(Received for publication, September 29, 1994; and in revised form, January 13, 1995)
From the
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In eukaryotes, the N-end rule pathway is a ubiquitin-dependent, proteasome-based system that targets and processively degrades proteins bearing certain N-terminal residues. Arg-DHFR, a modified dihydrofolate reductase bearing an N-terminal arginine (destabilizing residue in the N-end rule), is short lived in ATP-supplemented reticulocyte extract. It is shown here that methotrexate, which is a folic acid analog and high affinity ligand of DHFR, inhibits the degradation but not ubiquitination of Arg-DHFR by the N-end rule pathway. The degradation of other N-end rule substrates is not affected by methotrexate. We discuss implications of these results for the mechanism of proteasome-mediated protein degradation.
The surface of a protein molecule bears a number of peptide bonds, which are potential cleavage sites for proteases. Nonetheless, only some intracellular proteins are short-lived in vivo, indicating that most of these solvent-exposed bonds cannot be cleaved by intracellular proteases. Features of a protein that make it short-lived in vivo or in vitro are called degradation signals or degrons (Varshavsky, 1991). The resistance of a long- lived protein to proteases located in the same compartment is due in part to the sequence selectivity of proteases, which require the presence of a sequence motif or at least a specific residue in a substrate. In addition, the rate of peptide bond cleavage by even a relatively nonspecific protease depends on conformational flexibility of the motif that the protease recognizes. For example, only some of the potential cleavage sites on the surface of a globular protein are cleaved efficiently by the bacterial metalloendoprotease thermolysin, and these preferred cleavage sites are located in exposed segments of the polypeptide chain which have the highest spatial mobility (Fontana et al., 1986).
Thus, even if a motif recognized by a protease is present on the surface of a protein, conformational rigidity of this potential degron may preclude its efficient utilization by the protease. Conversely, a conformationally destabilized protein may acquire degrons that are masked in an unperturbed version of the protein (Parsell and Sauer, 1989). This can happen not only through conformational relaxation of a previously rigid (and therefore cryptic) surface degron but also through exposure of degrons in previously buried regions of the protein. The mechanistic connection between segmental mobility of a polypeptide chain and its susceptibility to proteolysis stems from a scarcity of local chain conformations that can lend themselves to an optimal transition-state intermediate without a conformational adjustment (Fontana et al., 1986; Creighton, 1992; Hubbard et al., 1994).
Dynamic aspects of a substrate's conformation are likely to play a major role in the functioning of intracellular proteolytic systems such as those that involve the multisubunit, multicatalytic protease called the proteasome (for reviews, see Lupas et al., 1995; Rechsteiner et al., 1993; Goldberg and Rock, 1992; Orlowski, 1990). A salient feature of these ATP-dependent systems is their processivity: once the degradation of a protein begins, it proceeds to completion. Thus, a proteasome-mediated system should be able to perturb the conformation of a globular protein substrate before or during its processive degradation by the proteasome. The ``conformational'' problem to be solved by the proteasome is analogous to the problem faced by a protein translocation system: components of a transmembrane channel must unfold a protein before or during its ``threading'' across membrane, except that in this case the protein is transported rather than destroyed (Blobel, 1980; Eilers and Schatz, 1986; Sanders and Schekman, 1992; Arkowitz et al., 1992).
In the present work, we show that a
proteasome-based proteolytic system called the N-end rule pathway is
remarkably sensitive to alterations in the conformational stability of
its substrates. The N-end rule relates the in vivo half-life
of a protein to the identity of its N-terminal residue (for review see
Varshavsky, 1992). Similar but distinct versions of the N-end rule
operate in all organisms examined, from mammals to bacteria (Bachmair et al., 1986; Gonda et al., 1989; Tobias et
al., 1991). In eukaryotes, the N-end rule-based degradation
signal, called the N-degron, comprises two determinants: a
destabilizing N-terminal residue and an internal lysine (or lysines) of
a substrate (Bachmair and Varshavsky, 1989; Johnson et al.,
1990; Hill et al., 1993). The Lys residue is the site of
formation of a multiubiquitin chain (Chau et al., 1989).
Ubiquitin (Ub) ()is a protein whose covalent conjugation to
other proteins (often in the form of a multi-Ub chain) plays a role in
a number of processes, primarily through routes that involve protein
degradation (for reviews, see Ciechanover and Schwartz, 1994; Parsell
and Lindquist, 1993; Vierstra, 1993; Jentsch, 1992; Varshavsky, 1992;
Finley, 1992; Hochstrasser, 1992).
The recognition of an N-end rule
substrate is mediated by a protein called N-recognin or E3.
The binding of N-recognin to a substrate's destabilizing
N-terminal residue is followed by the formation of a multi-Ub chain
linked to an internal lysine of the substrate (the second determinant
of its N-degron). A substrate-linked multi-Ub chain is required for the
degradation of at least some N-end rule substrates (Chau et
al., 1989; Dohmen et al., 1991). In both yeast and
mammals, the 200 kDa N-recognin and a specific
20-kDa
Ub-conjugating (E2) enzyme (one of several such enzymes in a
cell) are physically associated, forming a part of a larger targeting
complex (Madura et al., 1993; Hershko and Ciechanover, 1992).
A substrate bearing a multi-Ub chain is transferred (presumably while
still bound by the targeting complex) to the 26 S proteasome, a
2,000-kDa multicatalytic protease that contains about 40 distinct
subunits (Rechsteiner et al., 1993). The ensuing processive
degradation of the substrate yields short (
10-residue) peptides
and regenerates Ub from a multi-Ub chain. All Ub-dependent proteolytic
systems, including the N-end rule pathway, apparently share many
components of the 26S proteasome. Differences among these pathways
include their distinct targeting complexes, whose recognins (associated
with specific E2s) bind to degradation signals other than
N-degrons (Varshavsky, 1992).
Previous studies (Gonda et al., 1989; Reiss et al., 1988) showed that the N-end rule pathway is active in ATP-supplemented extract from rabbit reticulocytes. In the present work we used this in vitro system and methotrexate (MTX; a folic acid analog and inhibitor of the enzyme dihydrofolate reductase (DHFR)), to determine whether noncovalent conformational stabilization of a protein affects its degradation by the N-end rule pathway.
Figure 1:
Test proteins. The proteins shown (Ub-Met-e-DHFRha, Ub-Arg-e
-DHFRha, Ub-Met-
e-DHFRha, and Ub-Arg-
e-DHFRha) were expressed in E. coli, purified, and
used as test substrates in ATP-supplemented reticulocyte extract (see
``Materials and Methods''). DHFRha is DHFR whose C terminus
was extended with a 14-residue sequence containing the hemagglutinin
(ha) epitope (see ``Materials and Methods''). The 43-residue
region (derived from E. coli Lac repressor) between the Ub and
DHFRha moieties in Ub-Met-e
-DHFRha and Ub-Arg-e
-DHFRha is denoted by e
(Bachmair and
Varshavsky, 1989; Johnson et al., 1992). The residues are
numbered from a residue X at the Ub-e
junction to
the last residue of the ha epitope in Ub-X-e
-DHFRha. The sequence of DHFR-linked e
in the present work differs from that of the
gal-linked e
near the e
-reporter junction (Johnson et al., 1992). Lys-15 and Lys-17 residues are boxed in the
sequence of e
. In Ub-Met-
e-DHFRha and Ub-Arg-
e-DHFRha, e
was replaced by the sequence
Met/Arg-His-Gly-Ser-Gly-Ile-Met between Ub and Val, the wild-type N-terminal residue of mouse DHFR (Dohmen et al.,
1994).
E. coli JM101 and
DH5 (Ausubel et al., 1992) were used as hosts in plasmid
construction and in plasmid preparation for sequencing, respectively.
The final constructs were verified by restriction mapping and
nucleotide sequencing (Ausubel et al., 1992). E. coli BL21(DE3) (Studier and Moffat, 1986) was used for overexpression
of DHFR-containing fusions.
Figure 2:
Effect of MTX on the degradation of
DHFR-based N-end rule substrates in reticulocyte extract. Panel
A: lane a, purified, S-labeled
Ub-Met-e
-DHFRha (the amount of substrate added to this lane
and analogous lanes in other panels was larger than the initial amounts
of substrate in lane b and analogous lanes). Lane b,
Ub-Met-e
-DHFRha was added to ATP-depleted reticulocyte
extract and incubated for 10 min at 37 °C (time zero sample). Lanes c-e, same as lane a, but the samples were
withdrawn and analyzed by SDS-PAGE 15, 30, and 60 min after the
addition of ATP at time zero (see ``Materials and Methods''). Panel B: same as panel A but with
Ub-Arg-e
-DHFRha. Panel C: same as panel
B, but the Arg-Ala dipeptide was added to reticulocyte extract (to
the final concentration of 10 mM) together with
Ub-Arg-e
-DHFRha. Panel D: lanes
a-e, same as panel B, but the data are from another
experiment. Lanes f-i, same as lanes b-e,
but the assay was carried out in the presence of 20 µM MTX
(see ``Materials and Methods''). Panel E: lane
a, purified,
S-labeled Ub-Arg-
e-DHFRha. Lanes b-d, same as lanes b-d in panel
A, but the assay was carried out with Ub-Arg-
e-DHFRha, and
the incubation times were 0, 10, and 30 min (after the addition of
ATP). Lanes e-h, same as lanes a-d, but
the assay was carried out in the presence of 20 µM MTX. Arrowheads and arrows indicate, respectively, the
bands of 36-kDa Ub-X-e
-DHFRha fusions and their
28-kDa deubiquitinated derivatives, X-e
-DHFRha
(Ub-Arg-
e-DHFRha and Arg-
e-DHFRha in panel E). Asterisks indicate the band of 8-kD
a Ub, produced by
deubiquitination of
S-labeled
Ub-X-e
-DHFRha and Ub-Arg-
e-DHFRha. Half-open square brackets denote the bands of
multiubiquitinated Arg-e
-DHFRha. The incubation times are
indicated above the lanes.
The initial concentration of an S-labeled
Ub-X-DHFRha in the extract was
5 µg/ml; control
experiments (not shown) indicated that this concentration was not
rate-limiting for the degradation of either Arg-e
-DHFRha or
Arg-
e-DHFRha. MTX (where indicated) was added from a 2 M stock solution in 0.2 M NaOH to Ub-X-DHFRha in a
reaction tube and incubated for 5 min at 0 °C before assembling the
reaction mixture as described above. The final MTX concentration was 20
µM. Two equal samples were withdrawn from reaction tubes
at the times indicated in the legends to Fig. 2and Fig. 3. One sample was assayed for the amount of 5%
trichloroacetic acid-soluble
S, and the other was examined
by a Tricine buffer-based SDS-PAGE (9% acrylamide, 0.24% bisacrylamide)
(Schägger and Jagow, 1987). The gels were analyzed
using fluorography or PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). Trichloroacetic acid-soluble
S was determined as
follows. 0.1 ml of a solution of bovine serum albumin (50 mg/ml) was
added to a 10-µl sample; 0.11 ml of 10% trichloroacetic acid was
then added; the sample was incubated on ice for 10 min, followed by
centrifugation at 12,000
g for 5 min.
S
in the supernatant was determined using a water-compatible
scintillation mixture and a scintillation counter. In each of the
graphs of Fig. 3, the amount of trichloroacetic acid-soluble
S in a zero-point sample (before the addition of ATP;
5-10% of total
S) was subtracted from the values for
samples withdrawn at later times.
Figure 3:
Degradation of DHFR-based N-end rule
substrates in reticulocyte extract, measured by determining
acid-soluble S. Panel A:
,
Met-e
-DHFRha;
, Arg-e
-DHFRha;
,
Arg-e
-DHFRha in the presence of 1 mM Arg-Ala
dipeptide;
, Arg-e
-DHFRha in the presence of 20
µM MTX. Panel B:
, same as in panel A but from another experiment with Met-e
-DHFRha;
, Arg-e
-DHFRha;
, Arg-
e-DHFRha;
,
Arg-
e-DHFRha in the presence of 20 µM MTX. Each decay
curve was determined at least three times, in independent experiments,
with the results differing by less than 15% for each of the time
points. See ``Materials and Methods'' for the definition of a
zero time point and other details.
Linear Ub fusions are rapidly cleaved in vivo or in
cell-free extracts after the last residue of Ub, making possible the
production of otherwise identical proteins bearing different N-terminal
residues (Bachmair et al., 1986; Baker et al., 1992).
In one application of this method, Gonda et al. (1989)
incubated fusions of Ub to E. coli -galactosidase
(Ub-X-
gal) in reticulocyte extract, producing X-
gal test proteins bearing different N-termina
l
residues. Depending on the identity of a residue X, an X-
gal is either short-lived or metabolically stable in
ATP-supplemented extract (Gonda et al., 1989), similar to the in vivo findings with the same X-
gals in the
yeast Saccharomyces cerevisiae (Bachmair et al.,
1986). Mutational analysis has shown that either one of two lysines
(Lys-15 or Lys-17) in a non-
gal N-terminal region of an X-
gal test protein was also required for rap
id
degradation of X-
gal by the N-end rule pathway (Fig. 1) (Bachmair and Varshavsky, 1989; Johnson et
al., 1990). The function of these lysines was revealed by the
finding that Lys-15 or, alternatively, Lys-17, is the site of formation
of a multi-Ub chain (Chau et al., 1989). The non-
gal,
40-residue extension at the N terminus of
gal was derived in
part from an internal sequence of E. coli Lac repressor
(Bachmair and Varshavsky, 1989).
In the present work, the strategy
used by Gonda et al. (1989) with gal-based substrates was
employed to examine the degradation of similarly designed DHFR-based
substrates by the N-end rule pathway in reticulocyte extract. The
40-residue N-terminal extension of DHFR, derived from X-
gal test proteins (Fig. 1) (Bachmair and
Varshavsky, 1989), is denoted below as e
(extension (e)
containing lysine (K)) (Johnson et al., 1992). In the
constructs of Fig. 1, the C terminus of
Ub-X-e
-DHFR was extended with a 14-residue
sequence containing ha, an epitope tag derived from the influenza virus
hemagglutinin, which could be recognized by a monoclonal antibody
(Field et al., 1988; Johnson et al., 1992). Two
fusion proteins, Ub-Met-e
-DHFRha and
Ub-Arg-e
-DHFRha (Fig. 1), bore, respectively, Met
and Arg, a stabilizing and a destabilizing residue at the junction
between Ub and the rest of a fusion (Varshavsky, 1992). These proteins
were overexpressed in E. coli, labeled in vivo with
[
S]methionine, and purified by affinity
chromatography, using anti-ha antibody (see ``Materials and
Methods'').
Ub-Met-e-DHFRha and
Ub-Arg-e
-DHFRha were rapidly deubiquitinated upon addition
to ATP-depleted reticulocyte extract, yielding, respectively,
Met-e
-DHFRha and Arg-e
-DHFRha (Fig. 2, A and B, lanes a
and b), analogous
to the previously characterized deubiquitination of
Ub-X-e
-
gal fusions under the same conditions
(Gonda et al., 1989). Both Met-e
-DHFRha and
Arg-e
-DHFRha remained metabolically stable in ATP-depleted
extract (data not shown), but the addition of ATP resulted in a much
faster degradation of Arg-e
-DHFRha than
Met-e
-DHFRha, which remained long lived in ATP-supplemented
extract (Fig. 2, A and B, lanes
b-e, and Fig. 3A). The metabolic fates of
DHFR-based test proteins were monitored by SDS-PAGE (Fig. 2) and
also by measuring the amount of acid-soluble
S released
during the incubation of an
S-labeled protein in
ATP-supplemented extract (Fig. 3).
As shown in Fig. 3A, 33% of the initially present S-labeled Arg-e
-DHFRha was degraded to
acid-soluble fragments in ATP-supplemented reticulocyte extract after a
30-min incubation. By contrast, only 6% of the otherwise identical
Met-e
-DHFRha, bearing an N-terminal Met (stabilizing
residue in the N-end rule), was degraded after a 30-min incubation (Fig. 3A). Analysis by SDS-PAGE showed a transient
accumulation of multiply ubiquitinated Arg-e
-DHFRha
derivatives and a decrease in intensity of the band of unmodified
Arg-e
-DHFRha in the course of its incubation in
ATP-supplemented extract (Fig. 2B). By contrast,
Met-e
-DHFRha was neither ubiquitinated nor significantly
degraded in ATP-supplemented extract (Fig. 2A), in
agreement with the findings about similarly designed X-
gal substrates (Gonda et al., 1989).
Previous work has shown that an amino acid derivative such as a
dipeptide that bears a destabilizing N-terminal residue can inhibit the
degradation of a gal-based N-end rule substrate either in
vitro (reticulocyte extract) (Gonda et al., 1989) or in vivo (yeast cells) (Baker and Varshavsky, 1991). As shown
in Fig. 2C and Fig. 3A, the degradation
of a DHFR-based N-end rule substrate such as Arg-e
-DHFRha
in reticulocyte extract was almost completely inhibited by the Arg-Ala
dipeptide. Dipeptides bearing destabilizing N-terminal residues inhibit
the N-end rule pathway by competing with N-end rule substrates for
binding to N-recognin (Reiss et al., 1988; Gonda et
al., 1989; Baker and Varshavsky, 1991). Arg-Ala not only precluded
the degradation of Arg-e
-DHFRha (Fig. 3A)
but also inhibited its ubiquitination (Fig. 2C),
indicating that Arg-Ala blocks a step in the N-end rule pathway which
precedes the ubiquitination step.
We asked whether the folate analog
MTX, a high affinity DHFR ligand (K of
10
M) and a competitive inhibitor of
DHFR (Matthews et al., 1985), would affect the degradation of
a DHFR-based N-end rule substrate. Remarkably, the presence of MTX in
ATP-supplemented reticulocyte extract resulted in a nearly complete
inhibition of Arg-e
-DHFRha degradation (Fig. 3A). The inhibitory effect of MTX was confined to
the actual proteolysis of Arg-e
-DHFRha: its
multiubiquitination was in fact enhanced by MTX, in contrast to the
effect of Arg-Ala, which inhibited both the degradation and
ubiquitination of Arg-e
-DHFRha (Fig. 2D;
compare with Fig. 2C).
We also determined the effect
of MTX on degradation of Arg-e-DHFRha (derived from
Ub-Arg-
e-DHFRha), which lacked most of the
40-residue,
lysine-containing e
extension of Arg-e
-DHFRha (Fig. 1). Previous work ha
s shown that Arg-
e-DHFRha is much
longer lived than Arg-e
-DHFRha in the yeast S.
cerevisiae at 30 °C (t
of more than 4 h versus
10 min, respectively); it has also been shown that
a major reason for the increased metabolic stability of
Arg-
e-DHFRha is the absence of Lys-15 and Lys-17 residues:
Arg-e
-DHFRha, which contains Arg instead of Lys at
positions 15 and 17 of the otherwise unaltered e
extension,
is nearly as long-lived in yeast as Arg-
e-DHFRha (Bachmair and
Varshavsky, 1989). In a qualitative agreement with these in vivo data, Arg-
e-DHFRha was degraded more slowly than
Arg-e
-DHFRha in ATP-supplemented reticulocyte extract (Fig. 3B). The degradation of Arg-
e-DHFRha was
mediated by the N-end pathway, inasmuch as Met-
e-DHFRha, bearing a
stabilizing N-terminal residue, was degraded at a much lower rate than
Arg-
e-DHFRha (data not shown).
Similarly to the findings with
Arg-e-DHFRha (Fig. 2D and Fig. 3A), the addition of MTX almost completely
inhibited the degradation of Arg-
e-DHFRha in ATP-supplemented
reticulocyte extract (Fig. 3B). In contrast to the
extensive ubiquitination of Arg-e
-DHFRha prior to its
degradation (Fig. 2, B and D), the degradation
of Arg-
e-DHFRha was not accompanied by a significant accumulation
of its multiubiquitinated derivatives, and no enhancement of
multiubiquitination of Arg-
e-DHFRha could be detected in the
presence of MTX as well (Fig. 2E).
The effect of MTX
was confined to DHFR-based substrates: in parallel assays with
gal-based N-end rule substrates such as Arg-e
-
gal
(Gonda et al., 1989), the addition of MTX did not alter the
kinetics of Arg-e
-
gal degradation (data not shown).
The MTX-DHFR assay has been used previously to address the mechanistic and kinetic aspects of protein translocation across membranes (Eilers and Schatz, 1986, 1988; Vestweber and Schatz, 1988; Wienhues et al., 1991; Arkowitz et al., 1992). The present work extends the applications of MTX-DHFR to the problem of Ub-dependent protein degradation. We report the following results.
1) Arg-e-DHFRha (Fig. 1) is degraded by the N-end
rule pathway in ATP-supplemented reticulocyte extract in the absence
but not in the presence of MTX, a folic acid analog and a competitive
inhibitor of DHFR which binds to mammalian DHFRs with a K
of
10
M (Fig. 3A).
2) The effect of MTX is confined to
DHFR-based substrates, in that the degradation of other N-end rule
substrates such as Arg-e-
gal is not inhibited by MTX.
3) The degradation of Arg-e-DHFRha (this work) and other
N-end rule substrates (Gonda et al., 1989) in reticulocyte
extract is highly processive: no degradation intermediates could be
detected by SDS-PAGE in the course of proteolysis.
4) MTX inhibits
the degradation but not ubiquitination of Arg-e-DHFRha,
whose multiubiquitinated derivatives become more abundant in the
presence of MTX (Fig. 2D). Previous work (Chau et
al., 1989; Bachmair and Varshavsky, 1989) has shown that the
degradation of N-end rule substrates such as Arg-e
-
gal
and Arg-e
-DHFRha requires (and is preceded by) the
formation of a multi-Ub chain linked to one of two lysines (Lys-15 or
Lys-17) in the
40-residue extension (e
) at the N
terminus of Arg-e
-DHFRha.
5) Arg-e-DHFRha, which
lacks the e
extension, is also degraded (at a lower rate
than Arg-e
-DHFRha) by the N-end rule pathway in
reticulocyte extract, and this degradation is also inhibited by MTX (Fig. 3B). Only traces of multiubiquitinated
Arg-
e-DHFRha derivatives were observed during its degradation by
the N-end rule pathway, and the relative content of these derivatives
was further decreased in the presence of MTX (Fig. 2E and data not shown).
The extensive ubiquitination of
Arg-e-DHFRha prior to its degradation indicates that
ubiquitination is not rate-limiting for the degradation of
Arg-e
-DHFRha in reticulocyte extract. By contrast, the
scarcity of ubiquitinated derivatives of Arg-
e-DHFRha suggests
that ubiquitination of Arg-
e-DHFRha is among the slowest steps
that precede its degradation by the N-end rule pathway. A likely
explanation for these differences between Arg-e
-DHFRha and
Arg-
e-DHFRha is the difference in locations of multi-Ub chains
attached to these substrates: in Arg-e
-DHFRha, a multi-Ub
chain is linked to one of the sterically accessible lysines (Lys-15 or
Lys-17) in a flexible N-terminal extension (e
) located
outside of the folded DHFR globule (Fig. 1) (Bachmair and
Varshavsky, 1989). By contrast, in the case of Arg-
e-DHFRha a
multi-Ub chain must form on one of the numerous but relatively immobile
and (presumably) unfavorably located lysines of the folded DHFR globule
(Dohmen et al., 1994), thus accounting for both the lower
overall rate of Arg-
e-DHFRha degradation and the low steady-state
content of its ubiquitinated derivatives. This interpretation also
accounts for the observed difference in the effect of MTX on the
relative abundance of multiubiquitinated Arg-e
-DHFRha and
Arg-
e-DHFRha. Indeed, a block to Arg-e
-DHFRha
degradation due to a conformational stabilization of its DHFR moiety by
MTX would not be expected to perturb the formation of a multi-Ub chain
linked to the e
extension of DHFR, as observed. By
contrast, a multi-Ub chain linked to Arg-
e-DHFRha has to form on a
lysine of the folded DHFR moiety. This is a slow step, whose rate is
further decreased in the presence of MTX, which stabilizes the
conformation of DHFR (Matthews et al., 1985; Eilers and
Schatz, 1988). (It is assumed that the rate of formation of a multi-Ub
chain linked to Arg-
e-DHFRha is limited by the rate of chain
initiation at one of the lysines of DHFR.)
A model that accounts for
the findings of this work and is consistent with other evidence is
shown in Fig. 4. A major assumption is that the initiation of
processive degradation of an N-end rule substrate such as
Arg-e-DHFRha requires at least a local conformational
perturbation of the folded DHFR moiety which can be
``utilized'' by the proteasome. The probability of this
perturbation would be decreased in the presence of MTX, which
stabilizes the folded conformation of DHFR. Since MTX inhibits the
degradation but not multiubiquitination of Arg-e
-DHFRha,
the relevant conformational perturbation must be a step that occurs
after but not before the ubiquitination step (Fig. 4). It is not
specified whether this ``sufficient'' perturbation of DHFR is
a thermally driven fluctuation or is at least in part the result of
DHFR interactions with components of the N-end rule pathway, including
a DHFR-linked multi-Ub chain; the model is consistent with either
possibility. Another assumption of the model is that an interaction
between a ubiquitinated Arg-e
-DHFRha substrate and the
proteasome-based proteolytic machine is reversible, in that a
substrate-proteasome complex dissociates (or enters a state that cannot
result in proteolysis; see below) after a stochastically determined
time interval (Fig. 4).
Figure 4:
On the mechanism of MTX effect. Each of
the four depicted transitions (I-IV) in the N-end rule
pathway is actually a multistep reaction (Varshavsky, 1992). The shapes
of structures are arbitrary. Among the protein-protein complexes shown
in this diagram, the interactions between N-recognin (N-r) and
the Ub-conjugating enzyme (E2), and between N-recognin and a
DHFR-based N-end rule substrate were demonstrated directly (Bartel et al., 1990; Madura et al., 1993). By contrast, a
direct interaction between N-recognin and proteasome is a conjecture
that remains to be verified. The path of a multi-Ub chain (black
ovals) in a complex between proteasome and a multiubiquitinated
substrate is unknown. However, if the E2 enzyme that initiates
the formation of a substrate-linked multi-Ub chain is also responsible
for the chain elongation, the growing tip of the chain must be in
proximity to this E2, as shown in the diagram. Superimposition
of the substrate and proteasome denotes a complex (of unknown
structure) between them. A DHFR-based N-end rule substrate such as
Arg-e-DHFRha (Fig. 1), bearing a destabilizing
N-terminal residue (d) and a mobile lysine residue (K) outside of the folded DHFR globule, is bound by a complex
of N-recognin and E2 (step I). Formation of a
lysine-linked multi-Ub chain (the chain's length is arbitrarily
set at five Ub moieties) and the binding of a multiubiquitinated
substrate by the proteasome take place at step II. In
vitro, the multiubiquitination of an N-end rule substrate can
occur in the absence of proteasome; however, it is possible that the
binding of a targeted substrate by the proteasome accompanies or even
precedes the substrate's multiubiquitination in vivo. At step III, a local or a global conformational perturbation of
DHFR in a complex with the proteasome occurs, resulting in proteolysis
(the irreversible step IV), which yields short fragments of
DHFR and regenerates Ub from a multi-Ub chain. Formation of the
MTX
DHFR complex stabilizes the folded DHFR conformation,
decreasing the probability of a conformational perturbation of DHFR (step III) which can be utilized by the proteasome and thereby
inhibiting the degradation of a DHFR-containing substrate. In the case
of a protein such as Arg-
e-DHFRha (Fig. 1), which lacks a
targetable lysine residue outside of the DHFR globule, the binding of
MTX and the resulting conformational stabilization of DHFR suppress
both ubiquitination and degradation of Arg-
e-DHFRha. See
``Materials and Methods'' for further
details.
Given these assumptions, the near cessation of degradation of DHFR-based N-end rule substrates in the presence of MTX is explained as follows. In the absence of MTX, the mean time interval between the formation of a substrate-proteasome complex and a relevant conformational perturbation of DHFR within the complex is either close to or shorter than the mean lifetime of the complex. In the presence of MTX, its binding to DHFR (and the resulting conformational stabilization of the DHFR globule) decreases the probability of a relevant perturbation of DHFR within the substrate-proteasome complex but does not influence the mean lifetime of the complex (Fig. 4). Specifically, the time interval before perturbation of the substrate is postulated to become significantly longer than the time interval before dissociation of the substrate-proteasome complex, resulting in abortive cycles of targeting and multiubiquitination but little net degradation of a DHFR-based N-end rule substrate, as observed.
The understanding of proteasome mechanics is still rudimentary, and therefore the model of Fig. 4is vague about details of the MTX effect. To cite just one example, the postulated ``dissociation'' of a substrate-proteasome complex may actually be less than a complete dissociation: for the model to be relevant, it is sufficient that the complex can enter a state in which the proteasome becomes unable to initiate the degradation of a DHFR-based substrate upon an otherwise sufficient perturbation of DHFR.
The degradation of DHFR-based N-end
rule substrates in vivo (in S. cerevisiae cells) was
found to be at most weakly inhibited by the addition of MTX to the
growth medium. ()However, in contrast to the above in
vitro assays ( Fig. 2and Fig. 3), in which the N-end
rule pathway targets a previously synthesized, folded DHFR, in the in vivo assays the binding of MTX to a nascent DHFR-based
substrate takes place in kinetic competition with the targeting of the
same substrate by the N-end rule pathway. Moreover, while the binding
of MTX requires the folded DHFR conformation, the targeting by the
N-end rule pathway is expected to occur at any time after the
(cotranslational) formation of a destabilizing residue at the N
terminus of a substrate. A strategy in which the N-end rule pathway is
repressed at first (in a specially constructed yeast strain) and then
induced should eliminate the above kinetic competition and allow the
effects of MTX to be tested in vivo under conditions in which
a DHFR-based substrate is folded and MTX-bound before it is targeted by
the N-end rule pathway. These experiments are in progress.
Interestingly, the in vivo degradation of a DHFR-based N-end
rule substrate whose DHFR moiety was fused to an unrelated protein such
as S. cerevisiae Cdc28p (Dohmen et al., 1994) could
be inhibited by MTX, in contrast to the degradation of an otherwise
identical substrate lacking the Cdc28p domain.
The role of
a DHFR-linked protein in conferring the sensitivity to inhibition by
MTX remains to be understood.
The ability to suppress the in
vivo degradation of a short lived protein with its
cell-penetrating, low M ligand would have a number
of applications, including the possibility of constructing new types of
conditional mutants (Dohmen et al., 1994).