Role of ubiquitin in proteasomal degradation of mutant
1-antitrypsin Z in the endoplasmic
reticulum
Jeffrey H.
Teckman1,2,
Reid
Gilmore3, and
David H.
Perlmutter1,2,4
Departments of 1 Pediatrics and
4 Cell Biology and Physiology, Washington
University School of Medicine, and 2 Division
of Gastroenterology and Nutrition, Children's Hospital, St. Louis,
Missouri 63110; and 3 Department of Biochemistry
and Molecular Biology, University of Massachusetts, Boston,
Massachusetts 01655
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ABSTRACT |
A delay in
intracellular degradation of the mutant
1-antitrypsin
(
1AT)Z molecule is associated with greater retention
within the endoplasmic reticulum (ER) and susceptibility to liver
disease in a subgroup of patients with
1AT deficiency.
Recent studies have shown that
1ATZ is ordinarily
degraded in the ER by a mechanism that involves the proteasome, as
demonstrated in intact cells using human fibroblast cell lines
engineered for expression of
1ATZ and in a cell-free
microsomal translocation assay system programmed with purified
1ATZ mRNA. To determine whether the ubiquitin system is
required for proteasomal degradation of
1ATZ and whether
specific components of the ubiquitin system can be implicated, we have
now used two approaches. First, we overexpressed a dominant-negative
ubiquitin mutant (UbK48R-G76A) by transient transfection in the human
fibroblast cell lines expressing
1ATZ. The results
showed that there was marked, specific, and selective inhibition of
1ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER
degradation of
1ATZ. Second, we subjected reticulocyte
lysate to DE52 chromatography and tested the resulting
well-characterized fractions in the cell-free system. The results
showed that there were both ubiquitin-dependent and -independent
proteasomal mechanisms for degradation of
1ATZ and that
the ubiquitin-conjugating enzyme E2-F1 may play a role in the
ubiquitin-dependent proteasomal mechanism.
1-antitrypsin deficiency; liver disease; emphysema; quality- control apparatus; protein degradation; endoplasmic reticulum
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INTRODUCTION |
HOMOZYGOUS PIZZ
1-antitrypsin
(
1AT) deficiency is an autosomal recessive disease with
an approximate frequency of 1 in 1,800 live births (reviewed in Ref.
33). It is the most common genetic cause of liver disease in children.
It is also associated with chronic liver disease and hepatocellular
carcinoma in adults. Adults with
1AT deficiency may
develop pulmonary emphysema at an early age. The deficiency is
characterized by a single amino acid substitution of Glu342
to Lys (reviewed in Ref. 33). This substitution causes an aberration in
folding such that
1ATZ is retained in the endoplasmic
reticulum (ER) rather than secreted into the extracellular fluid, where its function is to inhibit neutrophil elastase.
Most studies of the pathogenesis of target organ injury in
1AT deficiency indicate that emphysema is caused by
decreased
1AT in the lung, permitting uninhibited
elastolytic attack on the lung parenchyma (33). In contrast, liver
disease is thought to be caused by the retention of the mutant,
presumably hepatotoxic,
1ATZ molecule in the ER of liver
cells (reviewed in Ref. 18). However, only 10-15% of PIZZ
individuals develop clinically significant liver disease (30, 31). In
previous studies, we tested the hypothesis that this subgroup of PIZZ
individuals is susceptible to liver injury by virtue of additional
unlinked genetic traits or environmental factors that delay degradation
of the mutant
1ATZ molecule after it is retained in the
ER (39). With the use of fibroblast cell lines from PIZZ patients with
liver disease (susceptible hosts) compared with those from PIZZ
individuals without liver disease ("protected" hosts), we found
that more efficient ER degradation of retained mutant
1ATZ correlated with protection from liver disease. We
now know that ER degradation of
1ATZ in fibroblast cell
lines from protected hosts and in a cell-free microsomal translocation
system is mediated at least in part by the proteasome (27). Degradation
in each system is absolutely dependent on ATP and inhibited by
lactacystin. Studies of a number of other mutant and unassembled
secretory and membrane proteins in several different systems have now
indicated that the proteasome is involved in ER degradation and
constitutes a key component of the quality-control apparatus of the ER
(5, 13, 15, 21, 26, 27, 36-38).
It is not yet clear exactly how the proteasome gets access to these
substrates or how the substrates get access to the proteasome, particularly the luminal secretory proteins. However, a process called
retrograde translocation, or dislocation, appears to be involved in at
least a few cases (5, 13, 26, 37, 38). It is also not yet entirely
clear whether the ubiquitin system is involved in the ER degradation
pathway for all of these substrates, even though it appears to be
involved in the degradation of most substrates of the proteasome.
Ubiquitin-conjugating activity is apparently required for ER
degradation of CFTR
F508 in mammalian systems (29, 36) and for mutant
carboxypeptidase Y in yeast (5, 7, 13, 26). However, ubiquitin does not
appear to be required for ER degradation of
3-hydroxy-3-methylglutaryl-CoA reductase (21). In the
cell-free mammalian microsomal translocation system, we found that
degradation of
1ATZ required stable binding to the
transmembrane ER chaperone calnexin and that there was polyubiquitination of calnexin only in microsomes which had
translocated
1ATZ. Moreover, in the presence of
adenosine 5'-O-(3-thiotriphosphate) (ATP
S) and
lactacystin, which inhibit deubiquitination and proteasomal degradation, respectively, an
1ATZ-polyubiquitinated
calnexin complex was directly demonstrated (27). Wild-type
1AT was not degraded in the cell-free microsomal
translocation system and did not induce polyubiquitination of calnexin
(27). These data provided evidence that the ubiquitin system and a
ubiquitinated intermediate were, at least in part, involved in ER
degradation of
1ATZ. However, in the current study we
used two additional strategies to definitively determine that the
ubiquitin system is required and to examine the possibility that
specific components of the ubiquitin system that are involved in ER
degradation of
1ATZ can be identified. First, we
examined the effect of dominant-negative ubiquitin molecules on
degradation of
1ATZ in intact cells. Second, we examined
the effect of fractionation and reconstitution of proteolysis-primed
lysate on degradation of
1ATZ in the cell-free microsomal translocation system.
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EXPERIMENTAL PROCEDURES |
Materials.
Rabbit reticulocyte lysate was purchased from Promega (Madison, WI).
[35S]methionine was purchased from
Amersham (Arlington Heights, IL) in purified form and from
ICN Radiochemical (Irvine, CA) in crude form
(Tran35S-label). The
m7G(5')ppp(5')G was obtained from Pharmacia
(Uppsala, Sweden). Canine pancreatic microsomal vesicles (RM),
salt-washed microsomal vesicles (KRM), and puromycin-treated
salt-washed microsomal vesicles (PKRM) were prepared by previously
described techniques (28, 35). The vesicles were extensively washed to
remove the salt and puromycin before their use in the
translation/translocation reaction. Antibody to
1AT was
purchased from Dako (Santa Barbara, CA). Purified ubiquitin was
purchased from Sigma (St. Louis, MO). Lactacystin was provided by Dr.
E. J. Corey (Boston, MA).
Cell lines.
Fibroblast cell lines that had been transduced with amphotropic
recombinant retroviral particles bearing
1ATZ cDNA have
been described previously (27, 32, 39). For the experiments described here, we used the CJZ and SMOZ cell lines, which are derived from protected hosts and are relatively efficient in ER degradation of
transduced
1ATZ. Transient expression of the wild-type
and K48R and K48R-G76A dominant-negative ubiquitin mutant genes (10) in
the fibroblast cell lines was accomplished by subcloning the corresponding cDNAs (a kind gift from Dr. Daniel Finley, Harvard University, Cambridge, MA) into the pZeoSV expression vector
(Invitrogen, Carlsbad, CA). The fibroblast cell lines previously
transfected with retroviral particles to constitutively express
1ATZ were then transiently transfected to express either
the wild-type or mutant ubiquitin cDNAs in the pZeoSV vector using the
Superfect transfection system (Qiagen, Santa Clarita, CA). The
conditions for transient transfections with wild-type and mutant
ubiquitin vectors were first optimized by measuring the
-galactosidase activity of cells transiently transfected with the
pZeoSV LacZ vector. To ensure that results of the experiments comparing
the wild-type and mutant ubiquitin vectors could not be altered by differences in transfection efficiency, transient transfections were
done with both the ubiquitin vectors and the pZeoSV LacZ vector and
aliquots of the cell lysates were used for
-galactosidase assays as
well as pulse-chase experiments.
Metabolic labeling, immunoprecipitation, and analytical gel
electrophoresis.
Cell lines were subjected to pulse-chase radiolabeling as previously
described (27, 32, 39). For the pulse period, the cells were incubated
at 37°C for 2 h in 250 µCi/ml Tran35S-label in DMEM
lacking methionine. The cells were then rinsed rigorously and incubated
in DMEM with excess unlabeled methionine for time intervals up to 6 h
as the chase period. At the end of each chase period, the extracellular
medium was harvested and the cells were lysed in PBS, 1% Triton X-100,
0.5% deoxycholic acid, 10 mM EDTA, and 2 mM phenylmethylsulfonyl
fluoride. The radiolabeled cell lysates were subjected to clarification
and immunoprecipitation, and immunoprecipitates were analyzed by
SDS-PAGE/fluorography exactly as described previously (39). Aliquots of
the radiolabeled cell lysates were also subjected to TCA precipitation
and scintillation counting of the TCA precipitates to ensure that there
was equivalent incorporation between cell lines under comparison.
Results were quantified by scanning of PhosphorImager plates (Storm
System; Molecular Dynamics, Sunnyvale, CA) exposed to the radiolabeled gels. Quantification is reported as means ± SD.
Cell-free translation and translocation.
The pGEM-4Z vector (Promega) containing either
1ATM cDNA
or
1ATZ cDNA was linearized beyond the 3'end of
the cDNA using Hind III. SP6 RNA polymerase was used for in
vitro transcription in the presence of
m7G(5')ppp(5')G to generate
m7G-capped mRNA following the protocol
provided by Promega and previously described (27).
1ATM
and
1ATZ polypeptides were synthesized in the
reticulocyte lysate cell-free system according to the protocol provided
by Promega. The cell-free reaction mixture (50 µl) contained 35 µl
of micrococcal nuclease-treated rabbit reticulocyte lysate and was
supplemented with the following final concentrations of additional
components: 20 µM 19-amino acid mixture minus methionine, 0.8 U/µl
RNase inhibitor RNasin, 0.8 µCi/µl
[35S]methionine, 4 A260/ml canine
pancreas microsomal vesicles, and 20 µg/ml appropriate mRNAs. The
cell-free translation and translocation assay was performed for 1 h at
30°C. The products were analyzed by 10% SDS-PAGE under reducing
conditions and visualized by fluorography.
Proteolysis assay in microsomal vesicles.
After the translation reaction, the microsomal vesicles that contained
either
1ATM or
1ATZ polypeptide were
isolated by centrifugation at 15,000 g for 15 min at 4°C.
The pelleted microsomal vesicles were resuspended in fresh
proteolysis-primed lysate contained in a final volume of 50 µl,
containing 40 mM Tris · HCl, pH 7.5, 5 mM
MgCl2, 2 mM dithiothreitol, 0.5 mM ATP, 10 mM
phosphocreatine, 15 µg of creatine phosphokinase (350 U/mg at
25°C; Boehringer-Mannheim, Indianapolis, IN), and fresh
reticulocyte lysate, followed by incubation at 37°C for the
indicated time intervals. Fractionation of the reticulocyte lysate by
DE52 (Whatman) chromatography was carried out as described (3, 12, 24).
Characterization of the resulting fractions has been described
previously (3, 12, 24).
Recombinant ubiquitin-conjugating enzyme E2-F1 (UbcH7) was generated in
a bacterial system as described using the UbcH7 cDNA cloned into the
pET3 vector (kindly provided by Dr. Martin Scheffner, Heidelberg,
Germany) (23). Overnight, 0.5-liter cultures of Escherichia
coli BL21(DE3) bearing the pET3 containing UbcH7 were incubated at
37°C for 3 h after the addition of 0.4 mM
isopropyl-
-D-thiogalactopyranoside to induce UbcH7
expression. The bacteria were then centrifuged to pellet and dissolved
in 15 ml of PBS-1% Triton X-100 and lysed by sonication. Supernatants
containing the E2-F1 protein were then harvested from the clarified
bacterial lysates.
Degradation assays.
Assays for degradation of 125I-lysozyme and
125I-ornithine decarboxylase (ODC) were performed exactly
as described (6, 20). Briefly, reaction mixtures contained
10 µl reticulocyte lysate or Fraction (Fr) II, 40 mM
Tris · HCl, pH 7.6, 5 mM MgCl2, 2 mM dithiothreitol, and 0.05-0.1 µg of 125I-labeled
protein substrate (~70,000 cpm) in a final volume of 50 µl. The
reactions were initiated and continued at 37°C for 240 min.
Aliquots were submitted for precipitation with 20% TCA, and the
TCA-soluble fraction was analyzed by gamma counting. Degradation values
were expressed as a percentage of the radioactivity released, with
100% representing the radioactivity in the TCA-insoluble fraction of
the original substrate.
 |
RESULTS |
Effect of dominant-negative ubiquitin mutants on ER degradation of
1ATZ in intact cells.
First, we examined the effect of the K48R and K48R-G76A
dominant-negative ubiquitin mutants on the degradation of
1ATZ in the CJZ cell line. When conjugated to
substrates, the K48R ubiquitin molecule prevents attachment of
additional ubiquitin molecules and thereby inhibits elongation of the
polyubiquitin chain and subsequent target protein degradation. This
ubiquitin mutant has been shown to be a moderately powerful inhibitor
of ubiquitin-dependent proteasomal proteolysis (10, 36). The G76A
mutation of ubiquitin inhibits the ability of the host cell to remove
the mutant ubiquitin from the chain and thus, when combined with K48R
in a double mutant molecule, results in an even more potent inhibitory
effect on ubiquitin-dependent proteolysis (10).
These dominant-negative mutants and wild-type ubiquitin were expressed
in the CJZ cell line by transient transfection. The cells were then
subjected to pulse-chase biosynthetic radiolabeling experiments (Fig.
1). The results show that CJZ cells that
were sham transfected or transfected with wild-type ubiquitin
synthesize a 52-kDa
1ATZ polypeptide. This polypeptide
progressively disappears over the first 2 h of the chase period. Our
previous studies in this cell line have shown that
1ATZ
is synthesized as a 52-kDa polypeptide precursor with high-mannose
oligosaccharide side chains as defined by endo H sensitivity and that
it is degraded between 2 and 4 h of the chase period (27, 39). Thus
this initial experiment showed that expression of exogenous wild-type
ubiquitin did not affect the synthesis or kinetics of degradation of
1ATZ. Next we examined the kinetics of degradation of
1ATZ in CJZ cells transfected with K48R-G76A ubiquitin
(Fig. 1, bottom). Here,
1ATZ is
also synthesized as a 52-kDa polypeptide and retained within the cells,
but, in this case, there is significantly less and significantly slower
disappearance of this polypeptide during the entire chase period. There
is still ~80% of the initial
1ATZ-specific radioactivity present at 2.5 h of the chase period. The K48R-G76A ubiquitin mutant inhibited degradation of
1ATZ in six
separate experiments in two different cell lines (data not shown). The half-time for the degradation of
1ATZ was
1.75 h in the presence of the wild-type ubiquitin and 3 h in the
presence of the double-mutant ubiquitin. There was no increase in
1ATZ released into the extracellular fluid
in the presence of K48R-G76A ubiquitin compared with that in the
presence of wild-type ubiquitin [i.e., negligible amounts of
1AT were secreted in each case (data not shown)].
The dominant-negative ubiquitin molecule did not affect cell viability
or metabolic activity as assessed by morphology, trypan blue exclusion,
and assay of total TCA-precipitable radioactivity (data not shown). The
difference in rate of degradation of
1ATZ in the
presence of K48R-G76A ubiquitin and wild-type ubiquitin could not be
explained by differences in transfection efficiency because there was
no difference in the
-galactasidase activity of the cells when
cotransfected with pZeoSV LacZ vector and the relevant ubiquitin
vector. Approximately 60% of the cells stained positively for
-galactasidase activity in each case. The single K48R ubiquitin
mutant did not inhibit degradation of
1ATZ (data not
shown). We did not analyze whether this lack of effect was due to lower
transfection efficiency or less potent inhibition of the
ubiquitin-dependent proteosomal degradation pathway. Together, these
initial results indicate that functional ubiquitin chain elongation is,
at least in part, required for the degradation of
1ATZ
retained in the ER of intact cells.

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Fig. 1.
Effect of wild-type and dominant-negative K48R-G76A mutant ubiquitin
molecules on degradation of 1-antitrypsin (AT)Z in the
CJZ cell line. The CJZ line was sham transfected (Mock) or transiently
transfected to coexpress either wild-type ubiquitin gene (WT Ubi) or
K48R-G76A double mutant dominant-negative ubiquitin gene (K48R-G76A
Ubi) as described in EXPERIMENTAL PROCEDURES. After 48 h,
cells were subjected to pulse-chase radiolabeling. Cell lysates were
analyzed by immunoprecipitation/SDS-PAGE/fluorography as described in
EXPERIMENTAL PROCEDURES. Migration of intracellular 52-kDa
1ATZ polypeptide is indicated by arrows at
left.
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Degradation of
1ATZ in a cell-free
mammalian microsomal translocation system.
Next we examined the possibility of using a cell-free microsomal
translocation system to determine the requirement for the ubiquitin
system and perhaps specific components of the ubiquitin system for
degradation of mutant
1ATZ. Our previous studies have shown that
1ATZ is degraded in the cell-free system by a
process that is dependent on ATP and inhibited by lactacystin (27).
First, we reexamined the specificity of degradation of
1ATZ in this system (Fig.
2). Mutant
1ATZ and
wild-type
1ATM mRNA, generated by in vitro
transcription, were used to program a rabbit reticulocyte system
supplemented with canine pancreatic microsomal vesicles. After 1 h at
30°C in the presence of [35S]methionine,
the microsomal vesicles were pelleted by centrifugation and resuspended
in fresh proteolysis-primed lysate containing ATP, an ATP regenerating
system, and excess unlabeled methionine, conditions previously
described in many studies of ubiquitin-dependent proteosomal
proteolysis (3, 12, 24). The reaction mixture was then incubated at
37°C for the chase period. At specified time intervals, equal
aliquots were collected, homogenized in Laemmli sample buffer, and
subjected to SDS-PAGE/fluorographic analysis (Fig. 2). The results show
that a 52-kDa radiolabeled polypeptide is generated from both
1ATZ and
1ATM mRNA. This polypeptide
corresponds to
1AT with high-mannose-type carbohydrate side chains. For mutant
1ATZ, this polypeptide is
rapidly degraded by 1 h of the chase period. For wild-type
1AT, there is very little disappearance over the 6-h
chase period. Rather, the
1AT polypeptide undergoes two
distinct cleavages to generate an ~48-kDa polypeptide, presumably the
result of endoproteolytic processing. These data indicate that there is
specific degradation of mutant
1ATZ in the cell-free
system that is used for all the subsequent experiments.

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Fig. 2.
Degradation of mutant 1ATZ and wild-type
1ATM in cell-free system. 1ATZ
and 1ATM mRNAs were translated for 60 min at 30°C in
rabbit reticulocyte lysate supplemented with canine pancreatic
microsomes. After translation, microsomal vesicles were harvested by
centrifugation. Pellets were resuspended in 50 µl proteolysis-primed
lysate and incubated at 37°C for various time intervals as shown.
Aliquots were taken at each time point and mixed with SDS-sample
buffer. All products were analyzed by SDS-PAGE/fluorography. Relative
mobility of molecular weight markers (Mr) is shown
at right.
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We also examined the degradation of
1ATZ in microsomes
that were salt-washed (KRM) or treated with puromycin and salt-washed (PKRM) to deplete the vesicles of signal recognition particle, ribosomes, and peripheral membrane proteins (Fig.
3). In each case, the vesicles were
extensively washed after treatment to remove the salt and puromycin
before the translocation assay. Although there was a modest decrease in
the amount of the 52-kDa
1ATZ polypeptide generated in
KRM and PKRM, the kinetics of disappearance of this polypeptide were
similar in all three types of vesicle preparations. There was
progressive disappearance between 20 and 40 min of the chase
period. The half-times for degradation were 28.1 ± 3.7 min for RM,
29.9 ± 4.4 min for KRM, and 33.2 ± 5.8 min for PKRM in four
separate experiments. These data indicate that the degradation of
1ATZ in the cell-free system does not depend on the full
complement of endogenous resident microsomal peripheral membrane
proteins (28, 35).

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Fig. 3.
Degradation of 1ATZ in native (RM), salt-washed (KRM),
and puromycin-treated salt-washed (PKRM) microsomal vesicles.
1ATZ mRNA was used to program translocation assays in
RM, KRM, and PKRM microsomes using protocol described in Fig. 2. Chase
time points are shown at bottom, and relative migration of
molecular mass markers (Mr) is indicated at
right.
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To provide further evidence for the requirement of the ubiquitin system
in degradation of
1ATZ and to identify specific
components of the ubiquitin system by reconstitution, we examined
fractions of rabbit reticulocyte lysate in the cell-free assay.
Previous studies have shown that fractionation of rabbit reticulocyte
lysate by DE52 chromatography results in two fractions: Fr I, which
contains ubiquitin and ubiquitin-conjugating enzyme E2-F1, and Fr II,
which contains several E2 enzymes, all known E3 enzymes, and the 26S proteasome (3, 12, 24). First, we examined the fate of
1ATZ generated in the cell-free system in the presence
of Fr I alone, Fr II alone, or Fr I and Fr II together (Fig.
4A). There is rapid degradation of
1ATZ by 30 min in the presence of both Fr I and Fr II
(Fig. 4A, left). In fact, the half-time for
disappearance of
1ATZ in both Fr I and Fr II in five
separate experiments was 26.9 ± 3.4 min (Fig. 4B), almost
identical to that in unfractionated reticulocyte lysate (data not
shown). There was almost no disappearance of
1ATZ in Fr
I alone. More than 50% of the initial radioactivity was still present
by 180 min of the chase period. Although there is a higher rate of
degradation in Fr II alone, it is never as rapid as that in Fr I and Fr
II together or that in unfractionated reticulocyte lysate. There was no
difference in the results of this experiment when exogenous ubiquitin
was added to each condition (data not shown). There was no difference
in the rates of disappearance when the combined Fr I and Fr II reaction
was compared with Fr I alone and Fr II alone at equal volumes of each
fraction in the reaction or at equal total volumes per reaction (data
not shown). Thus the difference in disappearance of
1ATZ
in Fr I and Fr II together compared with Fr I alone or Fr II alone
could not be attributed to the concentration of components in each
fraction used for the comparison. Together, these data indicate that
there are ubiquitin-independent and ubiquitin-dependent components that contribute to proteasomal degradation of
1ATZ. Moreover,
the data indicate that the ubiquitin-dependent component of this
degradation pathway requires the proteasome in Fr II and some essential
factor, or factors, of Fr I.


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Fig. 4.
Degradation of 1ATZ in cell-free system using fractions
of rabbit reticulocyte lysate. A: microsomal translocation
assays were programmed with 1ATZ mRNA exactly as
described in Fig. 2, except that fractions (Fr) I or II of rabbit
reticulocyte lysate were used instead of unfractionated rabbit
reticulocyte lysate. In these experiments, the same volumes of Fr I and
Fr II were used in all 3 reactions, so that Fr I + Fr II sample had
twice as much lysate equivalent as Fr I-alone and Fr II-alone samples
but in same total reaction volume. In other experiments, there was no
difference in kinetics of degradation of 1ATZ if Fr I
and Fr II sample had half as much of Fr I and Fr II each compared with
Fr I alone and Fr II alone. B: phosphorimaging was used to
provide quantitative data as means ± SD for 5 separate experiments.
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Two separate approaches were used to examine the possibility that the
intermediate level of degradation in Fr II was due to ubiquitin
contamination (Fig. 5). First (Fig.
5A), we used Western blot analysis to detect ubiquitin and
found an ~8-kDa polypeptide in whole reticulocyte lysate and Fr I,
which comigrated with purified ubiquitin, but no ubiquitin polypeptide
was detected in Fr II even when twice as much was loaded. Second (Fig.
5B), we examined the degradation of 125I-lysozyme
and 125I-ODC in whole reticulocyte lysate and Fr II.
Degradation of lysozyme requires both ubiquitin and proteasome, but
degradation of ODC by the proteasome does not require ubiquitin (22).
The results show that there is degradation of 125I-lysozyme
in reticulocyte lysate but not in Fr II, whereas 125I-ODC
is degraded in a relatively similar manner by reticulocyte lysate and
Fr II. These results make it unlikely that ubiquitin contamination of Fr II accounts for the intermediate level of degradation that occurs in this fraction.


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Fig. 5.
Detection of ubiquitin in Fr II using Western blot analysis (A)
and degradation assays (B). A: purified ubiquitin (1 µg), reticulocyte lysate (3 µl of a 1:10 dilution), Fr II (3 µl
of a 1:10 dilution), Fr II (2×) (6 µl of 1:10 dilution), Fr I
(3 µl of 1:10 dilution), Fr I (2×) (6 µl of a 1:10 dilution)
were loaded on a 15% SDS-polyacrylamide gel next to molecular mass
markers as shown at right. Migration of monomeric ubiquitin is
indicated by arrow at left. B: degradation of
125I-lysozyme and 125I-ornithine decarboxylase
(ODC) was performed as described in EXPERIMENTAL
PROCEDURES.
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Next, we examined the possibility that the intermediate level of
degradation of
1ATZ that occurs in Fr II is mediated by the proteasome by determining whether it required ATP and whether it
was inhibited by lactacystin (Fig. 6). The
results show that the intermediate level of degradation in Fr II does
not occur when ATP and the ATP-regenerating system are not present or
when lactacystin is included (Fig. 6A). By 10 h of the chase
period (Fig. 6B),
1ATZ is not present in whole
reticulocyte lysate and is only barely detectable in Fr II alone but is
present in substantial amounts in Fr II without ATP, Fr II supplemented
with ATP
S, and Fr II supplemented with lactacystin. Together, these
results indicate that there is a component of the degradation of
1ATZ in the cell-free system that is mediated by the
proteasome independent of ubiquitin.


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Fig. 6.
Degradation of 1ATZ in Fr II in absence of ATP or in
presence of ATP S or lactacystin. A: microsomal translocation
assays were programmed with 1ATZ mRNA using reticulocyte
lysate or Fr II as described in Figs. 2 and 4, but in separate
reactions no ATP/ATP regenerating system was added (Fr II-ATP), both
ATP/ATP regenerating system and ATP S (5 mM) were added (Fr II + ATP S), or both ATP/ATP regenerating system and lactacystin (20 µm)
were added (Fr II + lactacystin). B: aliquots from assays after
10 h of chase period were analyzed.
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Previous studies have shown that the ubiquitin-conjugating enzyme E2-F1
is exclusively found in Fr I (12, 24). Thus we examined the possibility
that addition of recombinant E2-F1 could completely reconstitute
degradation of
1ATZ in Fr II (Fig.
7). For this experiment, exogenous
ubiquitin was added to each reaction. The results show that there is a
significant increase in the rate of disappearance of
1ATZ in Fr II supplemented with E2-F1 but no change in
Fr II supplemented with control proteins compared with Fr II alone. In
fact, the rate of degradation in Fr II plus E1-F1 is almost identical
to that in Fr I and Fr II together. This effect of E2-F1 was also
concentration dependent, mediating a progressive increase in rate of
disappearance up to a concentration of 0.5 µg/50 µl reaction, above
which there was no further increase (data not shown).


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Fig. 7.
Effect of E2-F1 on degradation of 1ATZ in the cell-free
system using fractions of rabbit reticulocyte lysate. A:
translocation assays were programmed with 1ATZ mRNA
using Fr I + Fr II, Fr II, Fr II + E2-F1 (0.5 µg/50 µl reaction)
and Fr II + control proteins. Recombinant E2-F1 was generated in a
prokaryotic expression system as described in EXPERIMENTAL
PROCEDURES. Control proteins were generated in prokaryotic
expression system with pET3 vector alone. All reactions for these
experiments were supplemented with ubiquitin (2 µg). B:
phosphor imaging was used to provide quantitative data as means ± SD
for 4 separate experiments.
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To exclude the possibility that E2-F1 had a nonspecific effect on
degradation of
1ATZ, we compared the fate of
1ATZ in Fr II supplemented with E2-F1 with that in Fr I
supplemented with E2-F1 (Fig. 8). The
results show that the addition of E2-F1 to Fr I does not affect the
rate of degradation of
1ATZ. That is, the rate of
disappearance is similar in Fr I plus E2-F1 and Fr I plus control
proteins but increases markedly in Fr II plus E2-F1 compared with Fr II
plus control proteins.

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|
Fig. 8.
Effect of E2-F1 on degradation of 1ATZ in the cell-free
system using Fr I and II. Translocation assays were programmed with
1ATZ mRNA using Fr I + Fr II, Fr II + control proteins,
Fr II + E2-F1 (1 µg/50 µl reaction), Fr I + control
proteins, and Fr I + E2-F1 (1 µg/50 µl reaction). All reactions
were supplemented with ubiquitin (2 µg). Results were
analyzed as described in Fig. 2.
|
|
 |
DISCUSSION |
In this study, two strategies were used to determine whether the
ubiquitin system was required for proteasomal degradation of the mutant
secretory protein
1ATZ. First, dominant-negative mutant
ubiquitin molecules were coexpressed in intact cells in which
1ATZ is retained/degraded in the ER. The results showed that ER degradation of
1ATZ is significantly inhibited
by coexpression of K48R-G76A mutant ubiquitin but not by wild-type
ubiquitin, indicating that the ubiquitin system is at least in part
required for ER degradation of
1ATZ. Second, degradation
of
1ATZ was examined in a cell-free microsomal
translocation system with fractions of rabbit reticulocyte lysate that
are known to contain different components of the ubiquitin system. The
results show that there are both ubiquitin-independent and
ubiquitin-dependent mechanisms for degradation of
1ATZ
in this system. The ubiquitin-independent mechanism was demonstrated by
the presence of some degradation of
1ATZ in Fr II, which
contains the proteasome but not ubiquitin. There was no increase in
degradation when Fr II was supplemented with exogenous ubiquitin.
Degradation in Fr II was dependent on ATP and inhibited by lactacystin,
indicating that it involves the proteasome. However, degradation in Fr
II was still significantly slower than that in Fr I and Fr II together
or in unfractionated reticulocyte lysate, indicating the presence of an
essential factor, or factors, in Fr I. In fact, the addition of
purified recombinant ubiquitin-conjugating enzyme E2-F1, which is
ordinarily found in Fr I, and ubiquitin to Fr II completely
reconstituted degradation of
1ATZ. These data indicate
that there is also a ubiquitin-dependent mechanism for degradation of
1ATZ and that E2-F1 may be one of the E2 enzymes
involved in this latter mechanism.
There are a number of previous studies in which the role of the
ubiquitin system in the ER degradation pathway has been examined. In
yeast, the ubiquitin system appears to be required for proteasomal degradation of the mutant secretory protein carboxypeptidase Y (5, 7,
13, 26) and mutant forms of membrane proteins Ste6p (17) and Sec61p (4,
25). Interestingly, the two enzymes that are required are UbcH6 and
UbcH7. The UbcH6 enzyme corresponds to a ubiquitin-conjugating enzyme
that is an integral ER membrane protein. The UbcH7 enzyme corresponds
to mammalian ubiquitin-conjugating enzyme E2-F1. In mammalian
cells/systems, the ubiquitin system is required for proteasomal
degradation of the naturally occurring mutant membrane protein
CFTR
F508 (29, 36) and an experimental model mutant luminal protein,
the truncated ribophorin (RI332) (9). However, degradation
of the resident ER membrane protein 3-hydroxy-3-methylglutaryl-CoA
reductase by the proteasome does not appear to require the ubiquitin
system (21). When expressed in heterologous cells by itself in the
absence of the other T cell-receptor (TCR) subunits, the TCR-
subunit is degraded in the ER by a pathway that involves the proteasome
(2, 40, 41). Although polyubiquitination of TCR-
can be demonstrated in transfected cells when the proteasome is inhibited with lactacystin (40), a TCR-
mutant lacking lysines and therein unable to undergo ubiquitin conjugation is degraded as rapidly as wild-type TCR-
(41),
suggesting that ubiquitination is not required for its proteasomal
degradation. A major portion of newly synthesized apolipoprotein B is
degraded in the ER by a process that involves the proteasome, and
polyubiquitination of apolipoprotein B can be demonstrated in cells in
which the proteasome is chemically inhibited (8, 16, 42). However, it
is not yet known if ubiquitination is required for proteasomal
degradation of apolipoprotein B.
Several mechanisms by which the ubiquitin system and the proteasome
gains access to membrane-bound and luminal substrates of the ER
degradation pathway have been discussed in the literature. The
retrograde translocation mechanism in which substrates are transported
from the ER lumen or membrane through the Sec61p translocon has
received the most attention. Substrates in mammalian systems, such as
major histocompatibility complex class I molecules (14, 38),
CFTR
F508 (1), TCR subunits (40, 41), apolipoprotein B (8, 16, 42),
and RI332 (9), and substrates in yeast, such as mutant
Ste6p (17), mutant carboxypeptidase Y (7, 26), and mutant
pre-pro-alpha factor lacking carbohydrate side chains
(25) have been found to interact with Sec61p and are then released into
the cytosol when the ubiquitin system or the proteasome is inhibited.
Recently, Mayer et al. (19) have shown that a chimeric ER membrane
protein may be dislocated and degraded directly at the ER membrane by a
membrane-bound assembly of the ubiquitin-dependent proteasomal system.
These authors have suggested that the dislocation process may be driven
by the action of the proteasome. Indeed, several previous studies in
which ubiquitination or proteasomes are inhibited have suggested that
the activity of ubiquitin-dependent proteasomal system is a
prerequisite for the dislocation, or the retrograde translocation, of
substrates of the ER degradation pathway (5, 7, 9). This
membrane-extraction mechanism may be particularly relevant to
degradation of
1ATZ because ER degradation of this
substrate appears to involve polyubiquitination on the cytoplasmic tail
of the transmembrane ER chaperone calnexin only when it has bound
1ATZ at the luminal surface of the ER membrane.
Studies in yeast have also provided some information about the
components of the ubiquitin-dependent proteasomal system that appear to
assemble at the ER membrane, the "ubiquitin-conjugation platform." Biederer et al. (5) have shown that this platform includes Ubc7 bound to Cue1 and working in concert with the
membrane-bound ubiquitin-conjugating enzyme Ubc6. It is not yet clear
that the ubiquitin-conjugating enzyme E2-F1, the human ubc7, is the
human ortholog of yeast ubc7 (11).
A recent study by Van Leyen et al. (34) has suggested the possibility
that a type of autophagic response, termed programmed organelle
degradation, allows access of cytoplasmic proteases to both luminal and
integral membrane proteins. This process appears to involve the highly
regulated recruitment of 15-lipoxygenase from the cytoplasm to the ER
membrane, where it presumably oxygenates membrane phospholipids, in
turn releasing proteins from the ER lumen and membrane. A mechanism
like this could possibly account for the ER degradation pathway of some substrates.
In addition to degradation by the ubiquitin-dependent proteasomal
mechanism and by ubiquitin-independent proteasomal mechanisms, there is
evidence that nonproteasomal mechanisms may contribute in part to ER
degradation of some substrates. Previous studies have shown that
multiple mechanisms may contribute to ER degradation of CFTR
F508
(15, 29, 36), as appears to be the case for
1ATZ from
the studies reported here. Moreover, there is now evidence that
different mechanisms may account for the fate in the ER of different
mutants of the same ER protein (17).
 |
ACKNOWLEDGEMENTS |
The authors are indebted to Mary Pichler for preparing the
manuscript, Pam Hale and Katherine Massa for technical assistance, and
to Alan Schwartz, Aaron Ciechanover, and Julie Trausch-Azar for advice.
 |
FOOTNOTES |
These studies were supported in part by National Institutes of Health
Grants HL-37784 (D. H. Perlmutter), DK-52526 (D. H. Perlmutter),
DK-02379 (J. H. Teckman), and GM-35687 (R. Gilmore) and the American
Digestive Health Foundation Miles and Shirley Fiterman Foundation Basic
Research Award (J. H. Teckman).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. H. Teckman, Dept. of Pediatrics, Washington Univ. School of Medicine, 1 Children's Place, St. Louis, MO 63110 (E-mail:
teckman{at}kids.wustl.edu).
Received 5 May 1999; accepted in final form 9 September 1999.
 |
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