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.     

Multiple functions of sterols in yeast endocytosis

Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of ergDelta mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2Deltaerg6Delta and erg3Deltaerg6Delta cells exhibit a strong internalization defect of the alpha-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. The lack of phosphorylation is not due to a defect in Ste2p localization or in ligand-receptor interaction. Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin. Furthermore, sterol structures are required at a postinternalization step of endocytosis. ergDelta cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole. Therefore, there are at least two roles for sterols in endocytosis. Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway. Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment.     

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.     

The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.     

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.     

Actin and fimbrin are required for the internalization step of endocytosis in yeast.

In Saccharomyces cerevisiae, alpha-factor is internalized by receptor-mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta-tubulin and actin mutant strains, we showed that actin plays a direct role in receptor-mediated internalization of alpha-factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta-tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin-binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha-factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.     

TEDS site phosphorylation of the yeast myosins I is required for ligand-induced but not for constitutive endocytosis of the G protein-coupled receptor Ste2p

The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.     

Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities.     

Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast

Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.     

Role for the Ubiquitin-Proteasome System in the Vacuolar Degradation of Ste6p, the a-Factor Transporter in Saccharomyces cerevisiae

Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.     

A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15

A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants.     

The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae

The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a- factor receptor, Ste3p. Mutation of this sequence prevented pheromone- stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast.     

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.     

Characterization of Vta1p, a class E Vps protein in Saccharomyces cerevisiae

We identified VTA1 in a screen for mutations that result in altered vacuole morphology. Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA. These results suggest a VPS defect for Deltavta1 cells. Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting. Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane. Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells. We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white. Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity. A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment. This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport. Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment.     

Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.     

Requirements for Recruitment of a G Protein-coupled Receptor to Clathrin-coated Pits in Budding Yeast

Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor Ste2p has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to reveal unappreciated molecular and spatiotemporal features of GPCR endocytosis in budding yeast. Here, we identify recruitment of Ste2p to preexisting clathrin-coated pits (CCPs) as a key step regulated by receptor phosphorylation and subsequent ubiquitination upon ligand binding. The yeast casein kinase I homologue Yck2p directly phosphorylates six serine residues located in the C-terminal tail of Ste2p, and mutation of these serine residues to alanine significantly decreased recruitment of Ste2p to CCPs. We also found that the clathrin adaptors Ent1p, Ent2p, and Ede1p work cooperatively to recruit ubiquitinated Ste2p to CCPs. In addition, ubiquitination has a role in ligand-independent constitutive recruitment of Ste2p to CCPs, although this process is much slower than ligand-induced recruitment. These results suggest that ubiquitination of Ste2p is indispensable for recruiting Ste2p to CCPs in both ligand-dependent and ligand-independent endocytosis.     

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.     

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.     

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.     

Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs

The actin filament (F-actin) cytoskeleton associates dynamically with the plasma membrane and is thus ideally positioned to participate in endocytosis. Indeed, a wealth of genetic and biochemical evidence has confirmed that actin interacts with components of the endocytic machinery, although its precise function in endocytosis remains unclear. Here, we use 4D microscopy to visualize the contribution of actin during compensatory endocytosis in Xenopus laevis eggs. We show that the actin cytoskeleton maintains exocytosing cortical granules as discrete invaginated compartments, such that when actin is disrupted, they collapse into the plasma membrane. Invaginated, exocytosing cortical granule compartments are directly retrieved from the plasma membrane by F-actin coats that assemble on their surface. These dynamic F-actin coats seem to drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments. Active Cdc42 and N-WASP are recruited to exocytosing cortical granule membranes before F-actin coat assembly and coats assemble by Cdc42-dependent, de novo actin polymerization. Thus, F-actin may power fusion pore resealing and function in two novel endocytic capacities: the maintenance of invaginated compartments and the processing of endosomes.