Bivalent Inhibitor of the N-end Rule Pathway*

Yong Tae KwonDagger , Frédéric Lévy§, and Alexander VarshavskyDagger

From the Dagger  Division of Biology, California Institute of Technology, Pasadena, California 91125 and the § Ludwig Institute for Cancer Research, 155, ch. Des Boveresses, CH-1066 Epalinges, Switzerland

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta -galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta -galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Among the targets of the N-end rule pathway are intracellular proteins bearing destabilizing N-terminal residues (1, 2). This proteolytic pathway is one of several pathways of the ubiquitin (Ub)1 system, whose diverse functions include the regulation of cell growth, division, differentiation, and responses to stress (3-6). Ub is a 76-residue eukaryotic protein that exists in cells either free or conjugated to other proteins. Many of the Ub-dependent regulatory circuits involve processive degradation of ubiquitylated proteins by the 26 S proteasome, an ATP-dependent multisubunit protease (7, 8).

The N-end rule is organized hierarchically. In the yeast Saccharomyces cerevisiae, Asn and Gln are tertiary destabilizing N-terminal residues in that they function through their conversion, by the NTA1-encoded N-terminal amidase, into the secondary destabilizing N-terminal residues Asp and Glu. The destabilizing activity of N-terminal Asp and Glu requires their conjugation by the ATE1-encoded Arg-tRNA-protein transferase (R-transferase) to Arg, one of the primary destabilizing residues (reviewed in Refs. 1 and 9). In mammals, two distinct N-terminal amidases specific, respectively, for N-terminal Asn or Gln mediate the conversion of these tertiary destabilizing residues into the secondary destabilizing residues Asp or Glu (10, 11). The set of secondary destabilizing residues in vertebrates contains not only Asp and Glu but also Cys, which is a stabilizing residue in yeast (9, 12, 13).

The primary destabilizing N-terminal residues are bound directly by N-recognin, the E3 (recognition) component of the N-end rule pathway. In S. cerevisiae, N-recognin is the UBR1-encoded 225-kDa protein that binds to potential N-end rule substrates through their primary destabilizing N-terminal residues: Phe, Leu, Trp, Tyr, Ile, Arg, Lys, and His (1, 14). The Ubr1 genes encoding mouse and human N-recognin (also called E3alpha ) have been cloned as well (15). N-recognin has at least two substrate-binding sites. The type 1 site is specific for the basic N-terminal residues Arg, Lys, and His. The type 2 site is specific for the bulky hydrophobic N-terminal residues Phe, Leu, Trp, Tyr, and Ile (1, 12, 16, 17). N-recognin can also target short-lived proteins such as Cup9p (18) and Gpa1p (19, 20), which lack destabilizing N-terminal residues. The Ubr1p-recognized degradation signals of these proteins remain to be characterized in detail.

The known functions of the N-end rule pathway include the control of di- and tripeptide import in S. cerevisiae through the degradation of Cup9p, a transcriptional repressor of the peptide transporter gene PTR2 (18, 21); a mechanistically undefined role in the Sln1p-dependent phosphorylation cascade that mediates osmoregulation in S. cerevisiae (22); the degradation of Gpa1p, a Galpha protein of S. cerevisiae (19, 20); and the conditional degradation of alphaviral RNA polymerase in virus-infected metazoan cells (23). Physiological N-end rule substrates were also identified among the proteins secreted into the cytosol of the host cell by intracellular parasites such as the bacterium Listeria monocytogenes (24). Short half-lives of these proteins are required for the efficient presentation of their peptides to the immune system (24). A partial inhibition of the N-end rule pathway was reported to interfere with mammalian cell differentiation (25) and to delay limb regeneration in amphibians (26). Recent evidence suggests that the N-end rule pathway mediates a large fraction of the muscle protein turnover (27) and plays a role in catabolic states that result in muscle atrophy (28).

Targeted mutagenesis has been used to inactivate the N-end rule pathway in Escherichia coli and S. cerevisiae (14, 29). Analogous mutants have recently been constructed in the mouse as well.2 These approaches notwithstanding, an efficacious inhibitor of the N-end rule pathway would be useful as well, especially with organisms less tractable genetically. The emerging understanding of the N-end rule pathway in mammals suggests that selective inhibition or activation of this proteolytic system may also have medical applications. Previous work has shown that millimolar concentrations of amino acid derivatives such as dipeptides bearing destabilizing N-terminal residues can selectively inhibit the N-end rule pathway in extracts from rabbit reticulocytes (12, 17) and Xenopus eggs (13), and in intact S. cerevisiae cells as well (16). However, the same dipeptides were observed to have at most marginal effects on the N-end rule pathway in intact mammalian cells.3 One limitation of dipeptide inhibitors is their apparently low affinity for the type 1 and the type 2 site of N-recognin (30).

In the present work, we explored the possibility that a bivalent ligand can bind simultaneously to the type 1 and type 2 sites of N-recognin (see Fig. 1A). Similarly to the previously characterized bivalent interactions that involve either macromolecules or small molecules (31, 32), the cooperativity of binding at two independent, mutually nonexclusive sites would be expected to increase the affinity between N-recognin and a bivalent inhibitor by orders of magnitude, in comparison with the affinity of a monovalent binding by the same compound. We show that a bivalent inhibitor of the N-end rule pathway is feasible and consider the implications of this advance.

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Strains and General Techniques-- The S. cerevisiae strains used were JD52 (MATa ura3-52 his3-Delta 200 leu2-3,112 trp1-Delta 63 lys2-801) and JD55 (MATa ura3-52 his3-Delta 200 leu2-3,112 trp1-Delta 63 lys2-801 ubr1 Delta ::HIS3) (19, 33). Cells were grown on rich (YPD) or synthetic medium containing either 2% dextrose (SD medium), 2% galactose (SG medium), or 2% raffinose (SR medium) (34). To induce the PCUP1 promoter, CuSO4 was added to a final concentration of 0.1 mM. Transformation of S. cerevisiae was carried out using the lithium acetate method (35).

Plasmids-- The high copy (2µ-based) plasmids pRDelta -beta gal-TRP1 and pRDelta -beta gal-HIS3, which expressed Arg-eDelta K-beta gal (Ub-Arg-eDelta K-beta gal) (see Fig. 2A) from the galactose-inducible PCYC1/GAL1 hybrid promoter (2), were produced by replacing the URA3 marker gene of pFL7 with either TRP1 or HIS3. pLDelta -beta gal-TRP1 and pLDelta -beta gal-HIS3, both of which expressed Leu-eDelta K-beta gal (Ub-Leu-eDelta K-beta gal), were produced by replacing the Ub-Arg domain of pRDelta -beta gal-TRP1 and pRDelta -beta gal-HIS3 with Ub-Leu domain of the pLL2 plasmid.4 The plasmid pFL7 was produced from pUB23-R (2) by converting the lysine codons 15 and 17 of the extension eK into arginine codons (36, 37), yielding a construct encoding the extension eDelta K in front of a beta gal moiety lacking the first 23 residues of wild type beta gal (see Fig. 2A). The low copy, pRS315 vector-derived (38) plasmid pR-eDelta KhaUra3-R3R7 expressed Arg-eDelta K-ha-Ura3pK3R,K7R (Ub-Arg-eDelta K-ha-Ura3pK3R,K7R) from the PCUP1 promoter. Arg-eDelta K-ha-Ura3pK3R,K7R (see Fig. 2B) is called Arg-Ura3p in the main text. In this N-end rule substrate, the residues Lys-3 and Lys-7 of the S. cerevisiae Ura3p were converted to arginines (see "Results and Discussion"). In addition, the ha epitope tag (39) was placed between eDelta K and Ura3pK3R,K7R (see Fig. 2B). The plasmid pR-eDelta KhaUra3-R3R7 was produced from pFL1 (encoding Ub-Arg-eDelta K-ha-Ura3p) through site-directed mutagenesis of the URA3 codons for Lys-3 and Lys-7. pFL1 was produced from pKM1235 (which encoded Ub-Arg-eK-ha-Ura3p)5 by converting the eK-coding sequence into the one encoding eDelta K.

Pulse-Chase and Plating Efficiency Assays-- Pulse-chase assays with S. cerevisiae in mid-exponential growth (A600 of ~1) utilized 35S-EXPRESS (NEN Life Science Products) and were carried out as described previously (10, 19), including the immunoprecipitation with anti-beta gal and anti-ha antibodies and quantitation with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To determine plating efficiency, S. cerevisiae strains JD52 (UBR1) and JD55 (ubr1Delta ) expressing Arg-Ura3p (Ub-Arg-eDelta K-ha-Ura3pK3R,K7R; see Fig. 2B) were co-transformed with plasmids indicated in the legend to Fig. 3. The transformants were cultured in the raffinose-based medium (SR) lacking Leu, His, and Trp for 20 h. The cultures were then diluted into the otherwise identical galactose-containing (SG) medium to a final A600 of 0.1. At an A600 of 0.4, cultures were either supplemented with 0.1 mM CuSO4 or left unsupplemented. At the A600 of 1.0, the cultures were diluted with SG (which lacks Leu, His, and Trp) either containing or lacking 0.1 mM CuSO4 and were plated on the plates of the same medium composition that also either contained or lacked uracil. The plating efficiency (%) was defined as the ratio of the number of colonies on SG (-Leu, -His, -Trp, -Ura) plates to the number of colonies on SG (-Leu, -His, -Trp) plates, at the same concentration of CuSO4. For each measurement, colonies on 15 plates were counted to yield the average number of colonies per plate.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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We constructed a bivalent N-end rule inhibitor (Fig. 1A) from the previously studied N-end rule substrates derived from E. coli beta gal (2). In eukaryotes, linear Ub-protein fusions are rapidly cleaved by deubiquitylating enzymes at the Ub-protein junction, making possible the production of otherwise identical proteins bearing different N-terminal residues, a technical advance that led to the finding of the N-end rule (2). A beta gal-based N-end rule substrate contains a destabilizing N-terminal residue (produced in vivo using the Ub fusion technique (1)); a ~45-residue, E. coli Lac repressor-derived N-terminal extension called eK (extension e bearing lysines K); and the beta gal moiety lacking its first 21 residues. The resulting X-eK-beta gal is a short-lived protein in both yeast and mammalian cells, whereas an otherwise identical protein bearing a stabilizing N-terminal residue such as Met or Val is metabolically stable (1, 2). An N-degron comprises a destabilizing N-terminal residue and a Lys residue (or residues), the latter being the site of formation of a multi-Ub chain (1, 36). (Ubr1p can also recognize a set of other, internal degrons, which remain to be characterized (18).) If Lys-15 and Lys-17 of the eK extension are replaced by the Arg residues (which cannot be ubiquitylated), the resulting X-eDelta K-beta gal (Fig. 2A) is long-lived in vivo even if its N-terminal residue is destabilizing in the N-end rule (1, 37).


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Fig. 1.   The concept of a bivalent inhibitor of the N-end rule pathway. A, the type 1 and type 2 sites of S. cerevisiae Ubr1p (N-recognin), which are specific, respectively, for the basic (Arg, Lys, and His) and bulky hydrophobic (Phe, Leu, Trp, Tyr, and Ile) N-terminal residues. In the diagram, the type 1 and type 2 sites are occupied by their ligands, the N-terminal Arg and Leu, borne by a heterodimeric bivalent inhibitor (actually, a tetrameric beta gal-based protein in the present work). A test substrate bearing Arg, a type 1 destabilizing N-terminal residue is shown as well. The test substrate, in contrast to the protein-based inhibitor, bears at least one internal Lys residue (not indicated in the diagram) that can function as a component of the N-degron. The type 1 and type 2 sites of N-recognin are shown located close together in the N-terminal region of the 225-kDa Ubr1p. The recent genetic dissection of the Ubr1p substrate-binding sites6 placed the type 1 and type 2 sites close together in the ~60-kDa N-terminal region of the 225-kDa Ubr1p. B, a diagram illustrating the expected frequencies of heterodimeric (Arg- and Leu-bearing) dimers within a beta gal-based bivalent inhibitor. Specifically, at equal levels of expression of the two beta gal-based polypeptide chains, 50% of beta gal tetramers would be expected to be heterotetramers in which at least one of the two dimers bears different (Arg and Leu) N-terminal residues. In the beta gal tetramer, the two N termini of each dimer are spatially close, exposed, and oriented in the same direction (40). See also "Results and Discussion."


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Fig. 2.   Designs of bivalent inhibitor and test substrate. A, the beta gal-based fusions (the residue X was either Arg or Leu) used to construct the Arg/Leu-bearing bivalent inhibitor. The Ub moiety of the fusions was cotranslationally removed in vivo by deubiquitinating enzymes (1). The ~45-residue, E. coli Lac repressor-derived sequence termed eDelta K (extension (e) lacking lysines (Delta K)), is described in the main text. The beta gal part of the fusion lacked the first 21 residues of wild type beta gal (2). B, the Ura3p-based N-end rule substrate, Arg-eDelta K-ha-Ura3pK3R,K7R, derived from Ub-Arg-eDelta K-ha-Ura3pK3R,K7R and denoted Arg-Ura3p, is described in the main text.

In the present work, we used the metabolically stable Arg-eDelta K-beta gal (produced from Ub-Arg-eDelta K-beta gal) and Leu-eDelta K-beta gal (produced from Ub-Leu-eDelta K-beta gal). These proteins retain the ability to bind, respectively, to the type 1 and type 2 sites of N-recognin but cannot be ubiquitylated (37), apparently because the most N-terminal Lys residue in X-eDelta K-beta gal, at position 239, is too far from the N terminus of the protein. In the beta gal tetramer, the two N termini of each dimer are spatially close, exposed, and oriented in the same direction (40). At equal levels of expression of the two beta gal-based polypeptide chains such as Arg-eDelta K-beta gal and Leu-eDelta K-beta gal, 50% of beta gal tetramers would be expected to be heterotetramers in which at least one of the two dimers bears different (Arg and Leu) N-terminal residues (Fig. 1B). If the type 1 and type 2 substrate-binding sites of the 225-kDa Ubr1p are appropriately located and oriented, they might be able to bind the Arg- and Leu-bearing subunits of the mixed beta gal tetramer, especially in view of the presumed flexibility of the eDelta K extension (1) (Fig. 1A).

The reporter N-end rule substrate in this study was Arg-eDelta K-ha-Ura3pK3R,K7R, denoted below as Arg-Ura3p (Fig. 2B). This ha-tagged, type 1 N-end rule substrate was produced from Ub-Arg-eDelta K-ha-Ura3pK3R,K7R through the cotranslational in vivo cleavage by deubiquitinating enzymes (1, 6, 41). The lysine-lacking eDelta K extension of Arg-eDelta K-ha-Ura3pK3R,K7R, and the replacement of the first two lysines of the Ura3p moiety with arginines were used to decrease the rate of degradation of Arg-Ura3p by the N-end rule pathway and also to reduce the slow but detectable degradation of Arg-Ura3p by yet another pathway, through a degron distinct from the N-degron.3 Several Lys residues of Ura3p other than Lys-3 and Lys-7 are also close to its N terminus, thus accounting for the absence, in this case, of the all-or-none effect on the reporter degradation that is observed when eK is replaced with eDelta K in an X-eK-beta gal substrate (37). The Lys-3 right-arrow Arg and Lys-7 right-arrow Arg modifications decreased the enzymatic activity of the Ura3p moiety.2 The reduced enzymatic activity of Ura3pK3R,K7R facilitated selection assays (Figs. 3 and 4).


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Fig. 3.   A bivalent, but not monovalent, inhibitor of Ubr1p confers Ura+ phenotype on cells expressing the short-lived N-end rule substrate Arg-Ura3p. S. cerevisiae JD52 (UBR1) expressing Arg-Ura3p (Ub-Arg-Ura3p), a short-lived reporter (Fig. 2B), were cotransformed, alternatively, with pRS423 (HIS3-based control vector) and pRS424 (PGAL1, TRP1-based control vector) (denoted as 423+424); with pRS424 and pRDelta -beta gal-HIS3, expressing Arg-eDelta K-beta gal (Ub-Arg-eDelta K-beta gal) (denoted as R+424; monovalent inhibitor); with pRS423 and pLDelta -beta gal-TRP1, expressing Leu-eDelta K-beta gal (Ub-Leu-eDelta K-beta gal) (denoted as 423+L); with pRDelta -beta gal-HIS3 and pRDelta -beta gal-TRP1, both expressing Arg-eDelta K-beta gal (denoted as R+R; monovalent inhibitor); with pLDelta -beta gal-HIS3 and pLDelta -beta gal-TRP1, both expressing Leu-eDelta K-beta gal (denoted as L+L); or with pRDelta -beta gal-HIS3 and pLDelta -beta gal-TRP1, expressing Arg-eDelta K-beta gal and Leu-eDelta K-beta gal (denoted as R+L; the bivalent inhibitor). Cells were streaked on SG medium containing 0.1 mM CuSO4 and lacking Leu, His, Trp, and Ura (upper left panel), on the otherwise identical medium lacking the added CuSO4 (upper right panel), or on the Ura-containing SG medium lacking Leu, His, and Trp (controls; lower left panel). Plates were incubated at 30 °C for 3 days.


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Fig. 4.   Plating efficiencies of S. cerevisiae expressing Arg-Ura3p in the presence of bivalent and monovalent inhibitors of the N-end rule pathway. A, S. cerevisiae JD52 (UBR1) and JD55 (ubr1Delta ) expressing Arg-Ura3p were cotransformed with the sets of plasmids described and denoted in the legend to Fig. 3. The transformants were cultured as described under "Experimental Procedures" and plated on either SG(-Leu, -His, -Trp, -Ura) plates or control plates SG(-Leu, -His, -Trp) containing 0.1 mM CuSO4. The plating efficiencies shown are the values produced by normalization against the absolute plating efficiency (92%) of the positive control: the ubr1Delta strain JD55 expressing Arg-Ura3p and bearing the vector pRS424. B, the same experiment was done using plates lacking the added CuSO4. The plating efficiencies shown are the values produced by normalization against the positive control used in A. Under these growth conditions (no added CuSO4), the absolute plating efficiency of the positive control was 26%.

The first bivalent inhibitor assay employed ura3 S. cerevisiae expressing Arg-Ura3p (Fig. 2B) from the uninduced PCUP1 promoter. The Ubr1p-mediated degradation of Arg-Ura3p (t1/2 of ~8 min) and its correspondingly low steady-state concentration rendered wild type (UBR1) cells phenotypically Ura-, whereas ubr1Delta strains expressing Arg-Ura3p were phenotypically Ura+ (Figs. 3 and 4 and data not shown). Cells expressing Arg-Ura3p were cotransformed with two control plasmids (vectors; 423+424 in Fig. 3). Alternatively, these cells were cotransformed with two plasmids (bearing different selectable markers) that expressed either Arg-eDelta K-beta gal alone (R+R in Fig. 3), Leu-eDelta K-beta gal alone (L+L in Fig. 3), or both of them together (R+L in Fig. 3; the bivalent inhibitor mode) from a galactose-inducible promoter. Pairs of alternatively marked plasmids were used to make certain that the conditions of expression and the total amounts of beta gal-based proteins produced remained the same in all of these settings. The transformants were streaked on SG medium lacking uracil.

Remarkably, only those Arg-Ura3p-expressing cells that expressed both Arg-eDelta K-beta gal and Leu-eDelta K-beta gal became Ura+ under these conditions (Fig. 3). The cells that expressed either Arg-eDelta K-beta gal alone or Leu-eDelta K-beta gal alone remained Ura-, as did the cells that received control plasmids (Fig. 3). (The same cells grew equally well in the control SG medium containing uracil (Fig. 3, bottom left panel).) Note that the monovalent inhibitors were ineffective despite the fact that the concentration of either the Arg-based N terminus alone or the Leu-based N terminus alone was twice the concentration of the same N termini in the case of the bivalent inhibitor.

To quantify the effect of coexpressing Arg-eDelta K-beta gal and Leu-eDelta K-beta gal on the rescue of the Ura+ phenotype, a plating efficiency assay was carried out with the same transformants. Equal amounts of cells were plated on SG(+Ura) and SG(-Ura) plates, and the numbers of colonies were determined. When the Arg-Ura3p reporter was expressed at a sufficiently low rate (uninduced PCUP1 promoter), cells became Ura+ (through metabolic stabilization of Arg-Ura3p) only in the presence of both Arg-eDelta K-beta gal and Leu-eDelta K-beta gal (Fig. 4B). A weak stabilizing effect of Arg-eDelta K-beta gal alone could be detected only at a ~20-fold higher level of Arg-Ura3p expression (induced PCUP1 promoter) (Fig. 4A). No stabilization of Arg-Ura3p was observed in the presence of Leu-eDelta K-beta gal under any conditions (Fig. 4), confirming the specificity of inhibition in regard to the type (basic or bulky hydrophobic) of the primary destabilizing N-terminal residue of the reporter. Higher sensitivity of this assay at the higher level of Arg-Ura3p expression results from a higher steady-state level of the short-lived Arg-Ura3p, so that even its marginal stabilization suffices to render a small fraction of cells Ura+ (Fig. 4A; compare with Fig. 4B).

To analyze directly the in vivo degradation of Arg-Ura3p in the presence of different combinations of X-eDelta K-beta gal proteins, the transformants of Figs. 3 and 4 were subjected to pulse-chase analysis, with immunoprecipitation of both Arg-Ura3p and the (long-lived) X-eDelta K-beta gals (Fig. 5). Quantitation of the resulting electrophoretic patterns (Fig. 5C) confirmed and extended the conclusions reached through phenotypic analyses (Figs. 3 and 4). Specifically, the normally short-lived Arg-Ura3p (Fig. 5A, lanes 1-3) was strongly (but still incompletely) stabilized in the presence of both Arg-eDelta K-beta gal and Leu-eDelta K-beta gal (Fig. 5A, lanes 4-6; compare with lanes 1-3 and 7-9). This stabilization was manifested especially clearly as an increase in the relative amount of Arg-Ura3p at the beginning of chase (time 0), indicating reduced degradation of Arg-Ura3p during the pulse (Fig. 5C). This latter degradation pattern, termed "zero point effect," is caused by the previously demonstrated preferential targeting of newly formed (as distinguished from conformationally mature) protein substrates by the N-end rule pathway (16, 42). The increased steady-state level of Arg-Ura3p in the presence of both Arg-eDelta K-beta gal and Leu-eDelta K-beta gal accounted for the results of phenotypic analyses (Figs. 3 and 4). The much smaller but detectable stabilization of Arg-Ura3p by Arg-eDelta K-beta gal alone (Fig. 5C) was consistent not only with the inability of Arg-eDelta K-beta gal to confer the Ura+ phenotype on cells expressing Arg-Ura3p from uninduced PCUP1 promoter but also with the partial rescue of the Ura+ phenotype by Arg-eDelta K-beta gal in cells expressing Arg-Ura3p from the induced PCUP1 (Figs. 3 and 4 and data not shown).


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Fig. 5.   Metabolic stabilization of Arg-Ura3p in the presence of bivalent N-end rule inhibitor. A, S. cerevisiae JD52 (UBR1) expressing Arg-Ura3p, a short-lived Ura3p-based reporter (Fig. 2B), from the induced PCUP1 promoter, were cotransformed, alternatively, with either pRS423 (HIS3-based control vector) and pRS424 (PGAL1, TRP1-based control vector) (denoted as 423+424), or with pRDelta -beta gal-TRP1 and pLDelta -beta gal-HIS3, expressing Arg-eDelta K-beta gal and Leu-eDelta K-beta gal (denoted as R+L; the bivalent inhibitor). Control JD55 (ubr1Delta ) cells expressing Arg-Ura3p were transformed with pRS424. Cells grown in either dextrose-containing SD medium (no expression of beta gal) or galactose-containing SG medium were labeled with [35S]methionine/cysteine for 5 min at 30 °C, followed by a chase for 0, 10, and 30 min, extraction, immunoprecipitation, and SDS-10% polyacrylamide gel electrophoresis. B, same as in A, but cells were also cotransformed with the plasmids pRDelta -beta gal-TRP1 and pRDelta -beta gal-HIS3, both expressing Arg-eDelta K-beta gal (denoted as R+R). The assays were carried out in SG medium. C, in vivo decay curves of Arg-Ura3p (Fig. 2B) in wild type (JD52) and ubr1Delta (JD55) cells. The patterns in A and B were quantified as described under "Experimental Procedures." The initial amounts of Arg-Ura3p were normalized against the amount in ubr1Delta cells (100%). Note that the inhibitors also altered the zero point effect (degradation of a reporter during the pulse (16, 42)). , degradation of Arg-Ura3p in UBR1 (JD52) cells in the presence of both Arg-eDelta K-beta gal and Leu-eDelta K-beta gal (cells were grown in SG medium); open circle , the same transformants were grown in SD medium where beta gal fusions were not expressed; black-down-triangle , JD52 cells expressing Arg-Ura3p were transformed with the two alternatively marked plasmids expressing Arg-eDelta K-beta gal and grown in SG medium; , JD52 cells expressing Arg-Ura3p were transformed with the two alternatively marked control vectors and grown in SG medium; black-square, ubr1Delta (JD55) cells expressing Arg-Ura3p were transformed with control vectors and grown in SG medium.

The Arg/Leu-eDelta K-beta gal-based bivalent inhibitor of the present work, although surprisingly potent (Fig. 4B), is obviously far from optimal even for a protein-based inhibitor; because beta gal is a homotetramer, only ~50% of the coexpressed Arg-eDelta K-beta gal and Leu-eDelta K-beta gal chains would exist as heterodimers within tetramers (Fig. 1B). (This estimate assumes a random assortment of Arg- and Leu-bearing beta gal chains in the formation of beta gal tetramers. The actual in vivo assortment is expected to be biased, to an unknown extent, in favor of homodimeric associations, because individual polysomes would produce beta gal chains bearing either Arg or Leu but not both.) In addition, although the eDelta K extension (Fig. 2A) is capable of supporting the desired effects, it is also unlikely to be optimal. In summary, the efficacy of this first and necessarily suboptimal bivalent inhibitor bodes well for the future of this design.

A bivalent inhibitor is strikingly more efficacious than an otherwise identical monovalent inhibitor (Figs. 3-5). In addition, our findings are the first evidence that the type 1 and type 2 sites of N-recognin are spatially proximal in the 225-kDa S. cerevisiae Ubr1p. While this work was under way, genetic dissection of S. cerevisiae Ubr1p identified amino acid residues that are required for the integrity of the type 1 site but not the type 2 site, and vice versa.6 These results provided independent evidence for both the separateness and spatial proximity of the two substrate-binding sites of the 225-kDa N-recognin, in agreement with the present data. Our results (Figs. 3-5) strongly suggest that small bivalent inhibitors of the N-end rule pathway are feasible, and moreover, are expected to be much more potent than their monovalent counterparts. Work to produce such inhibitors is under way.

    ACKNOWLEDGEMENTS

We thank members of the Varshavsky laboratory, especially A. Kashina and G. Turner, for helpful discussions and advice in the course of this work. We also thank I. Davydov, F. Du, F. Navarro-Garcia, H. Rao, and especially G. Turner for comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK39520 (to A. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Div. of Biology, 147-75, Caltech, 1200 East California Blvd., Pasadena, CA 91125. Tel.: 626-395-3785; Fax: 626-440-9821; E-mail: avarsh{at}its.caltech.edu.

2 Y. T. Kwon and A. Varshavsky, unpublished data.

3 F. Lévy and A. Varshavsky, unpublished data.

4 M. Ghislain and A. Varshavsky, unpublished data.

5 K. Madura and A. Varshavsky, unpublished data.

6 A. Webster, M. Ghislain, and A. Varshavsky, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Ub, ubiquitin; beta gal, E. coli beta -galactosidase; E3, ubiquitin-protein ligase; ha, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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