From the
The developmentally programmed cell death of abdominal
intersegmental muscles in the tobacco hawkmoth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene
as a hormonally regulated event (Schwartz, L. M., Myer, A., Kosz, L.,
Engelstein, M., and Maier, C. (1990) Neuron 5, 411-419).
Solid phase immunochemical assays measuring intersegmental muscle pools
of free and conjugated ubiquitin reveal that the induction of
polyubiquitin mRNA is accompanied by a proportional increase in total
ubiquitin polypeptide. Ubiquitin conjugate pools increase 10-fold at
eclosion, during which loss of muscle protein mass is maximum. A
smaller but measurable increase in ubiquitin conjugates is observed
earlier in pupal development coincident with a modest enhanced
degradation of myofibrillar proteins. Accumulation of ubiquitin
conjugates is accompanied by induction in the pathway for polypeptide
ligation, including the activating enzyme (E1), several carrier protein
(E2) isoforms, and ubiquitin:protein isopeptide ligase (E3). Both
accumulation of ubiquitin polypeptide and the enzymes of the
conjugation pathway are subject to regulation by declining titers of
the insect molting hormone 20-hydroxyecdysone, which signals onset of
programmed cell death in the intersegmental muscles. Thus, programmed
cell death within the intersegmental muscles is accomplished in part by
stimulation of the ubiquitin-mediated degradative pathway through a
coordinated induction of ubiquitin and the enzymes responsible for its
conjugation to yield proteolytic intermediates. This suggests enzymes
required for ubiquitin conjugation may represent additional genes
recruited for developmentally programmed death.
The multi-enzyme pathway of ATP, ubiquitin-dependent proteolysis
represents the principal cytosolic mechanism in eucaryotes for the
specific degradation of short-lived and structurally abnormal proteins
(1) . Within this pathway, proteins are targeted for degradation
through the ATP-coupled covalent ligation of the carboxyl terminus of
ubiquitin (8.6 kDa) to
Programmed cell death
offers some of the most dramatic examples of protein catabolism
observed in animals; for example, loss of the entire tadpole tail
during metamorphosis to the frog can occur in under 3 days
(15) . However, at present little is known about the role of
ATP, ubiquitin-dependent proteolysis during programmed cell death. It
is reasonable to suspect that ubiquitin-mediated degradation is
involved since dramatic increases in polyubiquitin message or protein
have been observed in specific examples of programmed cell death
(16, 17, 18) . One system that is amenable to a
biochemical analysis of programmed cell death is the intersegmental
muscle (ISM)
The trigger for both ISM atrophy and death is a decline in the
circulating titer of the insect molting hormone 20-hydroxyecdysone
(20-HE). An initial decline on day 14 of pupal/adult development
initiates the atrophy program, while a further decline early on day 18
commits the muscles to die following adult eclosion later that day
(21) . If day 17 animals are given exogenous steroid, ISM death
is delayed until the circulating titer of the hormone falls below the
required threshold. Once the cells are committed to die, they can no
longer be rescued by steroid administration. The ability of the ISM to
die requires the repression and activation of a number of
ecdysteroid-regulated genes
(20) . If animals are treated with
20-HE on day 17, both death and the anticipated changes in gene
expression are delayed. Two of the repressed genes encode actin and
myosin heavy chain
(25) , while two up-regulated sequences
encode apolipophorin III
(26) and polyubiquitin
(16) .
In all species, the polyubiquitin genes encode multiple head-to-tail
repeats of the ubiquitin coding sequence, which when translated results
in an elevation of free ubiquitin monomers
(27) . The
Manduca genome contains two polyubiquitin alleles that encode
9 and 18 repeats
(16) .
The current studies were initiated to
exploit existing immunochemical methods for the quantitation of free
and conjugated ubiquitin pools
(28, 29) within
intersegmental muscles during the final stages of metamorphosis. The
results indicate that total ubiquitin levels, predominantly in the form
of conjugates, increase exponentially at the time of cell death in
response to a coordinated induction of both polyubiquitin and the
enzymes required for conjugate formation. This developmental response
is regulated by the same hormonal trigger required for initiation of
cell death. The coordinated induction of the conjugation pathway
supports a role for ubiquitin-mediated degradation in developmentally
programmed death and suggests that other putative cell death genes may
represent enzyme(s) of the ubiquitin conjugation pathway.
Bovine ubiquitin and protein A (Sigma) were radioiodinated by
the chloramine-T procedure
(30) using carrier-free
Na
The second phase of the response was
characterized by a 10-fold increase in the total ubiquitin pool from
late in day 18 until 17 h post-eclosion, at which time tissue sampling
became difficult. This increase in total ubiquitin agrees temporally
and quantitatively with the 10-fold increase in polyubiquitin mRNA
previously reported
(16) . During the second phase of the
response, the fractional level of conjugation exhibits an abrupt
elevation to a final value of 93% by 17 h post-eclosion (Fig. 1).
In contrast, free ubiquitin levels remained relatively constant at 35
± 2 pmol/mg protein throughout both phases of the time course
shown in Fig. 1. Therefore, newly synthesized ubiquitin arising
from induction of the polyubiquitin gene partitions directly into the
conjugate pool, suggesting that the normal homeostatic mechanism for
maintaining the balance between free and conjugated ubiquitin is
disabled
(29) . Western blots of the samples obtained during the
second phase again revealed no change in the distribution of conjugates
(not shown). Qualitatively similar results were observed in parallel
experiments, although the exact timing of events varied due to
uncertainty in the initial staging of pupa.
A 1.5-fold
increase in conjugating activity is observed during day 15
(Fig. 2), which coincides with the elevation in fractional level
of conjugation observed during the initial phase of the response
(Fig. 1), after which conjugating activity remained relatively
constant until day 18. A significant increase in conjugating activity
occurred early in day 18 and continued through eclosion just prior to
the beginning of day 19. The latter stimulation in conjugating activity
preceded the increase in accumulation of ubiquitin conjugates occurring
during the second phase of the response (Fig. 1). The total
elevation in conjugating activity from late day 14 to the maximum at
eclosion was 5-fold (Fig. 2), in good agreement with the overall
10-fold increase in the conjugate pool from Fig. 1, suggesting
that the net accumulation of conjugates is kinetically driven.
Qualitatively similar temporal results were observed in two other
replicate experiments (not shown), although the absolute timing of
eclosion varied due to some uncertainty in the initial staging of pupa.
The small decline in conjugation rate at 4 h post-eclosion shown in
Fig. 2
was not consistently observed; indeed, two other
experiments showed increased activity at 4 h post-eclosion (not shown).
In the latter two experiments, the absolute pools of ubiquitin
conjugates were smaller than that shown in Fig. 1, suggesting
that the enzymes required for ubiquitin conjugation may also be subject
to autodegradation by the system in very actively atrophying muscles,
resulting in a net decline in rate of ubiquitin conjugation following
eclosion. Despite this discrepancy, there was a rough correlation among
the three experiments between rate of conjugation and size of the
conjugate pool following eclosion.
Manduca E1 thiol ester comigrates with the corresponding reticulocyte
activating enzyme intermediate, indicating that the insect enzyme is of
similar molecular weight to that of vertebrates. Intersegmental muscle
extracts contained a range of lower molecular weight thiol ester
adducts that probably represent E2 isozymes since several comigrated
with reticulocyte E2 isozyme standards (Fig. 3). Intersegmental
muscle extracts contained three major E2 thiol ester bands that
migrated with or slightly below rabbit E2
Intersegmental muscles at 4 h post-eclosion
showed a significant increase in both total ubiquitin and the
fractional level of conjugation compared to reference muscles harvested
late on day 17 (), in agreement with the results of
Fig. 1
. In contrast, animals injected early on day 17 with 20-HE
but assayed 4 h after the normal time of eclosion contained a total
ubiquitin pool and percent conjugation similar to that of the day 17
control muscles. Both the induction of total ubiquitin and the increase
in percent conjugation must be under the same hormonal regulation that
signals programmed cell death in this tissue since control muscles
injected with carrier exhibited ubiquitin pools and percent conjugation
statistically identical to untreated 4 h post-eclosion samples. The
increase in rate of in vitro E3-dependent conjugation that
preceded eclosion (Fig. 2) was also blocked by injection of 20-HE
but not by treatment with carrier alone (). Parallel
experiments similar to that of Fig. 3showed that 20-HE, but not
carrier, also blocked the marked increase in E1 and E2 isozyme thiol
esters observed between late day 17 and eclosion (not shown). The data
in indicate that the hormonal regulation for onset of cell
death extends to a coordinated induction of ubiquitin and the enzyme(s)
responsible for its ligation to intracellular protein targets.
Necrosis within eucaryotes is the passive lethal response to
cellular injury resulting from thermal or chemical stress, ischemia,
and metabolic inhibitors. This process usually involves disruption of
membrane integrity followed by ionic disequilibrium, cellular swelling,
and ultimately lysis. In contrast, the majority of cell deaths observed
within animals involves an active metabolic process that is dependent
on de novo gene expression
(37) . In most cases, this
process occurs with the morphology of apoptosis, which includes
cellular condensation, genomic DNA degradation, cell surface blebbing
to form membrane-bound apoptotic bodies, and ultimately phagocytosis
(48) . Death of the intersegmental muscles, as well as many
other cells including mammalian neurons, also depends on gene
expression but proceeds with a morphology that is distinct from
apoptosis
(25) . In Manduca, this form of programmed
cell death involves dramatic elevations in the expression of the
polyubiquitin gene
(16) .
The present study represents the
most detailed examination to date of the potential role of
ubiquitin-mediated degradation in cellular remodeling. We demonstrate
that induction of polyubiquitin mRNA following eclosion during the
programmed cell death of ISM is accompanied by a proportional
accumulation of the mature ubiquitin polypeptide ( Fig. 1and
). This new, expanded pool of ubiquitin partitions directly
into cellular conjugates since free polypeptide remains constant
throughout the late pupal stage and eclosion (Fig. 1). We have
previously shown that cultured cells and tissues regulate the ratio
between free to conjugated ubiquitin through poorly understood
mechanisms of homeostasis, except under conditions of stress or other
factors correlated with enhanced degradation
(42) . The sharp
increase in the fractional level of ubiquitin conjugates in ISM during
terminal development indicates that comparable regulation is either
absent or severely attenuated. Kinetic models based on experimental
observations require that degradative flux through the ubiquitin
pathway be proportional to the concentration of ubiquitin conjugates
(42) . Accordingly, the marked increase in these degradative
intermediates immediately prior to eclosion coincides with the enhanced
rate of protein loss within ISM. Correlation between the more modest
increase in conjugates during day 15 and an elevated rate of specific
myofibrillar protein degradation
(21, 22) suggests that
the ubiquitin-mediated pathway is also responsible for this
developmentally programmed atrophy, consistent with its role in the
proteolysis of myofibrillar proteins in rat skeletal muscles
(12, 43) .
A previously
unreported increase in net ubiquitin conjugating activity within ISM
precedes the marked accumulation of conjugates on day 19
(Fig. 2). Enhanced ubiquitin conjugation during day 18 is
attributable to induction of isopeptide ligase (E3) activity under
assay conditions for which neither E1 nor E2 was rate limiting.
Stoichiometric thiol ester assays indicate that increased E3 activity
is coincident with induction of E1 and several E2 isozymes, two of
which are novel isoforms of molecular weight less than that of the
major E2
Like the induction of polyubiquitin message
(16) and the
accumulation of ubiquitin protein, the increase in E3 measured as net
ubiquitin conjugating activity as well as E1 and E2 levels measured by
thiol ester formation are also regulated by decline in the titer of
20-HE ( and accompanying text). During this time, an
increase in multicatalytic protease activity, responsible for the
ATP-dependent degradation of ubiquitin conjugates, and the appearance
of four novel multicatalytic protease subunits is also
observed.
An increase in the expression of
polyubiquitin message or ubiquitin protein at the time of programmed
cell death is observed in a number of tissues, including Manduca neurons
(18) and tunicates
(17) . Additionally,
Delic et al. (44) have demonstrated that antisense
inhibition of polyubiquitin gene expression blocks the
Elevations in ubiquitin
expression are not only seen with programmed cell death but also with
certain pathological cell deaths
(50, 51) . When
cerebral neurons are subjected to transient ischemia, massive cell
death occurs with reperfusion. Mortality is accompanied by a dramatic
increase in ubiquitin expression in the surviving neurons
(52, 53, 54) . Cells capable of mobilizing the
ubiquitin-dependent proteolytic pathway may be better able to deal with
the generation of misfolded or otherwise abnormal proteins following
oxidative stress. In addition, ubiquitin is a major component of the
neurofibrillary tangles characteristic of Alzheimer's disease
(50, 55) . Other neurodegenerative diseases, such as
amyotrophic lateral sclerosis
(56) , motor neuron disease
(57) , Parkinsons disease
(57) , and dementia pugilistica
(58) also accumulate ubiquitin protein within cerebral
structures. These data argue that ubiquitin may serve a role in both
the normal and pathological death of cells, most notably neurons. Since
it is not possible to assess changes in the levels and activities of
the ATP, ubiquitin-dependent proteolytic pathway in individuals with
neurodegenerative disorders, the use of animal models may facilitate
the development of testable hypotheses that can be examined in a
clinical setting.
Ubiquitin-mediated proteolysis has previously been
associated with the degradation of specific proteins or a limited
subpopulation of targets. Such specific enhanced degradation results
from changes in the susceptibility ot the protein target to
ubiquitination and subsequent degradation without observable
alterations in the capacity of the cell to catalyze this pathway. The
present results demonstrate ubiquitin-mediated degradation can also be
recruited for the global atrophy of a tissue such as the Manduca intersegmental muscle during programmed cell death. Under the
latter conditions, general enhanced degradation arises by the concerted
induction of a constellation of genes coding for ubiquitin, the enzymes
responsible for conjugation of the polypeptide to target proteins, and
subunits of the 26 S multicatalytic proteasome required for degradation
of the resulting ubiquitin conjugates. Thus, the general enhanced
degradation typified by erythroid maturation
(58) ,
embryogenesis
(59) , and programmed cell death (this study)
requires the coordinated induction of enzyme activities representing
the complete degradative pathway for the respective developmental
transitions. This conclusion expands the potential roles for the
ubiquitin pathway in cellular regulation.
We are indebted to Patricia Reback for technical help
in the experiments reported here and to Dr. John Buckner for provision
of Manduca eggs.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amino groups of lysine residues present on
the protein substrate (discussed in Ref. 2). Enhanced exposure of
lysine residues appears to be the underlying determinant for the
specificity of ubiquitin ligation
(3, 4) .
Conformational changes that increase the availability of potential
sites of ubiquitin conjugation may arise by partial denaturation,
errors in intracellular trafficking, or limited endoproteolytic
cleavages that expose new destabilizing amino termini predisposed to
recognition by the system
(5, 6) . In addition to this
broad selectivity for abnormal proteins, ubiquitin-mediated degradation
exhibits a marked specificity for a select subset of regulatory
proteins for which covalent modification or alterations in ligand
binding is thought to expose otherwise cryptic sites for conjugation
(7) . The latter class includes a variety of regulatory proteins
such as cyclin B, the tumor supressor p53, c-Myc, c-Fos, and MAT 2
(8, 9, 10, 11) . ATP,
ubiquitin-dependent protein degradation has also been implicated in
more global cellular processes such as skeletal muscle atrophy
(12) , spermatogenesis
(13) , and antigen processing in
thymic stromal cells
(14) for which current evidence suggests a
general induction of the entire pathway.
(
)
of the tobacco hawkmoth
Manduca sexta (19, 20) . Groups of ISM span
each of the abdominal segments of the larva and are composed of single
syncytial cells that are each 5 mm long and up to 1 mm in diameter.
Following pupation, the muscles in the first two and last two segments
die. The ISM in the remaining four segments persist unmodified until
day 15 of the normal 18 days of pupal/adult development. On day 15, the
ISM begin to atrophy, resulting in a 40% loss of muscle mass without
significant changes in the physiological properties of the cell
(21, 22) . The ISMs are then required to perform the
eclosion (emergence) behavior of the adult moth on day 18, after which
the ISMs die during the subsequent 30 h
(23, 24) .
(
)
Translation of
these polyubiquitin genes provides the cell with a powerful
amplification system for increasing the molar concentration of
ubiquitin within the cell.
I obtained from Amersham Radiochemicals. Homogeneous
ubiquitin-conjugating enzymes were isolated by combined affinity and
fast protein liquid chromatography methods
(2) from rabbit
reticulocytes prepared by phenylhydrazine induction
(31) .
Affinity-purified rabbit polyclonal antibodies specific for conjugated
ubiquitin were those previously described
(28, 29, 32) . The insect molting hormone
20-hydroxyecdysone was purchased from Sigma. Protein was determined by
the method of Lowry et al. (33) , using bovine serum
albumin as standard.
Preparation of Intersegmental Muscle
Samples
Intersegmental muscles were rapidly dissected in
ice-cold saline from animals at various stages of pupal-adult
development before and after the onset of cell death
(21) .
Excised muscles were flash frozen in liquid nitrogen and stored at
-80 °C until analyzed to prevent degradation of the conjugate
pool and to preserve endogenous conjugating activities. After weighing
the samples, extracts were prepared from individual muscles by mincing
the frozen tissue on a glass plate chilled to -60 °C. The
minced tissue was disrupted by sonication for 40 s in 4 volumes of
ice-cold homogenizing buffer composed of 50 m
M Tris-Cl (pH
7.5), 0.25
M sucrose, 1% (w/v) SDS, 1 m
M
tosyl-
L-lysine chloromethyl ketone, 1 m
M
L-tosylamido-2-phenylethyl chloromethyl ketone, 5 m
M
EDTA, and 5 m
M N-ethylmaleimide
(34) . Crude
extracts were immediately boiled for 10 min and then centrifuged for 5
min at 14,000 g to remove insoluble debris. This
method of sample preparation has been previously shown to
quantitatively preserve conjugates in proteolytically active rabbit
reticulocyte
(28) and rat skeletal muscle extracts
(34) and was independently confirmed for the present samples. In
studies requiring activity assays for endogenous conjugating enzymes,
extracts were prepared from contralateral muscles using homogenizing
buffer supplemented with 1 m
M dithiothreitol from which SDS,
EDTA, and the protease inhibitors had been omitted.
Quantitation of Free and Conjugated Ubiquitin
Pools
The absolute content of free ubiquitin within the
intersegmental muscle extracts was determined by Western blotting using
affinity-purified rabbit polyclonal antibodies against ubiquitin
(29) . Conjugate levels were determined by a solid phase dot
blot assay against standards prepared as previously described
(28) . Samples were diluted with 50 m
M Tris-Cl (pH 7.5)
containing 0.15
M NaCl and 0.01% (w/v) SDS to be within the
empirically determined linear response range of the assay.
Immunospecifically bound antibody was detected by autoradiography
following incubation with I-protein A
(28) . The
resulting dot blot autoradiograms were quantitated densitometrically
using a BioTek EL308 microplate reader fitted with a 550-nm filter. The
latter refinement significantly improved the sensitivity and
reproducibility of the assay over previous methods
(28) .
Endogenous intersegmental muscle E1 and
E2 isozymes were assayed by their ability to form
I-Ubiquitin Assays for
Conjugating Enzymes
I-ubiquitin thiol ester
(2, 30) . Briefly,
extracts were incubated 1 min at 37 °C in a 50-µl final volume
containing 50 m
M Tris-Cl (pH 7.5), 1 m
M ATP, 10
m
M MgCl
, 1 m
M dithiothreitol, and 5
µ
M
I-ubiquitin (approximately 10,000
cpm/pmol). Stoichiometric formation of E1 and E2
I-ubiquitin thiol esters were quantitated after resolving
the incubations by non-reducing SDS-PAGE in the cold, cutting the
resulting bands from the dried gel and determining the associated
I-ubiquitin by
counting
(2, 30) .
Ubiquitin-protein ligase (E3) activity was determined in incubations
similar to those for thiol ester formation with the exception that
reactions additionally contained 10 m
M creatine phosphate and
10 IU/ml rabbit muscle creatinine phosphokinase as an ATP regenerating
system
(31) . Initial rates of
I-ubiquitin
conjugation to endogenous intersegmental muscle proteins were measured
by resolving the samples on SDS-PAGE, cutting the lanes from the gel,
and quantitating the associated radioactivity by
counting. The
concentration of
I-ubiquitin present in both the thiol
ester and conjugation assays was sufficient to obviate isotope dilution
by endogenous unlabeled ubiquitin in the extracts, based on prior pool
measurements.
Ubiquitin Pools Increase Following
Eclosion
Two distinct phases were consistently observed
when free and conjugated ubiquitin pools were quantitated in samples
obtained at the indicated times during the late pupal stage and
following eclosion (Fig. 1). In the first phase, total ubiquitin
remained constant from late in day 14 through early in day 18
( closed circles); however, there was a statistically
significant 2-fold increase in the absolute size of the conjugate pool
over the same time frame, resulting from redistribution of total
polypeptide from the free to the conjugated form. This shift between
pools was reflected in the increased fraction of total ubiquitin
conjugated, Fig. 1 (open circles), and coincided with the initial
increase in specific myofibrillar protein degradation occurring during
this time
(21, 22) . Western blot analysis of the
molecular weight distribution of ubiquitin adducts indicated a general
accumulation of the total population of adducts present late in day 14
(not shown). Within the limits of resolution for the Western blots,
this indicates that the same population of protein targets is subject
to enhanced conjugation.
Figure 1:
Intersegmental muscle ubiquitin pools
during late development. Free and conjugated ubiquitin pools within
intersegmental muscle extracts were quantitated as described under
``Materials and Methods'' from which total ubiquitin
( solid circles) and the percent conjugated ( open circles) were calculated. Each point represents
the mean ± S.E. of pooled muscles from five animals. Time of
adult eclosion is noted in the figure.
Coordinated Induction of Conjugating Activity
Precedes Eclosion
Extracts were prepared from muscles
contralateral to those used for the ubiquitin pool assays in
Fig. 1
to test whether induction of ubiquitin conjugating activity
coincided with the increase in ubiquitin adducts (Fig. 2). For each
time point, equal volumes of muscle extract were pooled and then
dialyzed (12-kDa exclusion) against 50 m
M Tris-Cl (pH 7.5)
containing 1 m
M dithiothreitol to remove metabolites and
endogenous free ubiquitin. Separate control studies indicated that
reticulocyte E1 and E2, when added to parallel pooled
extracts, were stable during dialysis so that loss of conjugating
activity by inactivation could be precluded (not shown). Initial rates
of conjugation by the dialyzed extracts were measured in the presence
of saturating
I-ubiquitin (5 µ
M) to obviate
isotope dilution by any remaining endogenous ubiquitin not removed
during dialysis. The resulting free and conjugated
I-ubiquitin in the incubations were resolved by SDS-PAGE
and then quantitated by cutting lanes from the dried gel and
determining associated radioactivity by
counting
(31) .
Replicate determinations for rates of conjugation for a given pooled
sample were generally within 5% of the mean value.
Figure 2:
Changes in I-ubiquitin
conjugation activity during late development. Initial rates of
conjugation were determined in pooled extracts prepared from
contralateral intersegmental muscles from those used in the study shown
in Fig. 1. Assays of 20 min at 37 °C were performed as described
under ``Materials and Methods'' using 360 µg of muscle
extract protein.
The assay results of
Fig. 2
reflected the ubiquitin-protein ligase (E3)-dependent step
in the cell-free extracts since rates of conjugation were unaffected by
addition of homogeneous rabbit reticulocyte E1 and E2or
by increasing the concentration of
I-ubiquitin (not
shown). Independent complimentation experiments confirmed that
reticulocyte E1 and E2
fully supported the intersegmental
muscle conjugating activity. Since the assays in
Fig. 2
necessarily utilize endogenous extract protein as
substrates for ligation, the data do not distinguish between increases
in net E3 activity and increases in the pool of proteins susceptible to
ubiquitin ligation. However, since the pattern of ubiquitin adducts
revealed by immunoblots was not qualitatively different during the time
of enhanced conjugating activity, it is unlikely the observed increase
in rate is attributable to appearance of a distinct pool of target
protein substrates. The results of Fig. 2do demonstrate that
conjugating activity increased prior to the second phase induction of
total ubiquitin at the time of eclosion and that a small but consistent
increase in conjugating activity was observed between days 15 and 16
(Fig. 2) that correlated with the initial phase increase in
fractional level of conjugation (Fig. 1).
Coordinated Induction of E1 and E2 Isozymes Parallels
Increased Rates of Conjugation
Endogenous levels of E1 and
E2 isozymes present in the pooled extracts were qualitatively estimated
autoradiographically by their stoichiometric formation of
I-ubiquitin thiol esters in brief incubations (Ref. 2 and
Fig. 3). This functional thiol ester assay for E1 and E2 in crude
extracts exploits the rapid formation of these intermediates compared
to the relatively slower rate of subsequent conjugation
(2) .
Thiol esters can be distinguished from conjugates by the former's
sensitivity to thiolytic cleavage by heating in the presence of
-mercaptoethanol
(2) . Most of the bands present in Fig. 3
are thiol esters since they were absent from parallel control gels run
under reducing conditions (not shown). The dense band at the top of each lane represents rapidly
formed high molecular weight conjugates resistant to boiling in the
presence of
-mercaptoethanol. Finally, control studies confirmed
that the dialyzed extracts contained no inhibitors of reticulocyte E1
or E2
thiol ester formation (not shown).
, the principal
isozyme present in reticulocytes
(2, 35) . Depending on
running conditions, thiol ester gels can reveal spurious additional
bands arising from unfolding artifacts peculiar to the non-reducing
SDS-PAGE system
(2, 36) . However, the two thiol ester
bands migrating just below the reticulocyte E2
standard
are probably not running artifacts since their autoradiographic
intensities are not proportional to that comigrating with the
reticulocyte marker (Fig. 3). Therefore, these two putative
isoforms may represent cell death-specific E2 species unique to ISM.
Figure 3:
Functional assay of E1 and E2 isozymes
during late development. Endogenous E1 and E2 isozymes present in
muscle extracts were quantitated by their stoichiometric formation of
I-ubiquitin thiol esters in incubations identical to
those of Fig. 2 with the exceptions that reactions were for 1 min and
the samples were resolved in the cold by non-reducing SDS-PAGE (see
``Materials and Methods''). To the right of the
figure are shown migration positions for ubiquitin, E1, and selected
rabbit reticulocyte E2 isozymes run in
parallel.
Thiol ester bands present in the day 14.8 pooled samples were just
at the limit of detection for the exposure of the original
autoradiogram from which Fig. 3was derived. Small increases in
thiol esters formed with E1, E2, and the two smaller
putative isoforms occurred during day 15 (Fig. 3). Induction of
these bands coincided with the increases in conjugating activity
(Fig. 2) and percent conjugation (Fig. 1) observed during
the initial phase stimulation of myofibrillar protein degradation.
Levels of these four thiol ester bands remained relatively constant
until late in day 17 when a second wave of induction occurred that
continued through eclosion. Additional bands of thiol esters are also
induced during this second stage, one pair of which comigrate with
rabbit E2
(Fig. 3). The second stage of induction
coincided with the larger second phase increase in conjugating activity
occurring over this same time frame (Fig. 2). The drop in
conjugating activity observed 4 h post-eclosion (Fig. 2)
parallels a reduction in the thiol ester bands in Fig. 3.
Therefore, the results in Fig. 3demonstrate that E1 and E2
isozymes coordinately increase during the same time frames in which
overall conjugating activity and fractional level of conjugation are
also elevated.
Induction of Ubiquitin Conjugates Is Under Hormonal
Regulation by 20-Hydroxyecdysone
As reviewed in the
Introduction, the onset for programmed cell death within intersegmental
muscles is triggered by a natural decline in the circulating titer of
20-HE occurring late on day 17 that induces transcription of a subset
of putative ``cell death genes''
(22) . Cell death and
induction of these genes, including that for polyubiquitin, can be
delayed by injection of exogenous hormone prior to the programmed
decline in its circulating titer
(16, 21) . The data of
Table I demonstrate that artificial manipulation of 20-HE also blocks
both the accumulation of ubiquitin conjugates and their rates of
formation. Pupa were injected with either 20-HE or an equal volume of
carrier 10% ethanol early in day 17, prior to the decline in the
natural titer of the molt hormone. At the indicated times,
intersegmental muscles were harvested, and the ubiquitin pools were
quantitated as in Fig. 1. Contralateral muscles were assayed for
I-ubiquitin conjugating activity as described in the
legend of Fig. 2.
(
)
polypeptide (Fig. 3). However, the
enhanced capacity to ligate ubiquitin does not result in a significant
accumulation of conjugates until induction of ubiquitin on day 19. The
concerted induction of E1 and E2 is probably required to ensure
substrate specificity of ubiquitin ligation since induction of E3 alone
could potentially result in shifting the rate-limiting step of
conjugation from E3 to an earlier reaction in the ligation pathway.
Because substrate specificity resides in the E3-catalyzed step, kinetic
specificity for target protein conjugation would otherwise be lost.
(
)(
)
Therefore, commitment
to ISM cell death represents a coordinated expansion in the capacity of
the cell to carry out ubiquitin-mediated degradation. This suggests
that other putative cell death genes probably will be found to be
additional components of this degradative pathway, particularly that
required for ubiquitin conjugation.
irradiation-induced death of human peripheral lymphocytes
(44) .
However, many cells can undergo programmed cell death without
detectable increases in ubiquitin expression, including Drosophila ommatidia (45) , rat sympathetic neurons
(46) , PC12 cells
(47) , and mouse T cell hybridomas
(41) . In these tissues, basal levels of ubiquitin may be
sufficient to meet the proteolytic demands of cell death.
Alternatively, since cells undergoing apoptosis are rapidly
phagocytosed
(48) , much of the degradative responsibility may
reside with phagocytic mechanisms rather than on the dying cell in
these latter cases; consequently, induction of the ubiquitin-mediated
pathway may not be a general feature of all examples of programmed cell
death. It should also be noted that some tissues constitutively express
very high levels of ubiquitin-protein conjugates without a commitment
to die, such as certain neurosecretory cells in Manduca (18) and rat
(49) . In these cases, elevation in
ubiquitin-dependent proteolysis may be required for lineage-specific
demands such as neuropeptide processing.
Table: Effect of 20-hydroxyecdysone on
intersegmental muscle ubiquitin pools and rates of conjugation
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.