(Received for publication, September 25, 1995; and in revised form, November 20, 1995)
From the
Targeting of substrates for degradation by the ATP,
ubiquitin-dependent pathway requires formation of multiubiquitin chains
in which the 8.6-kDa polypeptide is linked by isopeptide bonds between
carboxyl termini and Lys-48 residues of successive monomers. Binding of
Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory
complex commits the attached target protein to degradation with
concomitant release of free ubiquitin monomers following disassembly of
the chains. Point mutants of ubiquitin (Lys Arg) were used to
map the linkage specificity for ubiquitin-conjugating enzymes
previously demonstrated to form novel multiubiquitin chains not
attached through Lys-48. Recombinant human E2
catalyzed
multiubiquitin chain formation exclusively through Lys-11 of ubiquitin
while recombinant yeast RAD6 formed chains linked only through Lys-6.
Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each
bound to subunit 5 of partially purified human 26 S proteasome with
comparable affinities. Since chains bearing different linkages are
expected to pack into distinct structures, competition between Lys-11
and Lys-48 chains for binding to subunit 5 demonstrates that the latter
possesses determinants for recognizing alternatively linked chains and
precludes the existence of subunit 5 isoforms recognizing distinct
structures. In addition, competition studies provided an estimate of K
18 nM for the intrinsic
binding of Lys-48-linked chains of linkage number n > 4.
This result suggests that the principal mechanistic advantage of
multiubiquitin chain formation is to enhance the affinity of the
associated substrate for the 26 S complex relative to that of
unconjugated target protein. Complementation studies with
E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported
isopeptide ligase-dependent degradation only through Lys-48-linked
chains, while E2
retained the ability to target a model
radiolabeled substrate through Lys-11-linked chains. Therefore, the
linkage specificity exhibited by these E2 isozymes depends on their
catalytic context with respect to isopeptide ligase.
ATP-dependent conjugation of ubiquitin to protein targets is currently recognized to mediate a variety of cellular processes by signaling selective degradation of the latter through the 26 S proteasome pathway, reviewed most recently in (1) and (2) . Among the cellular targets serving as substrates for this unique post-translational modification are various proteins exhibiting either constitutive or conditional short half-lives including cyclins(3, 4, 5, 6, 7, 8) , various oncoproteins(9, 10, 11, 12) , p53(13, 14, 15) , transcriptional factors (16, 17, 18) , and proteins of abnormal structure(19, 20, 21) . In all cases, the signal for ubiquitination probably requires transient exposure of one or more lysines that can serve as sites for recognition and attachment of the polypeptide. For certain targets, enhanced steric accessibility of sensitive lysines arising by minute conformational changes (22, 23, 24) or more global folding transitions (21, 25) may be accompanied by unmasking of specific amino-terminal residues that dispose the protein to recognition by relevant isopeptide ligases (E3) that confer specificity(4, 26, 27, 28, 29, 30) . In the case of cyclins, discrete recognition signals are conserved among related isoforms and within unrelated proteins(6, 31) , although the precise mechanism by which these sequences contribute to substrate recognition by relevant conjugating enzymes has not been well elucidated.
Attachment of single ubiquitin moieties to target proteins effects a modest rate of degradation by the 26 S proteasome(32, 33, 34) ; however, more robust signals for degradative targeting require subsequent formation of multiubiquitin homopolymers by chain elongation from the initial polypeptide conjugate(32, 33, 35) . Considerable recent work has demonstrated that these multiubiquitin chains are formed by a repeating structure in which the carboxyl terminus of each ubiquitin is linked to Lys-48 of the preceding ubiquitin(33, 35) . The crystal structure of the resulting multiubiquitin chain exhibits considerable packing order and symmetry that is thought essential for recognition by the S5 subunit of the regulatory complex capping the 26 S proteasome (36, 37) . This model is supported by mutagenesis studies identifying essential ubiquitin residues required for both multiubiquitin chain binding to S5 and for subsequent degradative targeting(38) .
Ubiquitin-mediated proteolysis has been most
extensively studied in yeast and rabbit reticulocytes. Within these
systems, the almost quantitative inhibition of ATP-dependent
degradation accompanying substitution of rmUb ()or UbK48R
for wild type polypeptide demonstrates that a significant fraction of
degradative flux proceeds through conjugated intermediates bearing
Lys-48-linked multiubiquitin chains since neither rmUb nor UbK48R
supports chain elongation(33, 35) . However, mounting
evidence supports the existence of multiubiquitin chains bearing
linkage specificities distinct from Lys-48. Purified recombinant yeast
RAD6, a member of the ubiquitin carrier protein (E2) isozyme family,
catalyzes multiubiquitin chain formation to core histones in the
presence of only ubiquitin activating enzyme (E1) to maintain the E2
active site cysteine charged with ubiquitin thiolester(39) .
The linkage specificity for these chains does not require Lys-48 since
rmUb but not UbK48R blocks the characteristic ladder of conjugates
revealed by SDS-PAGE(39) . Similar results supporting
Lys-48-independent chains have more recently been obtained with
recombinant E2
, an isoform cloned from human
keratinocytes using autoantibodies obtained from pemphigous foliaceus
patients(64) . Finally, stable Lys-63-linked chains requiring
participation of RAD6 (UBC2) have been observed in yeast and proposed
to account in part for the DNA repair function of this E2
isoform(40) . Since RAD6 normally supports Lys-48
chain-dependent degradation in yeast within the N-end rule
pathway(41, 42) , the latter observation suggests
linkage specificity may be context specific and depend on either the
target protein or, more likely, the cognate E3 required for
conjugation.
The currently accepted paradigm for target protein
conjugation requires ubiquitin activating enzyme, a ubiquitin carrier
protein, and ubiquitin:protein isopeptide ligase. The metabolic
significance of E3-independent conjugation by certain members of the E2
family remains uncertain, although conjugates formed in the absence of
E3 by yeast RAD6 and CDC34 as well as the rabbit reticulocyte isoform
E2 are substrates for 26 S proteasome-mediated
degradation(32) . In the present studies, mutagenesis of the
lysine residues present on ubiquitin have allowed the assignment of
linkage specificity for multiubiquitin chain formation by RAD6 and
E2
. Other results indicate that these alternatively
linked chains are recognized by subunit 5 of the 26 S proteasome,
suggesting target proteins marked by such homopolymer structures may be
degradative intermediates. Finally, reconstitution experiments with
rabbit reticulocyte extracts demonstrate that alternatively linked
chains are competent in the overall degradative pathway. These results
define a functional role for alternatively linked chains and serve as a
basis for future mechanistic studies with the cognate E3 isoforms.
Inorganic pyrophosphatase (high pressure liquid
chromatography purified) was obtained from Sigma. Carrier-free
NaI and [2,8-
H]ATP were purchased
from DuPont NEN. Lysine 48-linked diubiquitin generated by recombinant
E2
(43) was the generous gift of Dr. Cecile
Pickart (Johns Hopkins University). The monomer concentration of
diubiquitin was determined spectrophotometrically from the extinction
coefficient of free polypeptide(44) . Homogeneous wild type,
di-, and mutant ubiquitins were radioiodinated by the chloramine-T
procedure(45) . Recrystallized BSA was obtained from Pentex and
used for the preparation of
I-rcmBSA(46) . Rabbit
reticulocyte-rich whole blood was generated by phenylhydrazine
induction and used to generate fraction II (45) . A portion of
fraction II was used to prepare apparently homogeneous E2
and E1/E2-depleted fraction II(39, 47) . Rabbit
liver E1 was purified to apparent homogeneity by adapting reported
affinity chromatography/fast protein liquid chromatography methods
reported previously (47) and quantitated by the stoichiometric
formation of ubiquitin [
H]adenylate(48) .
Homogeneous native histone H2B (generous gift of Dr. Vaughn Jackson,
Medical College of Wisconsin) and recombinant yeast CDC34 (UBC3) and
RAD6 (UBC2) were those reported previously(49) .
Figure 1: Confirmation of CDC34 linkage specificity. The linkage specificity for CDC34-catalyzed autoubiquitination was examined with radiolabeled wild type, reductively methylated, or mutant ubiquitins as described under ``Materials and Methods'' (even-numbered lanes). Incubations were for 30 min in the presence of 80 nm of rabbit liver E1 and 80 nM recombinant CDC34. Odd-numbered lanes were quenched with SDS sample buffer before addition of radiolabeled polypeptide to control for the presence of contaminants in the ubiquitin preparations. Linkage number for multiubiquitin chains is shown to the right.
Figure 2:
Determination of E2 linkage
specificity. The linkage specificity for E2
-catalyzed
autoubiquitination was examined in incubations similar to those
described in the legend to Fig. 1except that reactions were for
20 min in the presence of 20 nM E1 and 30 nM recombinant E2
. Linkage number for multiubiquitin
chains is shown to the right.
Figure 3: Determination of RAD6 linkage specificity. The linkage specificity for RAD6-catalyzed ubiquitination of histone H2B was examined in incubations similar to those described in the legend to Fig. 1except that reactions were for 20 min in the presence of 10 nM E1, 20 nM recombinant RAD6, and 12 µM H2B. Linkage number for multiubiquitin chains is shown to the right.
Figure 4: Subunit 5 of the 26 S proteasome binds alternatively linked multiubiquitin chains. Aliquots of partially purified 26 S proteasome (25 µg) were resolved by 10% SDS-PAGE and either stained with Coomassie Blue (left lane) or transferred to nitrocellulose and incubated with radiolabeled chains of the indicated linkage type as described under ``Materials and Methods.'' Positions of subunit 5 and the 100-kDa putative isopeptidase-T bands are indicated to the left. Positions of molecular weight markers are shown to the right.
The autoradiogram of Fig. 1represents the results obtained for CDC34-catalyzed
autoubiquitination(55) . In the presence of wild type I-ubiquitin, a ``ladder'' of ubiquitin
conjugate bands is observed that corresponds to the successive addition
of single ubiquitin molecules based on relative molecular weight. That
the pattern of bands represents formation of a multiubiquitin chain is
demonstrated by the absence of conjugates above that of CDC34-Ub
when
I-rmUb is substituted for wild type
polypeptide(39) . With the exception of
I-UbK48R,
the other six arginine mutants exhibit patterns of multiubiquitin chain
formation qualitatively similar to that of wild type polypeptide. The
absence of chain formation in the presence of
I-UbK48R
confirms our earlier report that CDC34 catalyzes the specific
E3-independent formation of Lys-48-linked multiubiquitin chains (39) and indicates that the other mutations fail substantially
to affect this process. The presence of mono- and diubiquitinated forms
of CDC34 with
I-rmUb and
I-UbK48R represent
the conjugation of single ubiquitin moieties to distinct lysines
present on CDC34(39) . In addition, the minor band migrating
with the CDC34-Ub
adduct for
I-rmUb and
I-UbK48R represents the slow rate of CDC34-catalyzed
conjugation of E1 present in the incubations.
In Fig. 1, the
CDC34-Ub band formed at steady state with wild type
I-ubiquitin is under-represented compared to the bands
above and below this species. Independent studies have shown this gap
to result from a kinetic effect on chain elongation in which the rate
of CDC34-Ub
formation is faster than than of
CDC34-Ub
, leading to a steady state depletion of the
tetramer. (
)In contrast, for multiubiquitin chains formed
with
I-UbK6R the tetramer band is present at a steady
state level comparable to that of the other bands while the
CDC34-Ub
adduct is under-represented (Fig. 1). This
result indicates that the K6R mutation leads to a change in relative
rates of chain elongation at this step, leading to a switch in the
steady state levels of the adducts, and suggests Lys-6 represents a
specificity determinant for binding of CDC34 to the growing chain
during elongation.
Yeast RAD6 is not subject to
autoubiquitination but does catalyze facile conjugation to core
histones when used as model substrates(39) . The autoradiogram
of Fig. 3represents the pattern of conjugates formed to histone
H2B for each of the radioiodinated ubiquitin polypeptides. With wild
type I-ubiquitin, a clear pattern of H2B conjugates is
observed extending to the Ub
adduct. The absence of
discrete bands above that of the diubiquitin species in the presence of
I-rmUb confirms our earlier report that RAD6 is also
capable of multiubiquitin chain formation(39) . That these
higher order bands are absent in the
I-UbK6R lane
confirms that multiubiquitin chain formation by RAD6 exhibits an
exclusive linkage specificity requiring isopeptide bond formation
through this residue (Fig. 3). This conclusion is supported by
observation that the other arginine mutants support multiubiquitin
chain formation indistinguishable from that of wild type polypeptide.
These results confirm our earlier observations that multiubiquitin
chain formation catalyzed by RAD6 does not require linkage through
Lys-48(39) .
The 26 S proteasome was partially purified from human erythrocytes (52) and then resolved by SDS-PAGE. The left lane in Fig. 4displays a pattern of Coomassie-stained bands typical of this complex; that is, a family of bands in the molecular weight range of 14-30 kDa representing the 20 S core degradative complex and a series of higher molecular weight bands localized to the 19 S regulatory complex that confers both ATP and ubiquitin conjugate dependence on degradation (52) . Parallel lanes were transferred to nitrocellulose and incubated with multiubiquitin chains formed from radioiodinated polypeptide in chemically defined reactions as described under ``Materials and Methods.''
Multiubiquitin chains linked through Lys-48, formed in the
autoubiquitination of CDC34 (Fig. 1) and having an average
linkage number n > 7 specifically bound to a band of 50 kDa
relative molecular mass (Fig. 4), previously identified as
subunit 5 (37) . At equivalent monomer concentrations, I-diubiquitin but not
I-ubiquitin also
bound to the same band (not shown), consistent with the binding
specificity of subunit 5 in recognizing multiubiquitin
chains(37) . However,
I-diubiquitin bound to
subunit 5 only at monomer concentrations substantially greater than
that of the CDC34 multiubiquitin chains, again in agreement with the
increased affinity exhibited by this proteasome subunit for binding
chains of n
4(37) . Similarly, Lys-6-linked
multiubiquitin chains formed to histone H2B in the reaction catalyzed
by RAD6 (Fig. 3) and possessing an average linkage number n > 7 bound to a band having the same relative mobility (Fig. 4). Multiubiquitin chains linked through Lys-11 formed in
the autoubiquitination of E2
(Fig. 2) and having
an average linkage number n > 7 were also found to bind to
the same subunit as those possessing Lys-48 and Lys-6 linkages (Fig. 4). In control studies (not shown), neither histone H2B
nor E2
at an equivalent concentration had any effect on
the binding of radiolabeled Lys-6- and Lys-11-linked chains,
respectively, indicating that these conjugates did not bind to S5
through the target protein moiety to which they were conjugated.
During these studies, we consistently observed multiubiquitin chains possessing all three linkage specificities to associate with an additional protein band having a relative molecular mass of ca. 100 kDa (Fig. 4). This molecular weight is consistent with that of isopeptidase T, a ubiquitin-specific protease believed responsible for the disassembly of multiubiquitin chains and the subsequent reutilization of monomeric polypeptide during the degradative cycle of the 26 S proteasome(57) . This observation suggests isopeptidase T may possess a broad specificity for multiubiquitin chain disassembling. Studies are currently in progress to test this hypothesis with purified isopeptidase.
Fig. 5shows that a 10-fold excess of diubiquitin
results in only a 25% inhibition in binding of
I-labeled
Lys-48- or Lys-11-linked chains. This result confirms observations of
Deveraux et al.(37) that subunit 5 exhibits a
significantly diminished affinity for diubiquitin compared to
Lys-48-linked chains of higher linkage number. In contrast, a
10
-fold excess of unlabeled Lys-48-linked chains (n > 7) results in a 60-65% inhibition of both Lys-48- and
Lys-11-linked radioiodinated chains. In parallel control studies,
neither free CDC34 nor E2
at similar concentrations was
capable of competing with its respective radiolabeled
auto-multiubiquitin chains (not shown), ruling out the possibility that
the apparent competition results from direct binding of the E2 isoforms
to S5. Competition between unlabeled Lys-48-linked chains and labeled
Lys-48- or Lys-11-linked chains suggests that a single subunit 5
species recognizes chains of alternate linkage specificity. In
addition, subunit 5 must possess comparable affinities for Lys-48- and
Lys-11-linked chains since unlabeled diubiquitin and Lys-48-linked
chains result in similar degrees of inhibition for both homopolymer
structures.
Figure 5:
Lys-48- and Lys-11-linked multiubiquitin
chains competitively bind to subunit 5. Partially purified 26 S
proteasome was resolved by 10% SDS-PAGE and then transferred to
nitrocellulose as described in the legend to Fig. 4. Regions
corresponding to subunit 5 were excised and incubated with
Tris-saline-powdered milk to block excess nitrocellulose binding sites
(see ``Materials and Methods''). Nitrocellulose sections were
then incubated with 1.4 nMI-ubiquitin present
as Lys-48-linked CDC34 multiubiquitin chains of n > 7
(Lys-48 linkage) and either 15 µM unlabeled diubiquitin
(+Ub
) or 0.2 µM unlabeled Lys-48-linked
CDC34 multiubiquitin chains of n > 7
(+Ub
). Parallel
nitrocellulose sections were incubated with 0.6 nM
I-ubiquitin present as Lys-11-linked E2
multiubiquitin chains of n > 7 (Lys-11 linkage) and
15 µM unlabeled diubiquitin (+Ub
) or 0.2
µM unlabeled Lys-48-linked CDC34 multiubiquitin chains of n > 7 (+Ub
). Data
are expressed as percent of label bound in the absence of competing
unlabeled chain (None). Chain concentrations are expressed in
terms of monomer ubiquitin.
Fig. 6(panel A) illustrates rates of net ATP-dependent degradation observed with intact fraction II when supplemented with wild type or variant forms of ubiquitin. A significant decrease in degradative capacity is observed when rmUb is substituted for wild type polypeptide, indicating that the majority of proteolysis proceeds through degradative intermediates bearing multiubiquitin chains(33) . That these chains predominantly contain Lys-48 linkages is demonstrated by the similar inhibition found with UbK48R(33) . A 50% inhibition was consistently observed when incubations were supplemented with UbK11R. Inhibition by UbK11R within this context probably does not indicate formation of chains containing Lys-11 linkages within intact fraction II since complete inhibition is observed only with UbK48R; however, the data do not rule out the potential for chains bearing mixed linkages. More likely, inhibition by UbK11R reflects an effect of this mutant on either the rate of Lys-48-linked multiubiquitin chain elongation within the E3-dependent reaction or a diminished binding of Lys-48-linked UbK11R chains to subunit 5.
Figure 6:
RAD6 and E2 support ATP,
ubiquitin-dependent protein degradation. Initial rates of
I-rcmBSA degradation were measured for complete
reticulocyte fraction II (panel A) or E1/E2-depleted fraction
II supplemented with rabbit liver E1 (40 nM) and 50 nM recombinant E2
(panel B), 120 nM recombinant RAD6 (panel C), or 80 nM recombinant
E2
(panel D). Incubations also contained 20
µM of wild type (Ub), reductively methylated (rm), or mutant ubiquitins. Data are expressed as net ATP,
ubiquitin-dependent degradation (panel A) or net ATP,
ubiquitin- and E2-dependent degradation (panels B-D) for
the mean of triplicate determinations ±
S.D.
Profiles of net E2-dependent degradation
obtained with depleted fraction II supplemented with recombinant
E2, the cognate isozyme for E3-dependent proteolysis
within reticulocyte extracts, are qualitatively similar to those
observed with intact extract (Fig. 6, panel B). That
E2
supplementation of depleted fraction II is capable of
quantitatively reconstituting the level of degradation observed with
intact extract demonstrates that the bulk of ATP, ubiquitin-dependent
proteolysis proceeds through an E2
-mediated pathway of
conjugation. Panel C illustrates that RAD6 is competent to
support protein degradation in depleted fraction II when supplemented
with wild type ubiquitin, although the absolute rate of degradation is
considerably attenuated(39) . That RAD6 can complement
degradation in fraction II is expected since this isoform is considered
the yeast homolog of E2
for N-end rule-dependent
degradation(58, 59) . The relative efficacy of RAD6 in
complementing degradation varied with different preparations of
fraction II from which depleted extract was prepared (not shown),
suggesting that additional component(s) required for this pathway also
show preparation-dependent variability. Notable in the data of panel C is that degradation via a RAD6-dependent pathway
requires formation of Lys-48-linked multiubiquitin chains rather than
those linked by Lys-6 since degradation is inhibited to base-line
values when the reactions are supplemented with either rmUb or UbK48R
but not UbK6R. Therefore, RAD6 displays a change in multiubiquitin
linkage specificity for E3-dependent conjugation compared to earlier
results obtained in the absence of ligase (Fig. 3).
In
contrast, depleted fraction II supplemented with recombinant E2 displays a dependence on formation of Lys-11-linked chains
similar to that found in the E3-independent reactions of Fig. 2(Fig. 6, panel D). The absolute ability of
E2
to support degradation was considerably less than that
found with intact extract (panel A) and varied with different
preparations of fraction II (not shown). That
E2
-catalyzed degradation requires Lys-11-linked
multiubiquitin chain formation is demonstrated by the complete
inhibition of proteolysis observed with rmUb and UbK11R but not UbK48R.
Therefore, unlike the results with RAD6, E2
retains its
Lys-11 linkage specificity within the context of E3-dependent protein
degradation.
Pickart and Rose first resolved the E2 isoforms of
reticulocytes and demonstrated their ability to catalyzed
E3-independent ligation of ubiquitin to a narrow range of model protein
substrates(60) . Subsequent studies have emphasized
similarities in sequence of the core catalytic domains among members of
the E2 family and distinctions in their participation in a variety of
the regulatory phenotypes characteristic of ubiquitin-mediated protein
degradation, their ability to conjugate ubiquitin to various test
proteins, and their catalysis of multiubiquitin chain formation bearing
discrete linkage specificities (39, 61) . In the
present studies, we have utilized Lys Arg point mutants of
ubiquitin to map the linkage specificities for multiubiquitin chain
formation catalyzed by two members of the E2 family previously
demonstrated to form Lys-48-independent chains and have characterized
interactions between these structures and downstream components of the
degradative pathway.
The autoradiogram of Fig. 2demonstrates
that recombinant human E2 forms multiubiquitin chains
exclusively through Lys-11 of the polypeptide since none of the other
six lysine mutants significantly effects the pattern of radiolabeled
conjugates resolved by SDS-PAGE. This is distinct from the absolute
Lys-48 linkage specificity previously characterized for the analogous
autoubiquitination reaction of CDC34 (Fig. 1). In contrast, RAD6
exclusively forms Lys-6 linkages during chain elongation from the
initial ubiquitin conjugated to histone H2B (Fig. 3). The
distinct linkage specificities catalyzed by the three E2 isozymes
probably arise in part from core domain sequence differences since the
peptide insertion within the core catalytic domain of CDC34 that has
been proposed to account for its Lys-48 linkage specificity is absent
in both E2
and RAD6(55) . Available evidence
suggests the carboxyl-terminal extension domains present on RAD6 and
CDC34 are not required for multiubiquitination since their deletion has
little effect on the ability to support degradation or
conjugation(29, 59) . However, these observations may
be a function of the cognate E3 isozymes examined. Conjugation of the
initial ubiquitin moiety during CDC34-catalyzed autoubiquitination
occurs intramolecularly between subunits of a transient
homodimer(63) ; in contrast, the first ubiquitin ligated upon
E2
autoubiquitination is within the monomeric
polypeptide(62) . The kinetic order for chain elongation is
presently unknown for CDC34 and E2
, although steric
constraints imposed by the growing multiubiquitin chain suggests this
step is intermolecular.
Multiubiquitin chains linked through Lys-48
yield a highly symmetric structure stabilized by defined packing
interactions between the monomeric units(36) . Multiubiquitin
chains possessing novel linkages through Lys-6 or Lys-11 probably yield
related but distinct symmetric structures stabilized by packing
interactions unique from those found in Lys-48-linked chains. Retention
of a defined linkage specificity during elongation of such novel
structures must arise from complementary interactions between groups
present on the growing multiubiquitin chain and the respective E2
isozyme. Moreover, fidelity in linkage specificity accompanying chain
elongation probably requires the E2 to bind across more than a single
ubiquitin unit and thus recognize a unique pattern of interaction
sites. Such a model posits that each unique linkage specificity should
be characterized by a distinct constellation of ubiquitin residues
specifying these interactions. For CDC34 chain elongation, the minimum
recognition unit requires three ubiquitin units in correct Lys-48
linkage. The hypothesis of discrete sites on ubiquitin
directing linkage specificity is supported by our recent observations
that mutation of ubiquitin residues directing the specificity for
Lys-48 linkages during CDC34- and E2
-catalyzed chain
elongation have no effect on multiubiquitin chain formation by RAD6 and
E2
.
Alteration in the kinetics of
CDC34-catalyzed Lys-48 chain elongation revealed by the shift in steady
state formation of CDC34-Ub
versus CDC34-Ub
intermediates (Fig. 1) suggests that Lys-6 contributes to
define this linkage specificity either by direct interaction with the
E2 or by stabilizing the incipient structure. The accumulated
observations do not rule out an alternative interpretation that
conjugation of the second ubiquitin in correct linkage during chain
elongation directs formation of subsequent linkages by sterically
blocking other available lysine residues present on the polypeptide.
This interpretation appears unlikely since in the Lys-48 tetraubiquitin
structure all lysine residues remain solvent exposed(36) .
Because chains bearing different linkage specificities are expected
to pack into unique structures, we were surprised to find that polymers
of similar length linked through Lys-6, Lys-11, or Lys-48 bound with
comparable apparent affinity to the S5 subunit of the 26 S proteasome (Fig. 4). Moreover, both Lys-48 and Lys-11 chains bound
competitively to S5 (Fig. 5), precluding the existence of
distinct isoforms of S5 able to discriminate between alternative
structures. ()Either the unique structures expected for
chains of different linkage pack to present the same ubiquitin surface
residues for interaction with S5 or, more likely, the proteasome
subunit contains subsets of interacting sites recognizing
differentially linked chains. In either case, recognition of the
alternatively linked chains by S5 must be of high affinity based on the
nanomolar concentrations of these species used in Fig. 4. The
competition experiments of Fig. 5allow us to estimate the K
for binding of Lys-48 chains to S5. If one
reasonably assumes that labeled and unlabeled Lys-48-linked chains bind
with equal affinity, then the 60% inhibition found for competition of
200 nM unlabeled chains with the 1.4 nM labeled
chains present in the incubation predicts a K
of
130 nM, expressed as monomer ubiquitin concentration. A
linkage number n
7 for both labeled and unlabeled chains
requires an intrinsic K
18 nM for
chain binding. Similar calculations reveal that the 25% inhibition of
Lys-48 chain binding by 15 µM diubiquitin requires an
intrinsic K
of 23 µM for the latter
having a linkage number of n = 2.
This estimate
probably represents a lower limit to the actual affinity since it is
unlikely that the S5 subunit retains absolute native conformation
following SDS-PAGE resolution, electrophoretic transfer, and binding to
the nitrocellulose membrane. However, the magnitude of this estimated K suggests that the principal mechanistic effect
of multiubiquitin chain formation is in increasing the affinity of the
proteasome for target substrates over that of the unconjugated protein.
This argument is consistent with the significant increase in rate of
degradation for model substrates bearing multiubiquitin chains compared
to those containing only single ubiquitin moieties(32) .
Enhanced affinity of the 26 S proteasome to bind multiubiquitin
chain-linked substrates together with the marked ability of the
ligation pathway to recognize minute conformational changes arising by
denaturation or exposure of discrete signals provides a formidable
targeting mechanism for selective protein degradation within the cell.
We have previously shown that RAD6 and E2 support ATP,
ubiquitin-dependent degradation in E1/E2-depleted reticulocyte fraction
II extracts when supplemented with exogenous activating enzyme (39, 64) and that RAD6 functions in the E3-independent
targeting of
I-labeled histone H3 for degradation by
purified human erythrocyte 26 S proteasome(32) . The
complementation studies of Fig. 6confirm our earlier
observations that RAD6 and E2
support degradation and
confirm in both cases that degradation proceeding through
multiubiquitinated intermediates is significantly attenuated in the
presence of rmUb. Although RAD6 exhibits Lys-6 linkage specificity in
E3-independent chain formation (Fig. 3), within depleted
fraction II degradation proceeds exclusively through Lys-48 chains (Fig. 6, panel C). Conversely, E2
retains
the Lys-11 linkage specificity in both E3-independent chain formation (Fig. 2) and when added to depleted fraction II (Fig. 6, panel D). Therefore, the linkage specificity of these E2
isozymes is determined in part by the catalytic contribution of E3. Two
lines of evidence suggest RAD6- and E2
-dependent
degradation within depleted fraction II proceeds through E3-catalyzed
chain formation. First, both RAD6 and E2
support
formation of a heterogeneous distribution of
I-ubiquitin
conjugates to endogenous proteins when added to E1-supplemented
depleted fraction II that is similar to that observed for intact and
E2
-supplemented extract (not shown). Second, both RAD6
and E2
exhibit extremely restricted substrate
specificities for conjugation of exogenous substrates in the absence of
E3 (39, 64) and are unable to catalyze a significant
rate of rcmBSA ligation (not shown). Therefore, these observations
support and extend earlier observations that the multiubiquitin chain
linkage formed with RAD6 is context specific with respect to the
identity of the E3 involved.
At present, only Lys-48- and
Lys-63-linked chains have been observed in
vivo(35, 40) . Formation of Lys-63-linked chains
within yeast do not challenge the conclusion that Lys-48 chains
represent the principal mechanism of degradative targeting since the
former appear to serve a regulatory rather than proteolytic
function(40) . Moreover, the present observations indicate that
detection of alternatively linked chains requires expression of the
responsible E2/E3 pair. In the case of E2, this isozyme
is abundant in only a limited number of cell types other than
keratinocytes. (
)We are currently screening these cell lines
for the presence of Lys-11-linked multiubiquitin chains. The functional
significance of alternative chains is obscure at present, particularly
since both Lys-11- and Lys-48-linked chains appear equally competent to
target degradation. Steady state concentrations of ubiquitin conjugates
and therefore their rate of subsequent degradation by the 26 S
proteasome depend on the relative rates of conjugation versus disassembly(47, 53) . If chains of different
linkage form or undergo disassembly at differential rates, then the
presence of alternative structures may represent modulation of
proteolysis for specific substrates or substrate subpopulations.
The present data provide additional evidence for the formation of multiubiquitin chains bearing linkage specificities distinct from that of Lys-48. In addition, these alternatively linked chains bind to the 26 S proteasome and, in the case of Lys-11- and Lys-48-linked chains, direct degradation by the complex. The results provide a framework for studies in progress assessing the role of various E2 isozymes in E3-dependent conjugation during the targeting of substrates for degradation by the 26 S proteasome.