i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi.     

Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase

The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.     

A Di-leucine signal in the ubiquitin moiety. Possible involvement in ubiquitination-mediated endocytosis

Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes upon ligand-dependent ubiquitination. However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial. In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins. The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently. Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis. We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu(43)-Ile(44). The Leu(43)-Ile(44) sequence mediated endocytosis and lysosomal sorting in a Leu(43)-dependent manner. These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.     

Phosphorylation and specific ubiquitin acceptor sites are required for ubiquitination and degradation of the IFNAR1 subunit of type I interferon receptor

Ubiquitination, endocytosis, and lysosomal degradation of the IFNAR1 (interferon alpha receptor 1) subunit of the type I interferon (IFN) receptor is mediated by the SCFbeta-Trcp (Skp1-Cullin1-F-box protein beta transducin repeat-containing protein) E3 ubiquitin ligase in a phosphorylation-dependent manner. In addition, stability of IFNAR1 is regulated by its binding to Tyk2 kinase. Here we characterize the determinants of IFNAR1 ubiquitination and degradation. We found that the integrity of two Ser residues at positions 535 and 539 within the specific destruction motif present in the cytoplasmic tail of IFNAR1 is essential for the ability of IFNAR1 to recruit beta-Trcp as well as to undergo efficient ubiquitination and degradation. Using an antibody that specifically recognizes IFNAR1 phosphorylated on Ser535 we found that IFNAR1 is phosphorylated on this residue in cells. This phosphorylation is promoted by treatment of cells with IFNalpha. Although the cytoplasmic tail of IFNAR1 contains seven Lys residues that could function as potential ubiquitin acceptor sites, we found that only three (Lys501, Lys525, and Lys526), all located proximal to the destruction motif, are essential for ubiquitination and degradation of IFNAR1. Expression of Tyk2 stabilized IFNAR1 in a manner that was dependent neither on its binding to beta-Trcp nor IFNAR1 ubiquitination. We discuss the complexities and specifics of the ubiquitination and degradation of IFNAR1, which is a beta-Trcp substrate that undergoes degradation via a lysosomal pathway.