Role of ubiquitin in proteasomal degradation of mutant alpha 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A delay in intracellular degradation of the mutant alpha 1-antitrypsin (alpha 1AT)Z molecule is associated with greater retention within the endoplasmic reticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha 1AT deficiency. Recent studies have shown that alpha 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 alpha 1ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha 1ATZ mRNA. To determine whether the ubiquitin system is required for proteasomal degradation of alpha 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 alpha 1ATZ. The results showed that there was marked, specific, and selective inhibition of alpha 1ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha 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 alpha 1ATZ and that the ubiquitin-conjugating enzyme E2-F1 may play a role in the ubiquitin-dependent proteasomal mechanism.

alpha 1-antitrypsin deficiency; liver disease; emphysema; quality- control apparatus; protein degradation; endoplasmic reticulum


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HOMOZYGOUS PIZZ alpha 1-antitrypsin (alpha 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 alpha 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 alpha 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 alpha 1AT deficiency indicate that emphysema is caused by decreased alpha 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, alpha 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 alpha 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 alpha 1ATZ correlated with protection from liver disease. We now know that ER degradation of alpha 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 CFTRDelta 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 alpha 1ATZ required stable binding to the transmembrane ER chaperone calnexin and that there was polyubiquitination of calnexin only in microsomes which had translocated alpha 1ATZ. Moreover, in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) and lactacystin, which inhibit deubiquitination and proteasomal degradation, respectively, an alpha 1ATZ-polyubiquitinated calnexin complex was directly demonstrated (27). Wild-type alpha 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 alpha 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 alpha 1ATZ can be identified. First, we examined the effect of dominant-negative ubiquitin molecules on degradation of alpha 1ATZ in intact cells. Second, we examined the effect of fractionation and reconstitution of proteolysis-primed lysate on degradation of alpha 1ATZ in the cell-free microsomal translocation system.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha 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 alpha 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 alpha 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 alpha 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 beta -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 beta -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 alpha 1ATM cDNA or alpha 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). alpha 1ATM and alpha 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 alpha 1ATM or alpha 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-beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of dominant-negative ubiquitin mutants on ER degradation of alpha 1ATZ in intact cells. First, we examined the effect of the K48R and K48R-G76A dominant-negative ubiquitin mutants on the degradation of alpha 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 alpha 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 alpha 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 alpha 1ATZ. Next we examined the kinetics of degradation of alpha 1ATZ in CJZ cells transfected with K48R-G76A ubiquitin (Fig. 1, bottom). Here, alpha 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 alpha 1ATZ-specific radioactivity present at 2.5 h of the chase period. The K48R-G76A ubiquitin mutant inhibited degradation of alpha 1ATZ in six separate experiments in two different cell lines (data not shown). The half-time for the degradation of alpha 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 alpha 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 alpha 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 alpha 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 beta -galactasidase activity of the cells when cotransfected with pZeoSV LacZ vector and the relevant ubiquitin vector. Approximately 60% of the cells stained positively for beta -galactasidase activity in each case. The single K48R ubiquitin mutant did not inhibit degradation of alpha 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 alpha 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 alpha 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 alpha 1ATZ polypeptide is indicated by arrows at left.

Degradation of alpha 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 alpha 1ATZ. Our previous studies have shown that alpha 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 alpha 1ATZ in this system (Fig. 2). Mutant alpha 1ATZ and wild-type alpha 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 alpha 1ATZ and alpha 1ATM mRNA. This polypeptide corresponds to alpha 1AT with high-mannose-type carbohydrate side chains. For mutant alpha 1ATZ, this polypeptide is rapidly degraded by 1 h of the chase period. For wild-type alpha 1AT, there is very little disappearance over the 6-h chase period. Rather, the alpha 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 alpha 1ATZ in the cell-free system that is used for all the subsequent experiments.


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Fig. 2.   Degradation of mutant alpha 1ATZ and wild-type alpha 1ATM in cell-free system. alpha 1ATZ and alpha 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.

We also examined the degradation of alpha 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 alpha 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 alpha 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 alpha 1ATZ in native (RM), salt-washed (KRM), and puromycin-treated salt-washed (PKRM) microsomal vesicles. alpha 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.

To provide further evidence for the requirement of the ubiquitin system in degradation of alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 1ATZ in cell-free system using fractions of rabbit reticulocyte lysate. A: microsomal translocation assays were programmed with alpha 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 alpha 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.

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.

Next, we examined the possibility that the intermediate level of degradation of alpha 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), alpha 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 ATPgamma S, and Fr II supplemented with lactacystin. Together, these results indicate that there is a component of the degradation of alpha 1ATZ in the cell-free system that is mediated by the proteasome independent of ubiquitin.



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Fig. 6.   Degradation of alpha 1ATZ in Fr II in absence of ATP or in presence of ATPgamma S or lactacystin. A: microsomal translocation assays were programmed with alpha 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 ATPgamma S (5 mM) were added (Fr II + ATPgamma 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.

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 alpha 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 alpha 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 alpha 1ATZ in the cell-free system using fractions of rabbit reticulocyte lysate. A: translocation assays were programmed with alpha 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.

To exclude the possibility that E2-F1 had a nonspecific effect on degradation of alpha 1ATZ, we compared the fate of alpha 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 alpha 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 alpha 1ATZ in the cell-free system using Fr I and II. Translocation assays were programmed with alpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, two strategies were used to determine whether the ubiquitin system was required for proteasomal degradation of the mutant secretory protein alpha 1ATZ. First, dominant-negative mutant ubiquitin molecules were coexpressed in intact cells in which alpha 1ATZ is retained/degraded in the ER. The results showed that ER degradation of alpha 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 alpha 1ATZ. Second, degradation of alpha 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 alpha 1ATZ in this system. The ubiquitin-independent mechanism was demonstrated by the presence of some degradation of alpha 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 alpha 1ATZ. These data indicate that there is also a ubiquitin-dependent mechanism for degradation of alpha 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 CFTRDelta 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-alpha subunit is degraded in the ER by a pathway that involves the proteasome (2, 40, 41). Although polyubiquitination of TCR-alpha can be demonstrated in transfected cells when the proteasome is inhibited with lactacystin (40), a TCR-alpha mutant lacking lysines and therein unable to undergo ubiquitin conjugation is degraded as rapidly as wild-type TCR-alpha (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), CFTRDelta 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 alpha 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 alpha 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 CFTRDelta F508 (15, 29, 36), as appears to be the case for alpha 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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