Ubc6p and Ubc7p Are Required for Normal and Substrate-Induced Endoplasmic Reticulum-Associated Degradation of the Human Selenoprotein Type 2 Iodothyronine Monodeiodinase
Diego Botero,
Balazs Gereben,
Carla Goncalves,
Lucia A. de Jesus,
John W. Harney and
Antonio C. Bianco
Thyroid Division (L.A.J., J.W.H., A.C.B.), Department of Medicine, Brigham and Womens Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Institute of Experimental Medicine (B.G.), Department of Neurobiology, Budapest H-1078, Hungary
Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Womens Hospital, Harvard Institutes of Medicine Building, Room 550, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail: abianco{at}partners.org.
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ABSTRACT
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The type 2 monodeiodinase (D2) is an endoplasmic reticulum-resident membrane selenoprotein responsible for catalyzing the first step in thyroid hormone action, T4 deiodination to T3. Its short half-life is due to ubiquitination and proteolysis by proteasomes, a mechanism that is accelerated by D2 interaction with T4. To identify proteins involved in D2 ubiquitination, a FLAG-tagged selenocystine133-to-Cys mutation of the human D2 (CysD2) was created and expressed in Saccharomyces cerevisiae using the GAL1 gene promoter. CysD2 activity was detected in the microsomes, indistinguishable from transiently expressed CysD2 in vertebrate cells. Treatment with 100 mg/ml cycloheximide or 30 µM T4 caused rapid loss of CysD2 (t1/2 =
30 min). Clasto-lactacystin ß-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T4. Immunoprecipitation with anti-FLAG antibody combined with Western analysis with antiubiquitin revealed that CysD2 is heavily ubiquitinated. Expression of CysD2 in yeast strains that lack the ubiquitin conjugases Ubc6p or Ubc7p stabilized CysD2 half-life by markedly reducing CysD2 ubiquitination, whereas no difference was detected in Ubc1p-deficient mutants. Similarly, expression of CysD2 in UBC6 and UBC7 mutants also impaired the substrate-induced loss of CysD2 activity and protein. In conclusion, Ubc6p and Ubc7p are required for normal and substrate-induced ubiquitination and proteolysis of D2.
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INTRODUCTION
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T3 IS A biologically active polyiodinated molecule containing a biphenyl ether structure that modulates gene expression through ligand-dependent transcription factors, the thyroid hormone receptors. The main product of thyroid secretion is T4, a prohormone that must be activated by conversion to T3 to initiate thyroid hormone action. This deiodination reaction occurs in the phenolic-ring and is catalyzed by two selenocystine (Sec)-containing deiodinases, i.e. D1 and D2 (type 1 and 2 monodeiodinase). Alternatively, both T4 and T3 can be irreversibly inactivated by deiodination of the thyrosyl-ring, a reaction catalyzed by D3, the third member of the selenodeiodinase group.
Because of its physiological plasticity D2, not D1, is considered the critical homeostatic deiodinase during adaptation to iodine deficiency, cold exposure, or changes in thyroid status. In constrast to D1, D2 has a relatively low Km (Michaelis-Menten constant; T4) and a short half-life, enabling it to respond within minutes to neural and endocrine stimuli (see Ref. 1 for review). D2 is a 32-kDa type 1, endoplasmic reticulum (ER)-resident membrane protein that has a short lumenal NH2 terminus and a single-transmembrane domain within its first 40 amino acids. The bulk of the enzyme is in the cytosol, including its Sec-containing catalytic active center (2). In immunocytochemical studies using confocal microscopy, its distribution is typically perinuclear and it colocalizes with ER-resident binding protein when transiently expressed in HEK-293 and NB-2A cells, and in a human mesothelioma cell line (MSTO-211) where it is endogenously expressed (2, 3).
D2 is an unstable protein with a half-life of approximately 45 min due to its susceptibility to ubiquitination and proteasomal degradation (4, 5, 6). Both D2 activity and protein levels are rapidly stabilized in GH4C1 rat pituitary tumor cells and MSTO-211 cells by treatment with proteasome inhibitors (3, 4). Proteasomal uptake of D2 requires accessibility to its COOH terminus because its fusion to the FLAG epitope (but not to the NH2 terminus) increases severalfold the pool of ubiquitinated D2 and prolongs its half-life by 3- to 4-fold (6). A unique feature of D2 is a further enhancement of its intrinsic metabolic instability after exposure to its substrates, T4 or rT3. This substrate-induced loss of D2 activity and protein was first demonstrated in vivo (7) and subsequently confirmed in a number of cell systems (3, 4, 8). Interaction with the Se-containing enzymes active center accelerates, by unknown mechanisms, D2 ubiquitination and subsequent proteolysis (5). There have been reports of proteasome-mediated, catabolite-induced enzyme inactivation (9, 10), but D2 displays this unique mechanism of substrate-induced selective proteolysis that regulates its own levels and hence thyroid hormone activation. In contrast, the proteasome system does not appear to play a significant role in the posttranslational regulation of the other two members of the selenodeiodinase family (6).
In eukaryotes, normal ER-resident proteins such as D2 are degraded by the ER-associated degradation (ERAD), which also functions as cellular protein quality control (11, 12). Selective proteolysis occurs by the 26S proteasome complex after retrograde transport to the cytosol and ubiquitination. ERAD involves a variety of components, among which enzymes of the ubiquitin system are pivotal. Selection of specific proteins for proteolysis is usually achieved at the level of ubiquitin conjugation to the target, a process that is coordinated by the combined actions of a series of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). This system is very well characterized in the yeast Saccharomyces cerevisiae where approximately a dozen E2s or E2-related proteins are known (13). A conserved catalytic domain of approximately 150 amino acids characterizes E2s. Individual E2s are involved in different cellular processes and, therefore, in the ubiquitination of different classes of substrate proteins. E3s, on the other hand, more abundant and with no overt sequence homology, are thought to be largely responsible for the high degree of specificity of protein ubiquitination (14). Few examples of E3s participating in ERAD are available but it is likely that more exist (15, 16, 17, 18, 19, 20).
In yeast, a number of ER-resident proteins that are ERAD substrates undergo ubiquitination, including Sec61p (21) and carboxypeptidase Y (22), as well as hydroxy-3-methylglutaryl-coenzyme A-reductase (23). Three E2s, namely Ubc1p, Ubc6p, and Ubc7p, have been implicated in ERAD (15). Deletion of UBC6 and UBC7 genes stabilizes a mutant Sec61p, Sss1p, carboxypeptidase Y, Pdr5, and uracil (URA) permease (21, 22, 24, 25). Ubc6p is a C-terminal anchored membrane protein whose catalytic site faces the cytosol (26). Unlike Ubc6p, Ubc7p lacks a membrane anchor but associates with an ER-bound protein, Cue1p (27). Ubc1p is part of an E2 group that includes Ubc4p and Ubc5p, essential for cell growth, viability, and in-bulk turnover of short-lived and abnormal proteins (16, 19, 28, 29).
The goals of the present investigation were to express a functional D2 in S. cerevisiae and identify which E2s are involved in the normal and substrate-induced D2 ubiquitination and selective proteolysis. Here we report that a functional Sec133D2Cys in S. cerevisiae retains its metabolic instability displayed in mammalian cells and is further destabilized upon exposure to T4. Both natural decay and substrate- induced D2-selective proteolysis are specifically blocked by clasto-lactacystin ß-lactone and depend on UBC6 and UBC7 but not on the gene coding for the other E2 ERAD component, Ubc1p.
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RESULTS
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Characterization of T4-to-T3 Conversion Activity in S. cerevisiae
Lysates of galactose-induced wild-type yeast cells transformed with pCysD2-FLAG displayed substantial T4 deiodinase activity. A formal time-response curve of galactose induction indicates that T4 deiodinase activity plateaus at approximately 60 fmol/min·mg protein during the 12- to 48-h interval (Fig. 1A
). Controls included uninduced transformed cells in which T4-to-T3 conversion was minimal at all times (not shown) and cells transformed with empty pYes2 vector in which deiodinase activity remained at background levels regardless of the presence of 2% galactose (Fig. 1A
).

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Figure 1. CysD2 Expression in S. cerevisiae
A, Time course of induction with 2% galactose. At the designated time points, cells were harvested, lysed, and processed for CysD2 activity. Raw numbers are shown to allow comparison of specific CysD2 activity (solid circles) with background detected in lysates of vector-transformed yeast cells (open circles). B, Cell lysates were fractionated by ultracentrifugation and processed for CysD2 activity as described in Experimental Procedures. C, Lineweaver-Burke plot of the substrate saturation curve of CysD2 activity. In all panels values are the mean ± SD of three to four measurements.
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Cell lysates were fractionated as described in Materials and Methods. Most of the D2 activity present in the cell lysate was localized in the microsomal fraction, compatible with CysD2 (FLAG-tagged Sec133-to-Cys mutation of the human D2) being a microsomal membrane protein in yeast, as is the case in vertebrate cells (Fig. 1B
). One of the properties of D2 that differentiates it from D1 is its insensitivity to inhibition by PTU. To verify D2 identity, all assays of deiodinase activity in yeast cell extracts were performed in the presence of 1 mM PTU, which only reduced total deiodinase activity 510%. CysD2 identity was also confirmed by the kinetics of the deiodination to T4 (Fig. 1C
). The calculated Km (T4) is approximately 2.5 µM, supporting that the T4 deiodinase activity found in the transformed yeast cells is authentic cysD2.
Evidence for Proteasomal Involvement in Basal and Substrate-Induced CysD2 Proteolysis in S. cerevisiae
To establish the relative CysD2 stability in S. cerevisiae, CysD2-expressing wild-type and cell wall- permeable Ise1 cells (30) were treated with 100 µg/ml cycloheximide (CX) for 30 min, harvested, lysed, and processed for CysD2 activity. Samples of similarly treated Ise1 cells were processed and analyzed by Western blot using anti-FLAG antibody to determine levels of FLAG-tagged CysD2 protein. Treatment with CX caused a rapid fall in CysD2 activity to 3550% of control values (Table 1
). We also tested whether the degradation of CysD2 in yeast is accelerated by substrate (30 µM T4). In both wild-type and in Ise1 cells D2 activity was rapidly lost after exposure to substrate (Table 1
and Fig. 2
) but not to 30 µM T3 (not shown). Loss of D2 activity is likely the result of CysD2 proteolysis because CysD2 protein levels fell accordingly during basal and substrate exposure conditions (Fig. 2
). In parallel experiments we used 30 µM Tetrac to induce CysD2 down-regulation, a T4 analog with reportedly greater permeability in yeast (31), but similar findings were obtained (not shown).

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Figure 2. CysD2 Is Stabilized by Clasto-Lactacystin ß-Lactone
D2-expressing wild-type yeast cells were incubated with 100 µg/ml CX for 30 min or 30 µM T4 during 2 h. Cells were harvested and processed for Western analysis with anti-FLAG antibody. As indicated, some cells were treated for 2 h with 20 µM clasto-lactacystin ß-lactone (Lac).
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To test whether the intrinsic D2 instability and its susceptibility to substrate were dependent on the proteasome function, such as in mammalian cells, D2-expressing Ise1 cells were treated with clasto-lactacystin ß-lactone during the last 2 h of induction with galactose, a specific proteasome inhibitor known to permeate the cell wall of these cells (30). Although treatment with clasto-lactacystin ß-lactone did not affect basal levels of D2 activity, galactose-induced D2 levels were significantly higher in the cells treated with the proteasome inhibitor (Table 1
) due to a prolongation of its half-life (Fig. 5A
). This is likely due to increased levels of D2 protein as indicated in the Western analysis (Fig. 2
). Treatment with clasto-lactacystin ß-lactone also blocked the CX- and T4-induced loss of D2 activity (Table 1
) and protein decay (Fig. 2
).

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Figure 5. Ubc6p and Ubc7p But Not Ubc1p Deficiency Stabilizes CysD2 Half-Life
CysD2-expressing cells were transferred to a galactose-free dextrose-containing CSM-URA media to stop Dio2 transcription. Cell samples were taken at the indicated time points thereafter and immediately frozen for later processing. In panel A, Ise1 cells were treated with 20 µM clasto-lactacystin ß-lactone (Lac) for 2 h before carbon source replacement. Results obtained with UBC1 cells are shown in panel B, UBC6 cells in panel C and UBC7 cells in panel D, along with wild-type cells as controls. Values are the mean ± SD of three to four independent experiments. Shown in panel E are typical changes in CysD2 levels by Western analysis in UBC1, UBC6, UBC7, and wild-type cells after transfer to dextrose-containing CSM-URA media.
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D2 Is Ubiquitinated in S. cerevisiae
The Western analysis of D2-expressing S. cerevisiae lysate show that in addition to the approximately 32-kDa D2 band, there is a large number of higher molecular mass bands (3290 kDa) that are more intense than the approximately 32-kDa band (Fig. 3
). These bands are specific to D2 because they are absent from the lane loaded with lysates of cells transformed with empty pYes2. The identity of these high molecular mass bands was investigated after the cell lysates were immunoprecipitated (IP) with anti-FLAG antibody and the pellets immunoblotted with anti-FLAG antibody or antiubiquitin antiserum after elution with an excess of FLAG peptide (Fig. 3
). In the IP lane probed with anti-FLAG antibody, there is a perfect match with the bands detected in the total cell lysate, confirming the specificity of the anti-FLAG antibody. When IP pellets were probed with the antiubiquitin antiserum, the CysD2 band almost disappeared but the staining of the highest molecular mass bands, which matched those visualized with the anti-FLAG antibody, was even more intense, compatible with their identity as ubiquitin-CysD2 conjugates. It is notable that these ubiquitin-CysD2 conjugates are smaller than those of 100- to 300-kDa forms in HEK-293 cells (Fig. 3
).

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Figure 3. Identification of Ubiquitin-CysD2 Conjugates
Total cell lysates were processed for Western analysis using anti-FLAG antibody (lanes B and E). Alternatively, total cell lysates were immunoprecipitated with anti-FLAG antibody, and the pellets were Western blotted with anti-FLAG antibody (C) or antiubiquitin antiserum (D). Controls included a total cell lysate prepared from HEK-293 cells transiently expressing CysD2 (A) and a total cell lysate prepared from yeast cells transformed with vector (E). Ub, Ubiquitin.
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CysD2 Degradation in S. cerevisiae Requires Ubc6p and Ubc7p But Not Ubc1p
In the previous sections we have established that D2 has a short half-life and undergoes ubiquitination in wild-type yeast cells. More importantly, the substrate-induced loss of D2 activity and protein, a typical D2 characteristic in mammalian cells and tissues, also occurs in yeast cells. In the next set of experiments we wished to identify potential E2 proteins involved in the ubiquitination of D2. We therefore used the UBC1, UBC6, and UBC7 yeast mutants, which lack Ubc1p, Ubc6p, and Ubc7p ER-associated E2 enzymes, respectively. In wild-type cells, basal D2 activity was 57 fmol T4/min·mg protein, not different from basal D2 levels in the Ubc mutants (Table 2
). Exposure to 2% galactose induced D2 activity by approximately 6.8-fold in wild-type cells but the induction in Ubc6p- and Ubc7p-deficient mutants was much higher, approximately 11- and 17-fold, respectively (Table 2
). No increased induction was observed in the Ubc1p-deficient yeast mutants. The Western analysis of these cell lysates using anti-FLAG antibody revealed that the expression of D2 protein is much higher in the UBC6 and UBC7 mutant strains, particularly in the latter (Fig. 4
), in agreement with the higher D2 activity observed in this strain. Interestingly, the intensity of the Ub-D2 bands is decreased in lysates of UBC6 and UBC7 mutants, supporting impaired D2 ubiquitination (Fig. 4
). On the other hand, D2 levels are normal, and the pattern of high molecular mass ubiquitin-D2 conjugates seen in wild-type cells is present in the lysate of UBC1 cells, suggesting that the deletion of the third Ubcp involved in ERAD, Ubc1p, did not stabilize CysD2 (Fig. 4
).

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Figure 4. CysD2 Ubiquitination Is Decreased in UBC6 and UBC7 Yeast Mutants
CysD2-expressing UBC1, UBC6, and UBC7 yeast mutants were lysed and processed for Western analysis with anti-FLAG antibody. The profile obtained with UBC1 total cell lysate (A) is similar to that of wild-type cells (Fig. 3 , lane B). The total cell lysates of UBC6 (B) and UBC7 (C) contain less ubiquitinated CysD2. As a control, a total cell lysate of HEK-293 cells transiently expressing CysD2 is shown in panel D. Ub, Ubiquitin.
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To confirm that ubiquitination is a critical step in determining the CysD2 half-life in yeast, we studied CysD2 activity half-life and protein levels in the UBC mutants after the Dio2 gene transcription was blocked by changing the carbon source to dextrose. To validate the system, we first demonstrated that treatment of wild-type yeast with lactacystin, a specific proteasome inhibitor, prolongs D2 half-life (Fig. 5A
). A similar result was observed in the mutant strains. In the first 10 min after dextrose addition, there was a transient 2040% increase in CysD2 activity possibly due to dextrose being a superior carbon source than galactose (Fig. 5
, BD). After that, however, CysD2 activity disappeared in the wild-type cells with a half-life of approximately 23 min. The levels of CysD2 protein, as detected by Western analysis using anti-FLAG antibody, were also rapidly decreased after Dio2 transcription was blocked (Fig. 5E
). Interestingly, in both UBC6 and UBC7 strains there was a prolongation of the D2 activity half-life to more than 60 min, whereas no change in activity half-life was detected when CysD2 was expressed in the UBC1 strain (Fig. 5
, BD). This correlates with the levels of CysD2 protein, stabilized in the UBC6 and UBC7 mutants after inhibition of Dio2 transcription when compared with wild-type and UBC1 cells. These results confirm that a prolongation in the CysD2 half-life caused by an abrogation of its ubiquitination and degradation is the cause of elevated CysD2 activity and protein levels in Ubc6p- and Ubc7p-deficient strains.
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DISCUSSION
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This is the first report of heterologous expression of a human iodothyronine monodeiodinase in yeast. The level of CysD2 expression achieved with the GAL promoter-driven expression vector resulted in measurable enzyme activity as well as quantifiable CysD2 protein (Figs. 1
and 2
). The CysD2 properties in yeast were similar to that in mammalian cells in a number of respects, indicating that this model system is physiologically relevant. First, CysD2 activity is localized in the microsomal fraction of yeast cell lysates, indicating membrane localization, and it displays kinetic properties indistinguishable from transiently expressed CysD2 in mammalian cells (Fig. 1
). Second, CysD2 protein levels correlate with enzymatic activity (Figs. 2
and 4
). Third, the short half-life of CysD2 activity and protein (
30 min), as assessed by either CX-induced arrest in protein synthesis or by dextrose-induced transcriptional suppression, are remarkably similar to the values found in mammalian cells (3, 5) (Figs. 2
and 5
). Under both conditions the rapid turnover rate is stabilized by treatment with proteasome inhibitors (Figs. 2
and 5
). Fourth, yeast CysD2 is susceptible to the unique and highly specific substrate-induced acceleration of proteolysis mediated by the proteasome pathway (Fig. 2
and Table 1
). These four points indicate that D2 ubiquitination and proteolysis in yeast is due to its intrinsic instability and substrate interaction, and not due to generalized nonspecific activation of the unfolded-protein response (UPR) due to the expression of a heterologous protein.
There is considerable evidence that D2 is degraded by proteasomes in mammalian cells (3, 4, 5, 6). The present study provides additional confirmation that proteasomes mediate selective D2 proteolysis. Galactose-induced CysD2 activity (Table 1
) and protein levels (Fig. 2
) are increased in Ise cells treated with lactacystin b-lactone, a yeast wall-permeable proteasome inhibitor. This increase in CysD2 activity is due to stabilization of D2 half-life, which was doubled in the presence of proteasome inhibitor (Fig. 5E
). Accordingly, treatment with this proteasome inhibitor also blocked the CX-induced loss of CysD2 activity (Table 1
).
Ubiquitination is required for the proteasomal degradation of D2 (6). Ubiquitin-D2 conjugates were first identified in HEK-293 cells transiently expressing CysD2-FLAG after immunoprecipitation with anti-FLAG antibody and Western analysis with antiubiquitin antiserum (6). In the present investigation, a similar strategy was used to establish the identity of the abundant FLAG-containing higher molecular mass bands detected in the Western analysis of the yeast cell lysates (Fig. 3
). It is interesting that, contrary to the ubiquitin-CysD2 bands seen in HEK-293 cells, these ubiquitin-CysD2-containing yeast bands are abundant and display a ladder pattern (Fig. 3
). The reasons for this discrepancy are not clear but could indicate that the proteasomal uptake of the ubiquitin-CysD2 conjugates is less efficient in yeast so that a larger more evident pool of these conjugates can build up.
Once the D2 yeast expression system was sufficiently characterized and convincing evidence of its similarity to the mammalian system and physiological relevance were obtained, mutant yeast strains that lack specific E2 enzymes of the ERAD were used to define which of these are rate limiting in D2 ubiquitination. In a previous study we employed a similar genetic analysis to establish the essential role of E1 and ubiquitination in D2 proteolysis (6). Two observations point to the involvement of Ubc6p and Ubc7p in regulating D2 proteolysis in yeast: the abundance of the ubiquitin-CysD2 conjugates is much lower and the levels of CysD2 protein are much higher than in wild-type or UBC1 cells (Fig. 4
). Both results are supported by higher D2 activity (Table 1
) and protein levels (Fig. 4
) documented in these cells. These findings are explained by a prolonged activity and protein half-life in both UBC6 and UBC7 mutants, documented in experiments in which Dio2 transcription was blocked with dextrose (Fig. 5
). This indicates that both Ubc6p and Ubc7p are directly involved in the normal turnover and ubiquitination of CysD2. Ubc1p, on the other hand, is not.
Substrate-induced loss of D2 activity and protein also occurs in yeast (Tables 1
and 2
and Fig. 2
), indicating that it is mediated by intrinsic properties of the D2 protein rather than by vertebrate cell-specific mechanisms. Even though we do not yet understand the details of the molecular mechanisms involved, it is clear that substrate interaction with the enzymes active center accelerates ubiquitination and hence proteasomal degradation (5, 6). It is remarkable that this unique mechanism is lost in UBC6 and UBC7 mutants (Table 1
). This is a clear indication that both Ubc6p and Ubc7p are involved in not only the normal turnover of D2 but also the substrate-induced CysD2 ubiquitination. On the other hand, CysD2 expression in the UBC1 mutant was indistinguishable from wild-type cells in terms of basal and galactose-inducible CysD2 activity, susceptibility to substrate exposure (Table 2
), and half-life (Fig. 5
). Because of the specific link between Ubc1p and UPR (16), this also supports the specificity of CysD2 proteolysis in our system, which is unrelated to UPR.
The rapid loss of D2 activity and protein could be explained by ubiquitination alone, with only a minor role played by the proteasomes. Previous studies indicate that D2 is inactive after conjugation to ubiquitin because the level of unconjugated, but not conjugated, D2 protein correlates with D2 activity (6). Given the large pool of ubiquitin-D2 conjugates, the loss of D2 activity and protein levels reflects ubiquitination and not proteolysis per se. This observation is supported by the increased D2 activity and protein in the UBC6 and UBC7 cells (Table 1
and Fig. 4
) that display impaired D2 ubiquitination.
Although we do not fully understand the mechanism by which substrate interaction increases ubiquitination, it is intriguing to speculate that other Ubc6p and Ubc7p substrates might also be subject to a similar regulatory mechanism. In the case of D2, this process is a potent homeostatic regulator of the biological activity of secreted thyroid hormone.
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MATERIALS AND METHODS
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Reagents
CX and clasto-lactacystin ß-lactone were obtained from Calbiochem (La Jolla, CA) and dissolved in dimethylsulfoxide. T4 (Sigma Co., St. Louis, MO) and PTU (United States Biochemical Corp., Cleveland, OH) were dissolved in 40 mM NaOH. Except when stated otherwise, all chemicals and reagents were from Sigma. Outer ring-labeled T4 (specific activity, 4400 Ci/mmol) was from NEN Life Science Products (Boston, MA).
Yeast Strains, Plasmids, Transformation, and Culture Media
The strains of S. cerevisiae used in the present investigation are described in Table 3
. All were generous gifts of Dr. Alfred Goldberg (Harvard Medical School, Boston, MA). UBC1 genotype was confirmed by PCR using both primers from the UBC1 locus (32).
For the yeast expression studies a Sec133-to-Cys mutation of the human D2 (CysD2) was created because yeast lack the aminoacyl transfer tRNA for Sec incorporation. This substitution increases the D2 Km (T4) by approximately 500-fold (33). The eight-amino acid FLAG sequence was fused to the COOH terminus of CysD2. The COOH-FLAG-CysD2-pYES2 construct (pCysD2-FLAG) was generated by inserting the EcoRI/XbaI fragment of the COOH-FLAG-CysD2 D10 (6) between the corresponding sites of the pYES2 vector, a high-copy episomal vector designed for inducible expression of recombinant proteins in S. cerevisiae using the GAL1 gene promoter (Invitrogen, Carlsbad, CA).
All media and related products were obtained from BIO101 (Carlsbad, CA). Yeast cells were made competent and transformed using the S.c. EasyComp Transformation Kit (Invitrogen) according to the instructions of the manufacturer. Transformed cells were grown in agar plates of synthetic defined medium (SD) containing complete supplement mixture (CSM-URA) and 2% dextrose. Single colonies were grown exponentially [optical density at 600 nm (OD600), 0.81.0] at 30 C in liquid CSM-URA containing 2% dextrose.
Experimental Procedures
For D2 induction studies, cells were grown exponentially at 30 C in SD containing CSM-URA and 2% galactose (
16 h to OD600
0.8). In the time course experiment, induction was stopped at specific time points. In the CX- and T4-induced decay experiments, approximately 15 OD600 log-phase wild-type and Ise1 cells were harvested and resuspended in 4 ml of prewarmed SD medium containing CSM-URA, 2% galactose, and 100 µg/ml CX or 30 µM T4. CX-treated cells were harvested after 30 min and T4-treated cells were harvested after 2 h. Some cells were treated for 2 h with 20 µM clasto-lactacystin ß-lactone. At the end of all treatments cells were either processed for D2 activity or Western analysis (see below). When exposed to T4, cells were washed once in media containing 20% horse serum to deplete the cell pellet of T4. In the dextrose-induced Dio2 gene shut-off experiments, approximately 15 OD600 log-phase cells were collected by centrifugation and then shifted to a prewarmed CSM-URA medium containing 2% dextrose to shut off Dio2 gene transcription. Aliquots were taken at the indicated times and either processed for D2 activity or Western analysis.
Cell Harvesting and Lysis
Cells were collected in an equal volume of 30 mM sodium azide on ice and harvested by centrifugation at 2500 rpm for 10 min. The cell pellets were immediately frozen in liquid nitrogen and stored at -80 C. For cell lysis, pellets were resuspended in 1:1 vol phosphate buffer, pH 7.4, containing 110 mM NaCl, 5 mM EDTA, 10 mM dithiothreitol (DTT), and a number of protease inhibitors (400 µg/ml aprotinin, 145 mM benzamide, 30 µM leupeptin, 100 µg/ml pepstatin A, 1 mM phenylmethylsulfonylfluoride). An equal volume of glass beads (0.5 mm, Sigma) was added to the cell suspension which was followed by agitation. Cell disruption was achieved after 10 cycles of 1 min of blending (vortex) and 1 min cooling in ice. Lysates were then centrifuged at 3,000 rpm for 10 min and the supernatant transferred to a new tube containing sucrose in lysis buffer to a final concentration of 0.25 M and stored at -80 C until further processing. In one experiment cell lysates were processed for subcellular fractionation. These were centrifuged at 3,000 x g for 10 min. The supernatant was then transferred to a new tube and centrifuged at 100,000 x g for 60 min at 4 C. The cytosol (supernatant) was stored and the microsomal pellet washed once with lysis buffer. Both cytosol and pellet were later processed for D2 activity.
D2 Assay
D2 activity was measured as described (3). Essentially, about 300 µg total cell lysate protein were incubated for 3 h in the presence of 0.51 nM [125I]5'-T4, 20 mM DTT, and 1 mM PTU. Specific T4-to-T3 conversion was calculated by subtracting nonspecific deiodination in tubes containing the same amount of protein cell lysate obtained from cells of the same strain transformed with empty pYes2 vector. The background activity of these samples was less than 2%. Deiodinase activity was expressed as femtomoles T4/min·mg protein.
IP and Western Blot Analysis
In these studies cell lysates were obtained similarly as described above except that lysis buffer contained 0.5% Triton X-100 and no DTT was added (34). Total cell lysates (
100 µg) were resolved by 12% SDS-PAGE and electrotransferred to a polyvinylidine difluoride membrane (Immobilon, Millipore Corp., Bedford, MA) for the Western analysis. Alternatively, cell lysates (
400 µl) were immunoprecipitated with 30 µl of M2-anti-FLAG agarose affinity gel (Sigma) for 4 h at 4 C. After three successive washes with lysis buffer FLAG-tagged proteins were eluted with 50 µl of 100 µg/ml FLAG peptide. The supernatant (40 µl) was then mixed with gel loading buffer and resolved by SDS-PAGE as above. Western analysis used the chemiluminescence kit of Roche Molecular Biochemicals (Indianapolis, IN), according to the instructions of the manufacturer. The blots were probed with an anti-FLAG M2 antibody (1:3333, Sigma) or with a polyclonal antiubiquitin antibody (1:1000, Chemicon, Temecula, CA). In the Western analysis, we used a lysate of HEK-293 cells transiently expressing a FLAG-tagged D2 as positive control as described previously (6). The negative control was a lysate of yeast cells transformed with empty pYes2 vector. Protein concentrations were measured by Bradford (35).
Statistical Analysis
Data are presented as mean ± SD throughout the studies. ANOVA was used for comparative analysis; 5% was the level of significance required to reject the null hypothesis.
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ACKNOWLEDGMENTS
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The authors are grateful to Dr. P. Reed Larsen for his support and for his review of this manuscript.
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FOOTNOTES
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This work was supported by NIH Research Grant DK-58538.
B.G. is a Magyary Zoltán postdoctoral fellow of the Hungarian Education Ministry and supported by an Felsooktatási Kutatási és Fejlesztési Pályázat grant.
* Present address: Division of Endocrinology, Childrens Hospital Boston, Boston, Massachusetts 02115.
Present address: Alameda Rio Claro, 189, Apartment 1, 01332-010 São Paulo SP, Brazil.
Abbreviations: CSM, Complete supplement medium; CX, cycloheximide; CysD2, FLAG-tagged Sec133-to-Cys mutation of the human D2; D1, D2, D3, type 1, type 2, and type 3 monodeiodinase; DTT, dithiothreitol; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; ER, endoplasmic reticulum; ERAD, ER-associated degradation; IP, immunoprecipitated; PTU, propylthiouracil; SD medium, synthetic defined medium; Sec, selenocystine; UPR, unfolded protein response; URA, uracil.
Received for publication April 8, 2002.
Accepted for publication June 3, 2002.
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