From the 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
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ABSTRACT |
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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 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 E3 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 G 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.
Strains and General Techniques--
The S. cerevisiae
strains used were JD52 (MATa ura3-52 his3- Plasmids--
The high copy (2µ-based) plasmids
pR 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- We constructed a bivalent N-end rule inhibitor (Fig.
1A) from the previously
studied N-end rule substrates derived from E. coli -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
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
) 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.
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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
200
leu2-3,112 trp1-
63 lys2-801) and JD55 (MATa
ura3-52 his3-
200 leu2-3,112 trp1-
63 lys2-801 ubr1
::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).
-
gal-TRP1 and pR
-
gal-HIS3, which expressed
Arg-e
K-
gal (Ub-Arg-e
K-
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.
pL
-
gal-TRP1 and pL
-
gal-HIS3, both of which expressed
Leu-e
K-
gal (Ub-Leu-e
K-
gal), were
produced by replacing the Ub-Arg domain of pR
-
gal-TRP1 and
pR
-
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 e
K in front of a
gal moiety lacking the first 23 residues of wild type
gal (see
Fig. 2A). The low copy, pRS315 vector-derived (38) plasmid
pR-e
KhaUra3-R3R7 expressed
Arg-e
K-ha-Ura3pK3R,K7R
(Ub-Arg-e
K-ha-Ura3pK3R,K7R) from the
PCUP1 promoter.
Arg-e
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
e
K and Ura3pK3R,K7R (see Fig.
2B). The plasmid pR-e
KhaUra3-R3R7 was produced from pFL1
(encoding Ub-Arg-e
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
e
K.
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 (ubr1
) expressing Arg-Ura3p
(Ub-Arg-e
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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
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
gal moiety
lacking its first 21 residues. The resulting X-eK-
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-e
K-
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 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
gal-based
bivalent inhibitor. Specifically, at equal levels of expression of the
two
gal-based polypeptide chains, 50% of
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
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 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 e
K (extension
(e) lacking lysines (
K)), is described in the
main text. The
gal part of the fusion lacked the first 21 residues
of wild type
gal (2). B, the Ura3p-based N-end rule
substrate, Arg-e
K-ha-Ura3pK3R,K7R, derived
from Ub-Arg-e
K-ha-Ura3pK3R,K7R and denoted
Arg-Ura3p, is described in the main text.
In the present work, we used the metabolically stable
Arg-eK-
gal (produced from
Ub-Arg-e
K-
gal) and Leu-e
K-
gal
(produced from Ub-Leu-e
K-
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-e
K-
gal, at position
239, is too far from the N terminus of the protein. In the
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
gal-based polypeptide chains such as
Arg-e
K-
gal and Leu-e
K-
gal, 50% of
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
gal tetramer, especially in view of the
presumed flexibility of the e
K extension (1) (Fig.
1A).
The reporter N-end rule substrate in this study was
Arg-eK-ha-Ura3pK3R,K7R, denoted below as
Arg-Ura3p (Fig. 2B). This ha-tagged, type 1 N-end rule
substrate was produced from
Ub-Arg-e
K-ha-Ura3pK3R,K7R through the
cotranslational in vivo cleavage by deubiquitinating enzymes
(1, 6, 41). The lysine-lacking e
K extension of
Arg-e
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
e
K in an X-eK-
gal substrate (37). The
Lys-3
Arg and Lys-7
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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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 ubr1
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-e
K-
gal alone (R+R in Fig. 3),
Leu-e
K-
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
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-eK-
gal and
Leu-e
K-
gal became Ura+ under these
conditions (Fig. 3). The cells that expressed either Arg-e
K-
gal alone or Leu-e
K-
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-eK-
gal and
Leu-e
K-
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-e
K-
gal and
Leu-e
K-
gal (Fig. 4B). A weak stabilizing
effect of Arg-e
K-
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-e
K-
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-eK-
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-e
K-
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-e
K-
gal and
Leu-e
K-
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-e
K-
gal and Leu-e
K-
gal accounted
for the results of phenotypic analyses (Figs. 3 and 4). The much
smaller but detectable stabilization of Arg-Ura3p by
Arg-e
K-
gal alone (Fig. 5C) was consistent
not only with the inability of Arg-e
K-
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-e
K-
gal in
cells expressing Arg-Ura3p from the induced PCUP1 (Figs. 3
and 4 and data not shown).
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The Arg/Leu-eK-
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
gal is a homotetramer, only ~50% of the coexpressed
Arg-e
K-
gal and Leu-e
K-
gal chains
would exist as heterodimers within tetramers (Fig. 1B).
(This estimate assumes a random assortment of Arg- and Leu-bearing
gal chains in the formation of
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
gal chains bearing either Arg or Leu but not both.) In
addition, although the e
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviations used are:
Ub, ubiquitin;
gal, E. coli
-galactosidase;
E3, ubiquitin-protein
ligase;
ha, hemagglutinin.
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