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J. Biol. Chem., Vol. 281, Issue 21, 14615-14621, May 26, 2006
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From the Department of Cell and Molecular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Received for publication, January 25, 2006 , and in revised form, March 24, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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50% of that of a wild-type homodimer (5).
On the basis of the high conservation of this 16-residue-long deiodinase dimerization domain (DDD), we generated a DDD-green fluorescent protein (GFP) fusion that substitutes for one of the binding partners in the assembly process (5). This soluble non-catalytic surrogate of the deiodinase subunit was found (i) to co-localize with both the D1 and SeD2 subunits, (ii) to form heterodimers with both the D1 and SeD2 subunits that could be isolated by immunoprecipitation, and (iii) to impair the catalytic activity of the assembled D1 and SeD2 heterodimers just like the heterodimers containing a catalytically inactivated full-length deiodinase subunit (6). Notably, the soluble DDD-GFP deiodinase subunit surrogate eliminates the potential untoward effects resulting from mutagenesis-induced conformation changes in the polypeptide regions flanking the DDD in the native deiodinase subunit.
In this study, we used alanine scanning mutagenesis to identify key residues in the DDD that are required for holoenzyme assembly. The results show that three residues (Ile152, Tyr153, and Ile154) are responsible for the assembly of a D1 homodimer and that tight binding of the D1 dimerization domain (
G = –60 ± 5 kJ/mol for the isolated DDD) prevents holoenzyme disassembly and/or the dynamic exchange of binding partners. Site-directed mutagenesis was then used to generate a dimer-deficient full-length D1 subunit. This dimer-deficient D1 subunit was catalytically active and did not form dimers. For SeD2, the assembly of the type II deiodinase homodimer required a broader region of the DDD and utilized residues at both ends of this domain. These data illustrate that the deiodinase family utilizes a common dimerization domain for the assembly of a fully functional enzyme.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Culture—LLC-PK1 and SeD2 cells (C6 cells constitutively expressing exogenous rat SeD2) (8) were grown in 25-cm2 flasks, unless stated otherwise, in a humidified atmosphere of 5% CO2 and 95% air at 37 °C as described previously (6, 8). The growth medium was composed of Dulbecco's modified Eagle's medium containing 15 mM sodium bicarbonate, 25 mM HEPES (pH 7.2), 25 mM glucose, 1 mM sodium pyruvate, 10% (v/v) bovine calf serum, 50 milliunits/ml penicillin, and 90 µg/ml streptomycin. Constitutive expression of SeD2 in C6 cells was maintained by adding 200 µg/mg G418 to the growth medium (8). HEK293 cells were grown as described above with 10% (v/v) fetal bovine calf serum substituted for bovine calf serum. All cell types were subcultured every 3–5 days by seeding 50,000 cells/cm2 into 25-cm2 flasks. The culture medium was changed three times weekly.
Alanine Scanning Mutagenesis of the p27 Dimerization Domain—Complementary synthetic oligonucleotides corresponding to nucleotides 445–496 of rat p27 (GenBankTM accession number X57999 [GenBank] ) were used to create a series of DDD constructs with serial alanine substitutions along the D1 dimerization domain. 72-mer complementary oligonucleotides based on the native DDD sequence (5'-TAGACCACCATGGTCGCTGACTTCCTCATCATTTACATTGAAGAAGCTCACGCCACAGATGGATGGGCTCTG-3' and 5'-GATCCAGAGCCCATCCATCTGTGGCGTGAGCTTCTTCAATGTAAATGATGAGGAAGTCAGCACCATGGTGGT-3') were annealed, yielding a double-stranded 72-bp fragment (see Ref. 5) with an N-terminal Met residue in a Kozak consensus start site and NheI and BglII overhangs, and ligated in-frame into pEGFP-N1 (Clontech), generating sequential alanine-substituted dimerization domain (ADD)-GFP fusion protein(s). Correct insertion was confirmed by DNA cycle sequencing.
Generation of ADD-GFP-expressing LLC-PK1 and SeD2 Cells—LLC-PK1 or SeD2 cells were grown in 25-cm2 flasks to 60% confluence and transfected with 5–10 µg of pEGFP-N1 (GFP control), pDDD-GFP, or individual members of the pADD-GFP family (A147DD to A163DD) using cationic liposomes (DOTAP®, Roche Applied Science, Basel, Switzerland) according to the manufacturer's instructions. As indicated, cells constitutively overexpressing DDD-GFP or individual ADD-GFP fusion proteins were generated by antibiotic selection with 1.5 or 1.0 mg/ml G418 for LLC-PK1 and SeD2 cells, respectively. G418-resistant cells were expanded without clonal selection, and individual cell populations were grown in the presence of 0.8–1.0 mg/ml G418 to maintain expression of the exogenous GFP fusion protein. Greater than 99% of the G418-selected LLC-PK1 and SeD2 cells expressed GFP fusion proteins as judged by fluorescence microscopy. The quantity of DDD-GFP fusion protein present in the LLC-PK1 and SeD2 cells that constitutively expressed the individual members of the ADD series was determined by measuring total GFP fluorescence in cell lysates using excitation at 484 nm and emission at 507 nm.
Generation of Dimer-deficient D1 Subunits (p27A152)—Dimerization-deficient p27 was generated by an I152A substitution. In brief, 24-mer synthetic oligonucleotides bracketing the Ile152 codon (nucleotides 450–473, 5'-CTTCCTCATCGCTTACATTGAAGA-3' ((+)-strand) and 5'-TCTTCAATGTAAGCGATGAGGAAG-3' ((–)-strand), with the mutagenic nucleotides underlined) and the QuikChange-XL® mutagenesis kit (Stratagene, La Jolla, CA) were used to create full-length p27A152 according to the manufacturer's instructions. The integrity of the dimerization-deficient D1 subunit was confirmed by DNA cycle sequencing of the entire coding region (nucleotides 7–780).
DNA Sequencing—Double-stranded DNA sequencing was performed following the dideoxynucleotide method of Sanger et al. (9) using cycle sequencing and iterative primers. All sequence information was confirmed by sequencing both strands.
Photomicroscopy—LLC-PK1 cells were seeded onto glass coverslips and grown to 60% confluence in 6-well culture plates. Cells were transfected with 0.5 µg of GFP (control), pDDD-GFP, or individual pADD-GFP plasmids as described above and grown at 37 °C for 24 h. Cells were then fixed with 4% paraformaldehyde and washed with 100 mM glycine, and the nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes, Eugene, OR). The subcellular distribution of GFP, DDD-GFP, and the individual ADD-GFP fusion proteins in LLC-PK1 cells was determined by fluorescence microscopy. Digital images were captured using a SPOT CCD camera (Meyer Instruments, Inc., Houston, TX) and processed using Adobe Photoshop software. The photomicrographs shown are representative of 20–30 independent fields.
Affinity Labeling and Sucrose Density Gradient Centrifugation of p27- and p27A152-expressing LLC-PK1 and HEK293 Cells—Six 75-cm2 flasks containing confluent monolayers of either LLC-PK1 or HEK293 cells were transiently transfected with 7.5 µg of pcDNA3/p27A152 (dimerization-deficient p27) or pcDNA3/p27 (control) by cationic liposomes (DOTAP®) according to the manufacturer's instructions and grown for 24–48 h. D1 was affinity-labeled with 2 nM N-bromoacetyl-L-[125I]thyroxine (BrAc[125I]T4) in situ as described previously (6, 10), and cell membranes were isolated on 16% Percoll as detailed previously (6, 10, 11). The cell membranes in fractions 10–12 were collected, and the Percoll was removed by centrifugation at 3.9 x 108 gmin for 30 min in a Beckman SW 60Ti rotor. The purified membranes were solubilized with 5 mM taurodeoxycholate, and cell debris was removed by micro-centrifugation. Aliquots (300 µl) of solubilized membrane protein were layered onto 3.5 ml of 4–12% linear sucrose gradients prepared in 20 mM HEPES (pH 7.4) containing 1 mM EDTA, 1 mM dithiothreitol, and 5 mM taurodeoxycholate. Gradients were centrifuged for 24 h at 3.9 x 108 gmin in the SW 60Ti rotor, and 100-µl fractions were collected from the top of the sucrose gradient.
Immunoprecipitation of D1 Monomers and Heterodimers—1 µl of anti-p27 antiserum (R3050) (6) and 10 µl of protein A beads (Repligen Corp., Waltham, MA) were added to individual sucrose gradient fractions and incubated for 90 min at 4 °C. Immune complexes were collected by centrifugation, and the protein A beads were washed with 10 volumes (v/v) of lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol) and then counted in a well-type 
-counter.
DDD Peptide Competitive Inhibition Assays—The binding properties of selected synthetic alanine-substituted DDD mutants and the DDD were determined by a fluorescence-based competitive binding assay. Reacti-BindTM streptavidin-coated polystyrene plates (96-well; Pierce) were incubated with a 3-fold molar excess (360 pmol) of the C-terminally biotinylated DDD peptide (ADFLVIYIEEAHATDGWILAILK-biotin; DDD-biotin) at room temperature for 1 h. Unbound peptide was removed by washing three times with 200 µl of phosphate-buffered saline (pH 7.2) at 70 °C, and the plate was kept at 4 °C until used.
Fluorescein isothiocyanate (FITC)-labeled DDD (ASADFLIIYIEEAHATDGWAFKNNVK-FITC; DDD-FITC) was dissolved in phosphate-buffered saline (final concentration of 154 nM at pH 7.2) and used as the reporter molecule. In brief, 100 µl of DDD-FITC was mixed with 100 µl of DDD or ADD peptide (A152DD, A153DD, or A154DD) at 0–300 µM; preformed DDD dimers were destabilized by heating to 70 °C (see "Results"); and 100-µl aliquots of each probe/peptide mixture were added to individual wells of the DDD-coated plate. Binding reactions were performed at 70 °C for 5 min. Unbound peptide was removed by aspiration; the wells were rinsed three times with 200-µl aliquots of phosphate-buffered saline heated to 60 °C; and 100 µl of phosphate-buffered saline at room temperature was then added to each well. Fluorescence was determined on a SpectraMax Gemini XS fluorometer (Molecular Devices Corp.) at wavelengths of 492 nm (excitation) and 520 nm (emission) and processed using SoftMax Pro Version 4 software.
Analytical Methods—D1 activity was determined in cell lysates by measuring the release of radioactive iodide from 10 µM 3',5',3-[125I]triiodothyronine (100 cpm/pmol) in 20 mM dithiothreitol (unless indicated otherwise) in a total volume of 100 µl. D1 assays were performed in triplicate, and reactions were conducted in universal deiodinase buffer (100 mM boric acid, 100 mM HEPES, 100 mM glycine (pH 5.5), and 1 mM EDTA) with 10–25 µg of cell protein and incubated at 37 °C for 60 min. Product formation was measured as described previously (10), and data are expressed as units/mg of protein, where 1 unit equals 1 pmol of iodide released per min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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LLC-PK1 cells expressing a catalytically functional D1 holoenzyme were transfected with DDD-GFP, individual members of the ADD-GFP series, or GFP alone, and cells constitutively expressing the individual GFP fusion protein were selected by G418 resistance. Expression of the DDD-GFP fusion protein was confirmed by visual inspection using fluorescence microscopy. After correcting for autofluorescence, the cellular levels of the ADD/DDD-GFP fusion protein in LLC-PK1 cell lysates ranged from a low of 94 ± 10 relative fluorescence units (RFU)/mg of cell protein in A149DD-GFP cells to a high of 209 ± 30 RFU/mg of cell protein in A160DD-GFP cells. By comparison, DDD-GFP-expressing cells had 89 ± 4 RFU/mg of cell protein, a value equivalent to that in the M4-GFP-expressing LLC-PK1 cells with a 10:1 molar ratio of fusion protein to p27 (5). Thus, the molar ratio of ADD-GFP fusion protein bait to the target p27 in the cells used in our studies ranged from 10:1 to 20:1. As reported previously (5) and shown in Fig. 1B, constitutive expression of the DDD-GFP fusion protein decreased D1 activity in LLC-PK1 cells by 50%, characteristic of the assembly of a p27/DDD-GFP heterodimer. Sequential alanine substitution of individual residues along the 16-residue-long DDD-GFP fusion protein showed that mutation of three non-polar residues (Ile152, Tyr153, and Ile154) reversed the loss of D1 activity associated with the formation of the p27/DDD-GFP heterodimer. Alanine substitution of the remaining 11 amino acids did not alter the loss of D1 catalytic activity associated with the formation of the p27/DDD-GFP heterodimers, suggesting that these residues do not participate directly in the dimerization interaction.
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50%, led to the appearance of punctate and ribbon-like fluorescent signals along the cell periphery identical to those of the p27/DDD dimer (5). These data suggest that the ability of the ADD-GFP fusion protein to bind to native p27 located on the basolateral membrane parallels its ability to impair the catalytic activity when partnered with native p27.
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70 °C. At all temperatures, the addition of a 20-fold molar excess of the DDD decreased the quantity of DDD-FITC bound to 9–14% of the total, indicating that nonspecific binding of DDD-FITC was 12% of the total fluorescent reporter. The relationship between temperature and specific binding of DDD-FITC to the immobilized DDD is shown in Fig. 3. Regression analysis of the Arrhenius plot yielded a free energy of association of –60 ± 5 kJ/mol for the noncovalent dimerization reaction. Based on these findings, all subsequent competitive binding studies were performed at 70 °C.
The data shown in Fig. 4A illustrate the concentration-dependent binding of DDD-FITC to the immobilized DDD. With increasing concentrations of DDD-FITC, there was a progressive increase in the quantity of DDD-FITC specifically bound that approached a plateau of 8 pmol of DDD-FITC/well when the concentrations exceeded 300 µM. Competition between DDD-FITC and three 26-residue-long ADD peptides with alanine substitutions at Ile152 (A152DD), Tyr153 (A153DD), and Ile154 (A154DD) was then evaluated. Compared with the DDD, which decreased DDD-FITC binding by >95%, the addition of a 20-fold molar excess of the individual ADD peptides decreased DDD-FITC binding by 50–65%, confirming that these individual residues (Ile152, Tyr153, and Ile154) contribute partially to the dimerization reaction (Fig. 4B).
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Characterization of the Dimer-deficient D1 Subunit p27A152—On the basis of the finding that substituting Ile152 with alanine prevents the DDD-GFP fusion protein from partnering with native p27, we generated a full-length D1 subunit with the I152A substitution (p27A152) and examined the consequences of this single residue exchange on its ability to dimerize with the native p27 selenoprotein in LLC-PK1 cells. D1 subunits in p27A152-expressing LLC-PK1 cells were affinity-labeled with BrAc[125I]T4; cell membranes were solubilized with taurodeoxycholate; and the membrane proteins were separated by sucrose density gradient centrifugation as described previously (6). The distribution of the p27A152 polypeptide was then identified by immunoprecipitation using antibodies directed against its C terminus. As shown in Fig. 6A, p27A152 was located at the top of the gradient between fractions 2 and 5 with an estimated molecular mass of 25–30 kDa, and little, if any, p27A152 was found in fractions 12–16, corresponding to the position of the native D1 homodimer in LLC-PK1 cells (4).
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We then examined the catalytic potential of dimer-deficient p27A152. As illustrated in Fig. 7A, HEK293 cells transfected with the p27A152 construct showed deiodinating activity (14.9 ± 0.3 units/mg of protein) that was 50–60% lower than that observed in cells transfected with equal amounts of the p27 plasmid (33.5 ± 2.3 units/mg of protein). Thus, the p27A152 mutant retained 50% of the catalytic activity of its wild-type parent.
Sucrose density gradient centrifugation of BrAc[125I]T4-labeled solubilized HEK293 cell membranes showed that immunoreactive p27A152 sedimented between fractions 2 and 7, whereas the immunoreactive p27 holoenzyme sedimented between fractions 12 and 18 (Fig. 7B), characteristic of the D1 homodimer (4). Taken together, these data confirm that (i) dimer-deficient p27A152 is catalytically active; (ii) p27A152 is selectively affinity-labeled with the alkylating D1 substrate analog BrAcT4; and (iii) p27A152 does not partner with the native p27 subunit or with itself.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Alanine scanning mutagenesis is a powerful tool used to explore the role of individual residues in the function of a protein, and this tool was exploited to identify the residue(s) in the 16-residue-long DDD that contribute to the assembly of the deiodinase holoenzyme. To eliminate the contribution of structural elements that flank the DDD in the full-length enzyme subunit, we made a DDD-GFP fusion protein that would serve as a surrogate for one of the binding partners. Because the expression of this soluble non-Sec-containing reporter is routinely 5–10-fold greater than that of the rare native Sec-containing deiodinase subunit (5), the dimerization reaction is driven toward heterodimer formation. By also limiting the functional component of the DDD to its core element (5) and using the altered catalytic activity of its native binding partner, changes in the binding of sequential alanine-substituted DDD-GFP fusion probes to full-length p27 are less influenced by misfolding artifacts that often occur in the mutant protein(s). For example, amino acid substitution for the acidic Glu156, the basic His158, and the nonpolar Trp163 residues leads to the loss of catalytic activity (15, 16), but does not prevent heterodimer assembly, thereby eliminating the possibility that altered enzyme assembly is responsible for the loss of catalytic activity by mutation of these residues (15, 17).
Three residues (Ile152, Tyr153, and Ile154) are essential for D1 assembly as judged by the inability of their alanine-substituted mutants to impair deiodination or to bind to membrane-bound p27 in LLC-PK1 cells. To characterize the participation of individual residues in the interaction(s) between DDD monomers (interactions that are based on noncovalent bond formation), we synthesized peptides composed of the 17-residue-long DDD and developed a competitive binding assay. Because the DDD peptide(s) were expected to self-associate, it was not surprising to find that dynamic interactions between the immobilized DDD "acceptor" and the DDD-FITC ligand required an elevated temperature. This finding is consistent with the strength of the binding between DDD-GFP and its membrane-bound binding partner, which allowed us to map this domain in cells. Melting curves generated by measuring the quantity of free DDD acceptor monomers yielded a free energy of activation for dimerization of –60 ± 5 kJ/mol for these "out-of-context" peptides and revealed the strong propensity for DDD peptides to self-associate. Although competition for the immobilized DDD monomers between the DDD-FITC reporter and a 20-fold molar excess of the DDD or individual ADD peptides confirmed the individual contributions of Ile152, Tyr153, and Ile154 to the dimerization reaction, each residue contributed only partially to the dimerization reaction. Unlike the inability of the dimer-deficient p27A152 point mutant to self-associate and the failure of a subset of ADD-GFP fusion proteins to partner with native p27, the competition between individual alanine-substituted peptides revealed the shared contribution of these three residues to the overall dimerization reaction. These context-dependent differences in the behavior of the DDD raise the possibility that the flanking regions exert sufficient strain on the DDD to prevent homodimer assembly. Alternatively, cooperation between the responsible residues in the dimerization reaction may differ when dynamic binding is assessed because the ability of single residue(s) to completely prevent dimerization is dependent of the ratio of the soluble DDD surrogate to the local concentrations of full-length p27 in the endoplasmic reticulum. More important, once a D1 homodimer forms, it is highly unlikely to exchange binding partners.
Similarly, analysis of SeD2 assembly showed that expression of the DDD-GFP fusion probe in SeD2-expressing C6 cells completely inactivates deiodination (5), indicating that both SeD2 subunits are required for catalysis. Expression of the ADD series in SeD2 cells revealed that the residues required for dimerization were more extensive that those used for D1 assembly and were found at both ends of the DDD. Single alanine substitutions at any one of a five-residue stretch from positions 153 to 157 at the beginning of the DDD, including two of the three residues required for D1 assembly, or in a three-residue region located at its C-terminal end (positions 160–162) eliminated the ability of DDD to impair SeD2 catalysis. These differences between the D1 and SeD2 subunits in the residues required for dimerization were unexpected, but may contribute to the unique catalytic properties of these two isoenzymes. Interestingly, the three amino acids that differ between the D1 and SeD2 dimerization domains, Ile151 and Leu155, Glu155 and Asp159, and the structurally more significant Ala160 and Pro163, do not appear to contribute to SeD2 holoenzyme assembly because alanine substitution at any of these positions did not affect DDD binding to the full-length SeD2 subunit.
The difference(s) in the functional outcome of impaired holoenzyme assembly between D1 and SeD2 appear to reside in elements outside of the DDD. Rat D1 and SeD2 share 31.5% amino acid identity, and hydrophobic cluster analysis suggests that the substrate-binding sites/active centers of D1 and SeD2 are similarly organized (16). However, the inability of the SeD2 subunit to catalyze deiodination by itself indicates that protein-protein interactions in the SeD2 homodimer are necessary to form a catalytically active enzyme. Although the responsible protein domains are unknown, three unique segment insertions are found in the SeD2 subunit. Segment A (residues 88–112; located 17 residues upstream of the active center Sec residue, segment B (residues 168–181; located adjacent to the DDD, and segment C (residues 107–205; located in the C terminus) are likely candidates for study.
On the basis of the ability of a single point mutation to prevent the DDD-GFP fusion protein from partnering with native p27, we constructed a "dimerization-deficient" D1 subunit by substituting alanine for Ile152 in full-length p27 (p27A152). Transient expression of p27A152 in LLC-PK1 cells revealed that the substrate-binding site of exogenous p27A152 was intact as judged by its interaction with the affinity label BrAcT4, but the point mutant did not partner with native p27 or an overexpressed Cys mutant of p27 (M4-GFP) and remained as a membrane-bound monomer. Because the abundant activity of native D1 in these cells overwhelmed that contributed by p27A152, we turned to transient expression of p27A152 in HEK293 cells, which lack native D1. Coexpression of M4-GFP and p27A152 in HEK293 cells confirmed that p27A152 does not partner with native p27. Interestingly, expression of p27A152 yielded a catalytically functional membrane-bound deiodinase that did not self-associate to form a homodimer, consistent with the findings in LLC-PK1 cells expressing this point mutant. Thus, the catalytic potential of the D1 enzyme is an intrinsic property of individual subunits that constitute the holoenzyme, and the assembly of a homodimer is not obligatory to generate a functional enzyme.
The generation of catalytically active monomeric D1 by the mutation of Ile152 in the DDD is inconsistent with the proposed role of intermolecular disulfide bridging in holoenzyme assembly (2, 14). Because the isolated DDD forms stable heterodimers with both the D1 and SeD2 family members (5) and because cysteine is not one of the residues required for holoenzyme assembly, it is unlikely that intermolecular disulfide bridging contributes to enzyme assembly. Nonetheless, it is clear that intramolecular disulfide bridging is essential for the proper folding of individual deiodinase subunits.
In summary, a small non-polar patch (152IYI154) located at the beginning of the 16-residue-long dimerization domain is required for the assembly of the D1 enzyme. For its family member SeD2, two subdomains located at the ends of this region are required for the assembly of a catalytically active holoenzyme. Family members also differ in the catalytic potential of individual subunits; D1 subunits are fully capable of catalyzing deiodination alone, whereas the SeD2 isoenzyme appears to require two functional subunits for catalytic activity. The strength of binding through noncovalent interactions between the DDD of each binding partner suggests that, once two enzyme subunits come in contact with each other, they assemble into a highly stable dimer complex. Thus, formation of hybrid deiodinase dimers composed of different family member subunits is driven by the relative concentrations of each subunit; and once formed, heterodimers have little, if any, chance to reassemble into a homodimer with its cognate partner.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Cell and Molecular Physiology, University of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA 01655. Tel.: 508-856-6687; Fax: 508-856-5997; E-mail: jack.leonard{at}umassmed.edu.
2 The abbreviations used are: Sec, selenocysteine; D1, type I deiodinase; SeD2, type II selenodeiodinase; DDD, deiodinase dimerization domain; GFP, green fluorescent protein; TEMED, N,N,N',N'-tetramethylethylenediamine; ADD, alanine-substituted dimerization domain; WT, wild-type; BrAcT4, N-bromoacetyl-L-thyroxine; FITC, fluorescein isothiocyanate; RFU, relative fluorescence units.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) and dimer-deficient p27A152 (•) constructs. BrAc[125I]T4-labeled cell membranes were isolated on 16% Percoll gradients and solubilized with 5 mM taurodeoxycholate. Clarified detergent extracts were loaded onto 4–12% linear sucrose density gradients and centrifuged at 3.9 x 108 gmin, and 100-µl fractions were collected beginning at the top of the gradient. Fractions was incubated with 10 µl of protein A beads and 1 µl of anti-p27 D1 antiserum for 90 min, and immune complexes were collected by centrifugation. Data are reported as the means of closely agreeing triplicate determinations.