INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Ubiquitin plays a central role in the endocytic uptake of a number of plasma membrane proteins in the yeast Saccharomyces cerevisiae (14). With its attachment to the cytosolic domains of the plasma membrane substrate, ubiquitin provides the sorting determinant that initiates uptake. This uptake mechanism has been best characterized for yeast, but evidence suggests that both ubiquitin and ubiquitination enzymes participate in the internalization of a variety of mammalian plasma membrane proteins as well (5).

Ubiquitin-dependent endocytosis appears to differ from the well-characterized clathrin-mediated uptake of mammalian cells. For clathrin-mediated uptake, substrates for uptake are recognized and sequestered within clathrin-coated pits through the binding of short peptidyl signals to the AP-2 adaptin complex (13, 24, 41). For ubiquitin-dependent uptake in yeast, the attached ubiquitin moiety provides the recognition determinant (14). Furthermore, while the key players of clathrin-mediated uptake are present in yeast (i.e. clathrin heavy and light chains, adaptin subunits, and dynamin homologues), a definitive demonstration of a role for any of these proteins in endocytosis has remained elusive. Strains with all of the genes encoding the adaptin subunits deleted remain fully competent for ubiquitin-dependent uptake (18). For clathrin, the situation is less clear. Mutation and/or deletion of the unique clathrin heavy- or light-chain genes have partial effects on ubiquitin-dependent endocytosis, slowing but not abolishing uptake (29, 45). The clathrin involvement, therefore, is either partial or indirect: uptake, at least in part, must be clathrin independent.

A number of mutations that do abolish uptake have been identified in yeast. The largest class of these endocytosis-defective mutants are those in which the block is exerted through derangement of the actin cytoskeleton. In addition to mutations in the actin structural protein, endocytosis-defective mutants identify a variety of other actin-associated proteins, including End3p, End4p, Sac6p, Vrp1p, and Pan1p (51). Other required proteins include the type I myosins Myo3p and Myo5p, which are expected to interact with actin, and calmodulin, a known regulator of nonmuscle myosins. Although the molecular nature of the actin role in endocytosis remains uncertain, it seems likely to function in the mechanical aspect of the internalization process. Indeed, a recent report for rat mast cells indicates that actin polymerization may provide the force that drives the newly formed pinocytotic vesicles away from the plasma membrane (25).

Given the central role for ubiquitin modification in yeast endocytosis, it follows that some of the enzymes that catalyze this modification are required participants in the uptake process. Generally, protein ubiquitination involves a sequential transfer of the ubiquitin moiety from one class of ubiquitination enzyme to another, from E1 to E2 to E3 and to the substrate protein. The diversity of E2 and E3 enzymes catalyzing this process is thought to reflect the diversity of the substrates within the cell that ultimately receive ubiquitin modification. For a variety of endocytic substrates, the E2 component appears to be the redundant triad Ubc1p, Ubc4p, and Ubc5p (14). Most often implicated as the E3 component for this process is hect domain-containing protein Rsp5p (14). The human homologue of Rsp5p, NEDD4, is required for the endocytic down regulation of the renal ENaC sodium channel (44).

Ubiquitin's role in endocytosis differs from its role in proteasomal turnover. First, the degradation associated with ubiquitin-dependent endocytosis is mediated by the vacuolar proteases, not by the proteasome. Second, the two processes differ in terms of the extent of ubiquitination required: while a multiubiquitin chain composed of four or more ubiquitin moieties is required for proteasomal recognition (17), monoubiquitination suffices to trigger endocytic uptake (34, 47).

The two yeast pheromone receptors, the - and the a-factor receptors, have been well studied as model substrates for ubiquitin-dependent endocytosis. These two G protein-coupled receptors mediate the pheromonal communication that precedes the sexual conjugation of the two yeast haploid mating types, the MATa and MAT cells. For the -factor receptor (Ste2p), challenge with its ligand, the peptide -factor, results in ubiquitination of Ste2p and its subsequent internalization to the vacuole (yeast lysosome), where it is degraded by the resident proteases (15, 40). The most prominent form of endocytosis associated with the a-factor receptor (Ste3p) is a constitutive or ligand-independent uptake which also delivers receptor to the vacuole for degradation (7). Ste3p constitutive endocytosis is rapid, and consequently, Ste3p is a short-lived protein, with a half-life (t_1/2) of only 15 min in cells growing at 30°C.

The signal for the rapid constitutive endocytosis of Ste3p is an extended 58-residue-long PEST-like sequence located at the C-terminal end of the receptor's 183-residue regulatory C-terminal cytoplasmic tail domain (CTD) (36). Consistent with its PEST-like nature, the Ste3p endocytosis signal functions primarily as a ubiquitination signal, with the three lysine residues that map within this sequence serving redundantly as the sites for ubiquitin attachment (34). Using the receptor-ubiquitin fusion methodology developed originally for Ste2p (47), we have shown that the translational fusion of a single ubiquitin moiety in place of the Ste3p PEST-like sequence can functionally substitute for this signal in endocytosis, indicating that the Ste3p PEST-like constitutive endocytosis signal functions solely in ubiquitination, both specifying ubiquitination and serving as the site for ubiquitin attachment (34).

While genetic analyses have identified a large number of functions that participate in the initial plasma membrane uptake step, essentially nothing is known about how these proteins collaborate to effect uptake. The endocytic phenotypes of these mutants are identical: all block uptake, trapping the endocytic substrate at the cell surface. Of particular interest are the early-acting endocytic functions that must prepare the endocytic substrate for ubiquitination. The present work identifies two functions which act before the ubiquitination step in the Ste3p constitutive endocytosis pathway: the redundant type I casein kinases Yck1p-Yck2p and the ankyrin repeat protein Akr1p. Yck1p-Yck2p appear to be required for phosphorylation of the Ste3p PEST-like sequence, likely activating it as a signal for ubiquitination. Akr1p, on the other hand, is required for the proper localization of the kinases to the cell surface. Instead of localizing to the plasma membrane, in akr1 cells, Yck2p localizes diffusely throughout the cytoplasm, showing a mutant localization similar to that seen with mutation of the Yck2p C-terminal dicysteinyl prenylation site.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. pND1092, which is GAL1-(3xHA)YCK2 carried on the CEN/ARS/URA3 vector plasmid pRS316 (43), was constructed by conventional methods from the wild-type YCK2 plasmid pL2.3 (33). A fragment containing the GAL1,10 promoter together with the RNA start and translational start plus three iterations of the hemagglutinin (HA) epitope was fused in frame to the second codon of the YCK2 open reading frame (ORF). pND1113 is identical to pND1092 except that the final two codons of the YCK2 ORF have been mutated from the Cys-Cys dicodon to a Ser-Ser dicodon.

Strains. Two different genetic backgrounds, isogenic either with NDY341 (35) or with LRB759 (28), were used in this work (Table 1). The NDY341 isogenic strain set derives from either the wild-type parent strain RH144-3D or the temperature-sensitive end4-1 mutant version RH268-1C (30). The founders for LRB759 background strains are the wild-type LRB759 and the temperature-sensitive yck1-1::ura3 yck2-2^ts derivative LRB757 (28). All other strains were constructed by standard gene replacement technologies from these four starting strains.

(i) STE3 alleles. The constructed strains have one of six different alleles present at the STE3 locus: either wild-type STE3, ste3::LEU2, GAL1-STE3, GAL1-STE3365, GAL1-STE3320-413, or GAL1-STE3-Ubiquitin(7KR, G76A). The first step in the introduction of the different GAL1-driven STE3 alleles was the replacement of wild-type STE3 by ste3::LEU2. For the ste3::LEU2 versions of LRB759 and LRB757, i.e., NDY547 and NDY548, the ste3::LEU2 allele from the plasmid pSL2165 was introduced into the STE3 strains via the two-step gene replacement strategy previously described (36). GAL1-STE3 was introduced into the chromosome using the GAL1-STE3-integrating plasmid pSL1904 (34) via the two-step gene replacement (36). The integrating plasmids used for introducing the GAL1-STE3365, GAL1-STE3320-413, and GAL1-STE3-Ubiquitin(7KR, G76A) alleles are identical to pSL1904 except for the deleted or fused sequences. The STE3365 allele corresponds to an in-frame deletion of Ste3p CTD residues 365 through 468 (effectively, a C-terminal truncation mutant, as the wild-type Ste3p sequence naturally terminates with residue 470) (7). The GAL1-STE3320-413 allele, an in-frame deletion of codons 320 to 413, was constructed by joining a SalI site created at codons 320 and 321 to an XhoI site created at codons 413 and 414 (36). The GAL1-STE3-Ubiquitin(7KR, G76A) allele has the C-terminal 72 codons of STE3 replaced by a mutant ubiquitin gene in which the seven ubiquitin Lys codons are replaced by Arg codons and in which the C-terminal ubiquitin Gly codon is changed to an Ala codon (34).

(ii) Disruption of endocytic functions. Three genes required for endocytosis, AKR1, END3, and VRP1, were disrupted in a variety of different strains. For the AKR1 disruption, an akr1::LEU2 allele was constructed on the URA3 integrating plasmid vector pRS306 (43). The LEU2 fragment replaces AKR1 codons 13 through 735 (the SalI-to-HindIII interval) of the 764-codon-long AKR1 ORF. Replacement of wild-type AKR1 by akr1::LEU2 utilized the previously described two-step gene replacement strategy (36). For the vrp1::URA3 disruption allele (27), URA3 replaces a complete deletion of the VRP1 coding sequence. Ura3⁺ gene replacements of chromosomal VRP1⁺ (37) were screened by PCR analysis of genomic DNA derived from the isolates. The end3::G418^r disruption allele was generated by PCR (11, 49). An upstream 60-mer oligonucleotide and a downstream 69-mer oligonucleotide were used. Nineteen bases at the 3' end of the upstream oligonucleotide and 22 bases at the 3' end of the downstream oligonucleotide served to prime PCR synthesis of the G418 marker from the plasmid template, pFA-kanMX2 (49). Forty-one and 47 bases at the 5' ends of the upstream and downstream oligonucleotides, respectively, were derived from the sequences immediately upstream or downstream of the END3 coding sequence. The resulting PCR product, therefore, has 41 and 47 bp of the END3 homologous sequences surrounding the G418^r selectable marker. G418^r yeast colonies were selected from transformed cells (11), and bona fide disruptants were culled from the G418^r transformants via PCR analysis of genomic DNA extracted from the isolates.

Immunoblot analyses. Turnover of Ste3p and Ste3-ubiquitin (Ste3-Ub) was assessed in the different end mutant backgrounds via a nonradioactive pulse-chase protocol (34) in which a "pulse" of receptor synthesis induced with the addition of 2% galactose to exponential cultures of GAL1-STE3 or of GAL1-STE3-Ub cells in YP-raffinose (2%) medium is followed by a "chase" in which continued synthesis is repressed with the addition of 3% glucose. Culture aliquots removed at various times during the glucose chase period were then subjected to an additional incubation with energy poisons as described previously (34). Briefly, culture aliquots collected by centrifugation were resuspended in 1.4 M sorbitol-25 mM Tris-Cl (pH 7.5)-10 mM sodium azide-10 mM potassium fluoride-2 mM MgCl₂. Following a 90-min incubation at 37°C, glass bead protein extracts were prepared as previously described (7). We have found the pretreatment with energy poisons prior to extract preparation to be of particular benefit for the analysis of Ste3-Ub protein turnover; the energy poison incubation apparently releases the Ste3-Ub protein from complexes which otherwise result in an extremely heterogeneous electrophoretic mobility (34). For other experiments, not involving the turnover of Ste3p or Ste3-Ub, the energy poisoning step was eliminated and glass bead extracts were directly prepared as previously described (7). The affinity-purified rabbit polyclonal antibodies used for detecting Ste3p were prepared as previously described (36).

Phosphatase treatment. For assessments of Ste3p ubiquitination and of Ste3320-413p phosphorylation, protein extracts were treated with potato acid phosphatase (Boehringer Mannheim Corp., Indianapolis, Ind.) at a final concentration of 0.06 U/ml prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis as described previously (35). Phosphatase treatment of in vivo-labeled protein extracts was done prior to the Ste3p immunoprecipitation as described previously (8).

Pulse-labeling and immune precipitation. Cells were pulse-labeled for 10 min with a mixture of [³⁵S]methionine and [³⁵S]cysteine and then chased with excess cold amino acids for an additional 10 min as described previously (35). Pulse-labeled cells were then treated for 15 min with a-factor or were mock treated using a one-sixth volume of the 20-fold-concentrated pheromone preparations as described previously (35). Cells were then collected by centrifugation, extracts were prepared via glass bead disruption, and the labeled Ste3p was immune precipitated as previously described (35).

Ligand-dependent endocytosis. To induce internalization of Ste3365p, cultures were treated with a half volume of a cell-free filtrate prepared from a saturated culture of EG123 cells transformed with the a-factor overproduction plasmid pKK16 (22). Mock a-factor preparations were obtained from the isogenic mfa1::LEU2 mfa2::URA3 strain SM1229 (26). Internalization was monitored via our standard whole-cell protease-shaving regimen. The treatment of intact cells with pronase (Calbiochem-Novabiochem Corp., La Jolla, Calif.) and the subsequent cell extraction protocol were described previously (7). As a control for the maintenance of cell integrity, the inaccessibility to the extracellular proteases of cytoplasmic phosphoglycerol kinase (Pgk1p) was routinely assayed by Western blotting with a Pgk1p-specific antiserum (provided by Tom Stevens, University of Oregon).

Indirect immunofluorescence. A 2-h period of expression of the epitope-tagged Yck2p was induced from cells carrying either the GAL1-(3xHA)YCK2 plasmid (pND1092) or the GAL1-(3xHA)YCK2(CCSS) plasmid (pND1113) with the addition of 2% galactose to cultures growing exponentially in YP-raffinose (2%) medium. Cells were fixed, spheroplasted, and otherwise prepared for indirect immunofluorescence as described previously (7) except that the overall time of fixation was reduced from 16 to 3 h. Detection of the 3xHA epitope-tagged Yck2 proteins utilized a 1-h incubation with 1:3,000-diluted HA.11 monoclonal antibody (MAb) (Berkeley Antibody Co., Berkeley, Calif.) followed by a 1-h incubation with 1:500 diluted Cy3-conjugated donkey anti-mouse immunoglobulin G secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.). Images were captured using a Eclipse 600 (Nikon Inc., Melville, N.Y.) equipped with a Micromax CCD camera (Roper Scientific Princeton Instruments Inc., Trenton, N.J.).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Akr1p and Yck1p-Yck2p act before Ste3p ubiquitination. To identify endocytic functions that act early in endocytosis, we have used the ubiquitination event as a point of demarcation for dividing early from late functions. Five endocytosis-defective mutants were used for this analysis: end3, end4-1, vrp1, akr1 mutants and a yck1 yck2^ts double mutant. Features of two of these mutants, the akr1 mutant and the yck1 yck2^ts double mutant, suggested possible early action points. The yck1 yck2^ts double mutant is defective for Ste2p phosphorylation as well as for Ste2p ubiquitination and endocytosis, leading Hicke et al. (16) to suggest that the YCK-dependent phosphorylation of Ste2p may be a prerequisite for ubiquitination and consequent endocytosis. Furthermore, the same yck mutant also has been shown to have a block in Ste3p constitutive uptake (28). For Akr1p, two findings suggest a possible early action point for this function. First, AKR1 was identified as a two-hybrid interactor with the Ste3p CTD (10). As the PEST-like constitutive endocytosis signal is contained within the CTD, Akr1p might participate in the initial recognition of this signal or in the presentation of the signal to the ubiquitination machinery. In addition, Akr1p also shows a differential involvement in the two Ste3p uptake modes: while it is required for constitutive endocytosis, it is dispensable for the mechanistically distinct ligand-dependent mode. As the signals which direct these two uptake modes differ (7, 36, 46), Akr1p may participate in the initial steps which select Ste3p for constitutive uptake. The other three functions, namely, END3, SLA2 (END4), and VRP1 (END5), all have demonstrated involvements with the actin cytoskeleton (51).

akr1

yck1 yck2^ts

end3

vrp1

pep4

end3

vrp1

pep4

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Effects of the endocytosis-defective mutants on Ste3p constitutive ubiquitination. Receptor ubiquitination was assessed in wild-type (wt) and mutant MAT GAL1-STE3 strains of two different genetic backgrounds. Strains isogenic to the wild-type strain NDY341 (left panel) include the temperature-sensitive end4-1 mutant (NDY342), as well as pep4 (NDY356), akr1 (NDY788), vrp1 (NDY1040), and end3 (NDY1046) mutants. In addition, the isogenic ste3 strain NDY343 was used as control for the specificity of Ste3p antibodies. Strains isogenic to NDY877 (wild-type MAT GAL1-STE3) (right panel) include the yck1 yck2ts double mutant (NDY913) and pep4 (NDY1080), akr1 (NDY1083), and end3 (NDY1118) strains. Protein extracts were prepared from cell cultures, following a 2-h Ste3p expression period, induced with galactose addition to log-phase cultures growing in raffinose medium. Fifteen minutes before cells were collected for extract preparation, cultures of cells isogenic to NDY341 (left panel) or to NDY877 (right panel) were shifted from 25°C to either 37 or 30°C, respectively. Protein extracts were treated with potato acid phosphatase (see Materials and Methods) to remove heterogeneity in gel migration which results from the heterogeneous phosphorylation of Ste3p and then subjected to SDS-PAGE and Western blotting with Ste3p-specific antibodies. Arrows indicate the positions of the mono- and diubiquitinated receptors; dashes indicate the positions of proteins that cross-react with the Ste3p antibodies.

end3

vrp1

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Turnover of an Ste3-Ub fusion protein in the endocytosis-defective mutants. A 2-h period of GAL1-driven expression of Ste3-Ub (A) or Ste3p (B) was induced in wild-type (wt) or end mutant yeast cultures with galactose addition and terminated with glucose addition. Fifteen minutes prior to glucose addition, cultures were shifted from 25 to 37°C. Coincident with the glucose addition (0-h time point) and at the indicated glucose chase time points, culture aliquots were removed and treated with energy poisons (see Materials and Methods). Extracts were then prepared, and the loss of the Ste3 antigen was monitored by Western blotting with Ste3p-specific antibodies. (A) Ste3-Ub turnover. Wild-type and end mutant MAT GAL1-STE3-UB strains from two different strain backgrounds were used. Strains in the end4-1 background (top panel) included wild-type (NDY990), end4-1 (NDY991), pep4 (NDY992), akr1 (NDY1011), vrp1 (NDY1042), and end3 (NDY1076) strains. NDY343 (ste3) was included as control for the specificity of Ste3p antibodies. Strains in the yck1 yck2ts background (bottom panel) included wild-type (NDY1012), end3 (NDY1074), yck1 yck2ts (NDY1090), akr1 (NDY1085), akr1 pep4 (NDY1218), and yck1 yck2ts pep4 (NDY1223) strains. The high level of Ste3-Ub protein apparent in NDY992 cells (pep4) likely reflects the augmented growth rate of this strain relative to the isogenic wild-type strain seen in raffinose- and/or galactose-containing culture media, not to the pep4 turnover blockade: the end mutants in which turnover is blocked (e.g., end3, end4, vrp1, yckts, and akr1 mutants) do not have similar levels of Ste3-Ub protein over accumulation. (B) Ste3p turnover in wild-type and end mutant cells. Four isogenic MAT GAL1-STE3 strains were used for this analysis, i.e., wild-type (NDY877), yck1 yck2ts (NDY913), akr1 (NDY1083), and end3 (NDY1118) strains.

Participation of the YCKs and AKR1 in Ste3p phosphorylation. Ste3p is subject to a constitutive level of phosphorylation, which increases when cells are stimulated by pheromone (35). Much of this phosphorylation maps to the Ser- and Thr-rich regulatory CTD (8). Isolated from resting cells unstimulated by pheromone, Ste3p displays a heterogeneous gel mobility consistent with a heterogeneous constitutive phosphorylation (Fig. 3A). When isolated from a-factor-treated cells, receptor gel mobility is slowed, consistent with increased phosphorylation. Both the constitutive and ligand-induced phosphorylations collapse to a single, faster-migrating species with phosphatase treatment (Fig. 3A).

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Effects of akr1 and yck mutations on Ste3p phosphorylation. The constitutive and a-factor-induced phosphorylation of Ste3p was monitored by assessing the effects of a-factor treatment on the gel mobility of newly synthesized Ste3p. MAT cells were pulse-labeled for 10 min with [35S]methionine-cysteine, chased for 10 min with excess cold amino acids, and then treated for an additional 15 min with a-factor or mock treated in parallel. This protocol allows the labeled Ste3p to be maximally available at the cell surface for binding the a-factor ligand. Protein extracts were prepared from the cells, and the Ste3p purified by immune precipitation was subjected to SDS-PAGE and autoradiography. (A) Constitutive and ligand-induced phosphorylation of Ste3p. Extracts from wild-type MAT cells (NDY414), treated with or without a-factor pheromone, were either digested with potato acid phosphatase (p'ase) or mock digested (no phosphatase added). The NDY414 strain used for this experiment has Ste3p expressed from the HIS3 promoter instead of from its natural promoter. The level of Ste3p expression from the HIS3 promoter is increased two- to threefold relative to the basal-level expression from its natural promoter. The increased expression results in no obvious alteration to the profile of phosphorylated species apparent by this pulse-chase analysis (compare with panels B and C; Ste3p for these panels is expressed from its natural endogenous promoter). (B) Effects of the akr1 and yck mutations on Ste3p phosphorylation for cells growing at 30°C. Wild-type (wt) MAT cells (LRB759) as well as isogenic akr1 (NDY1037) and yck1 yck2ts (LRB757) cells were cultured at 30°C and then labeled and treated with a-factor as described above. (C) Effects of the yck mutations on Ste3p phosphorylation for cells growing at 37°C. Wild-type (LRB759) and yck1 yck2ts mutant (LRB757) cells cultured at 25°C were shifted to 37°C 10 min prior to the start of pulse-labeling.

yck1 yck2^ts

akr1

yck1 yck2^ts

yck1 yck2^ts

yck1 yck2^ts

yck1 yck2^ts

yck1 yck2^ts

STE3320-413

end3

akr1

yck1 yck2^ts

end3

akr1

yck1 yck2^ts

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Effects of akr1 and yck mutations on the phosphorylation of Ste3320-413p. MAT GAL1-STE3320-413 cells, either wild-type (wt) (NDY1039), akr1 (NDY1061), yck1 yck2ts (NDY1045), or end3 (NDY1070), growing in raffinose medium at 25°C were shifted to 30°C 1 h prior to the initiation of galactose-induced expression. Following a 90-min period of galactose-induced expression, cell extracts were prepared and were treated with phosphatase (p'ase) (+) or mock treated (). Samples were then analyzed by SDS-PAGE and Western blotting with Ste3p-specific antibodies.

AKR1 is required for Ste2p phosphorylation. Yck1p and Yck2p are required for both the constitutive and -factor-induced phosphorylation of Ste2p, the -factor pheromone receptor (16). Given the common phenotypes noted above for akr1 and yck mutants, we wondered if Akr1p might also participate. Extracts prepared from either -factor-treated or untreated wild-type MATa cells and isogenic akr1 and yck1 yck2^ts mutants were analyzed by Western blotting as previously described (16). A 10-min -factor treatment of wild-type cells results in a striking change in Ste2p gel mobility; the bulk of the receptor population shifts to a more slowly migrating cluster (Fig. 5). We have previously shown through phosphatase treatment that this Ste2p mobility shift is primarily due to induced phosphorylation; the ubiquitinated Ste2p species induced with this pheromone treatment are less prominent and migrate to higher-molecular-weight positions (8). Consistent with the previous report (16), this induced phosphorylation is largely blocked in yck1 yck2^ts cells (Fig. 5); only a small fraction of the receptor population undergoes a demonstrable shift upon -factor treatment. Compared to wild-type cells, akr1 cells also are clearly defective for the -factor-induced phosphorylation (Fig. 5). We note, however, that the phosphorylation defect manifested by these akr1 cells is somewhat less severe than that found for the isogenic yck^ts strain (Fig. 5). Nonetheless, for both mutants, the bulk of the receptor protein is not subject to a major shift in gel mobility, indicating that in both mutants the -factor induced phosphorylation of Ste2p is strongly impaired. Thus, we conclude that Akr1p is required together with Yck1p-Yck2p for this phosphorylation.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Akr1p is required together with Yck1p-Yck2p for the -factor-induced phosphorylation of Ste2p. Cultures of three isogenic MATa strains, i.e., wild-type (wt) (LRB758), yck1 yck2ts (LRB756), and akr1 (NDY1215) strains, growing in rich medium at 25°C were shifted to 37°C for 15 min and then either treated with 106 M -factor (+) for 10 min or mock treated in parallel (). Extracts were prepared and analyzed by Western blotting with a Ste2p-specific antiserum (provided by James Konopka, SUNY at Stony Brook). Extracts prepared from the isogenic MAT strain LRB759, which does not express Ste2p, were included as a control for the specificity of the antiserum (con). The brackets at right indicate the positions of the hyperphosphorylated Ste2p species present following -factor treatment.

Ste3p ligand-dependent endocytosis in yck and akr1 cells. It has been reported that while being required for the constitutive endocytosis of Ste3p, Akr1p is dispensable for the alternative, Ste3p ligand-dependent uptake mode (10). We were interested to see if the yck^ts mutant might have the same phenotype as the akr1 mutant in this regard as well. The ligand-dependent mode of Ste3p uptake has been observed mainly under conditions which disable rapid constitutive endocytosis (7, 10). For instance, while the 365 truncated Ste3p mutant which lacks the PEST-like endocytosis signal is blocked for constitutive endocytosis, it remains capable of a ligand-induced endocytosis, which is apparent when Ste3365p-expressing cells are exposed to the a-factor ligand (7). Unlike the constitutive uptake mode, which is coupled to rapid degradation of the receptor in the vacuole, our recent evidence indicates that the Ste3p ligand-dependent pathway links to two distinct receptor fates: while some of the internalized receptor is subject to a slow PEP4-dependent degradation, the bulk of the internalized receptor recycles back to the cell surface (L. Chen and N. G. Davis, submitted for publication).

akr1

yck1 yck2^ts

end3

yck1 yck2^ts

end3

akr1

akr1

yck1 yck2^ts

akr1

View larger version (47K): [in this window] [in a new window]   FIG. 6.   Akr1p and Yck1p-Yck2p remain partially competent for Ste3p ligand-induced endocytosis. The a-factor-induced internalization of the 365 Ste3p mutant was assessed by monitoring its changing availability to added, extracellular proteases. Uptake of the 365 mutant was followed in four isogenic MAT GAL1-STE3365 strains, i.e., wild-type (wt) (NDY1072), end3 (NDY1117), akr1 (NDY1216), and yck1 yck2ts (NDY662) strains. Thirty minutes following a 90-min period of galactose-induced Ste3365p expression (terminated by glucose addition), cultures were shifted from 25 to 37°C (nonpermissive for the yckts mutant). Following an additional 15 min at 37°C, cultures were treated with a-factor or mock treated. At the indicated times following the start of the pheromone treatment, culture aliquots were removed and the intact cells were digested with proteases (see Materials and Methods). Extracts prepared from these cells were analyzed by Western blotting with Ste3p-specific antibodies.

AKR1 is required for the cell surface localization of Yck2p. The common phenotypes of akr1 and yck1 yck2^ts mutants suggest the possibility of functional collaboration. Akr1p could act together with the YCKs as a subunit of the active kinase complex. Alternatively, Akr1p might bind to the PEST-like sequence of Ste3p newly delivered to the plasma membrane and serve to present this signal to the YCKs for phosphorylation. A third possibility, tested below, is that Akr1p is required for proper localization of the YCKs to the plasma membrane. Plasma membrane localization of Yck1p and Yck2p depends upon prenylation of C-terminal dicysteine sequences (48).

akr1

yck1 yck2^ts

View larger version (65K): [in this window] [in a new window]   FIG. 7.   Akr1p is required for localization of Yck2p to the plasma membrane. The wild-type strain (NDY877) or the isogenic akr1 strain (NDY1083) transformed by one of two plasmids carrying GAL1-driven YCK2 constructs N-terminally tagged by the 3xHA epitope (either pND1092, which carries the tagged wild-type Yck2p, or pND1113, which carries the equivalently tagged Yck2p mutant which has its C-terminal Cys-Cys prenylation motif replaced by Ser-Ser [CCSS]) was used. Following a 2-h period of galactose-induced expression, cells were removed from culture and fixed, and localization of the wild-type or mutant tagged Yck2p was probed in an indirect immunofluorescence protocol using the mouse HA.11 MAb directed against the HA epitope, followed by Cy3-conjugated secondary antibody directed against mouse immunoglobulin-G. As a control for the cross-reaction of the HA.11 MAb, NDY877 cells not carrying an HA-tagged construct were fixed and processed in parallel (no HA). A Nomarski (differential interference contrast [DIC]) image of each cell grouping is shown just to the right of the fluorescent image (anti-HA).

akr1

akr1

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Akr1p and the YCKs act early for Ste3p constitutive endocytosis. Two functions have been identified as acting early, prior to the ubiquitination event, for the constitutive uptake of Ste3p: Akr1p and the redundant casein kinase pair Yck1p-Yck2p. In addition to being defective for receptor ubiquitination, mutants with mutations in these two functions also were found to be defective for the phosphorylation of Ste3320-413p. While having much of the Ste3p CTD together with the majority of the potential Ser-Thr CTD phosphorylation sites deleted, the 320-413 receptor mutant retains a functional PEST-like endocytosis signal together with its 15 potential Ser-Thr phosphorylation sites. As both uptake and phosphorylation of Ste3320-413p require Akr1p and Yck1p-Yck2p function, we suggest, as has been previously suggested for the -factor pheromone receptor (16) and the uracil permease (23), that phosphorylation likely is a prerequisite for ubiquitination of Ste3p and thus also for its constitutive endocytosis. Unlike the case for Ste2p, which shows a more complete dependence of receptor phosphorylation on the YCKs (16), our results suggest for Ste3p that the YCKs may be devoted specifically to phosphorylation of the PEST-like sequence. While Yck1p and Yck2p may act directly in phosphoryl activation of the PEST-like signal, the Akr1p requirement appears to be for proper localization of the kinases to the plasma membrane. In akr1 mutants, the YCKs are mislocalized to the cytoplasm and thus fail to efficiently find their plasma membrane-localized receptor substrate.

Common phenotypes of akr1 and yck mutants. In addition to early action in Ste3p constitutive endocytosis, other phenotypes link Akr1p and Yck1p-Yck2p. Both are required for Ste2p phosphorylation (Fig. 5). Indeed, the Akr1p requirement for Ste2p phosphorylation likely accounts for the reported Akr1p requirement for Ste2p endocytosis as well (10, 16). In addition, mutations in the two functions show similar effects on the ligand-dependent endocytosis of Ste3365p: both mutants remain partially competent for uptake, and for both, uptake appears to be decoupled from turnover (Fig. 6). In both mutants following a-factor challenge, a period of initially rapid ligand-dependent internalization is followed by a period in which continued receptor internalization is curtailed; a plateau to receptor internalization is achieved, with approximately 40% of the receptor internalized and the remaining 60% localized to the cell surface. In wild-type cells growing at 30°C (rather than the 37°C utilized for Fig. 6) an internalization plateau strikingly similar to that observed in the akr1 and yck mutant backgrounds (Fig. 6) was seen (Chen and Davis, submitted). Such plateaus are easily explained within the context of receptor recycling, reflecting an equilibrium where continued internalization of the receptor is balanced by the rate of its recycling return to the cell surface; at this equilibrium point, net internalization halts. In wild-type cells, two fates are coupled to Ste3p ligand-dependent uptake: while the bulk of internalized receptor recycles back to the cell surface, some receptor is delivered to the vacuole for turnover (Chen and Davis, submitted). For the akr1 and yck mutants, turnover appears to be blocked (Fig. 6), suggesting a defect specific for the degradative arm of this uptake pathway. Thus, while YCK-dependent phosphorylation may not be required for triggering the ligand-dependent uptake of Ste3p, Yck1p-Yck2p phosphorylation of the receptor or of other endocytic components may function at a downstream trafficking event to augment endosome-to-vacuole transport of the receptor.

akr1

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gcs1

Role of Akr1p in YCK localization. How does Akr1p support the plasma membrane localization of the YCKs? The plasma membrane localization of Yck1p and Yck2p depends upon a C-terminal Cys-Cys prenylation signal (32, 48). The Yck2p mislocalization in akr1 cells, we found, mirrored the diffuse cytoplasmic localization seen for the prenylation defective CCSS Yck2p mutant, suggesting that Akr1p may function in Yck2p prenylation. However, the enzymatic system responsible for protein prenylation has been extensively characterized (52), and Akr1p has yet to turn up as a component.