©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Topological Analysis of the Integral Membrane Protein, Type 1 Iodothyronine Deiodinase (D1) (*)

Nagaoki Toyoda , Marla J. Berry , John W. Harney , P. Reed Larsen (§)

From the (1) Thyroid Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Type 1 iodothyronine deiodinase (D1) is a microsomal selenoenzyme which catalyzes deiodination of thyroxine to 3,5,3`-triiodothyronine. Immunoblotting showed that endogenous hepatic, renal, and transiently expressed D1 remains in microsomes after pH 11.5 treatment. In vitro translation studies using pancreatic microsomes identified a single transmembrane domain with a cytosolic carboxyl-terminal catalytic portion. The transmembrane domain is located between conserved basic amino acids at positions 11 and 12 and a group of charged residues at positions 34-39. A transiently expressed D1 protein in which residues 2-25 were deleted was inactive and not integrated into membranes. Activity was not restored by replacing these residues with transmembrane domains from a cytochrome P450 or type 3 deiodinase enzyme despite their incorporation into membranes. Elimination of the positive charges at positions 11 and 12 reduced the amount of transiently expressed protein by 70%, but the enzyme formed was catalytically normal. Similar results were found after conversion of the Lys-27 in the transmembrane domain to Met or Glu. We conclude that the amino terminus of D1 contains uncleaved signal and stop transfer sequence properties. In addition, positively charged residues at positions 11, 12, and 27 are required for optimal formation of the protein but not for catalysis.


INTRODUCTION

Conversion of the prohormone thyroxine (T4)() to the biologically active hormone 3,5,3`-triiodothyronine (T3) is the first step in thyroid hormone action. The bulk of the circulating T3 is formed within the cytoplasm of hepatic, thyroid, and renal epithelial cells, and the reaction is catalyzed by the selenoenzyme type 1 iodothyronine deiodinase, here termed D1 (1, 2, 3, 4) . Previous subcellular fractionation studies of liver D1 have shown that it localized to the endoplasmic reticulum (ER) (5-7), while those of the kidney and LLC-PK1 cells suggest it is present in plasma membrane (8, 9) . Leonard et al.(9) recently reported that both n-bromoacetyl-[I]T3 (BrAc-[I]T3)-labeled and catalytically active D1 were resistant to trypsin added to intact LLC-PK1 (porcine kidney) cells, indicating a cytoplasmic orientation of the enzyme (9) .

We have taken a molecular approach to determine the intracellular location of this protein using the rat D1 cDNA (1) . A comparison of the deduced amino acid sequences of the rat (1) , human (10) , and dog D1 (11) proteins shows they are highly homologous. All of these cDNAs contain an in-frame UGA codon encoding selenocysteine and the required selenocysteine-insertion sequence (SECIS) element in the 3`-untranslated mRNA (12) . While rat D1 has two potential glycosylation sites (Asn-94 and -203), only Asn-203 is conserved in the human and dog. The hydropathy plots of the three proteins are quite similar (Fig. 1). These demonstrate that only a single domain, located between residues 1 and 35 of these proteins, is sufficiently hydrophobic to qualify as a transmembrane sequence (sequences in Fig. 2). The studies described herein show that D1 is a type I integral membrane protein with residues 13 to 34 constituting the transmembrane sequence. The catalytic portion of the protein containing the essential selenocysteine (13) , His-174 (14) , and Phe-65 (11) residues is thus in the cytoplasmic compartment accessible to T4, T3, 3,3`,5`-triiodothyronine (rT3), and other iodothyronine substrates. Neither the transmembrane region of the bovine 21-hydroxylase P450 enzyme (P45021) (15) nor the putative transmembrane domain from the Xenopus laevis type 3 deiodinase (Xeno D3) (16) can replace the transmembrane domain of D1 despite the fact that the chimeric proteins are integrated into microsomal membranes. This suggested an additional role of certain of these amino-terminal residues either in the synthesis of the protein or in its catalytic function. We found that eliminating the highly conserved positive charges at positions 11, 12, or 27 reduced D1 synthesis, but the protein which was produced had normal catalytic characteristics. Thus, those basic residues and perhaps others in this portion of the protein play an important role in the efficient formation of an active enzyme over and above their role in membrane integration.


Figure 1: Hydropathy analyses of rat, human, and dog D1 proteins. Hydropathy analyses of rat, human, and dog D1 were predicted using the Kyte-Doolittle algorithm (window = 21) by means of the PEPPLOT program of the University of Wisconsin Genomic Computer Group (25).




Figure 2: Comparison of the deduced amino acid sequences of the hydrophobic regions (1-50) of rat, human, and dog D1 enzymes. A plus sign indicates a lysine or arginine residue.




EXPERIMENTAL PROCEDURES

Materials

Horseradish peroxidase-coupled goat anti-rabbit antibody was obtained from Dako Laboratories. Enhanced chemiluminescence (ECL) detection reagents and prestained protein molecular weight markers were from Amersham. L-[S]Methionine (1, 140 Ci/mmol) was from DuPont NEN. Trypsin and -chymotrypsin were from Sigma. The rabbit reticulocyte lysate system and canine pancreatic microsomal membranes were from Promega Biotech. All other chemicals were of reagent or molecular biology grade.

Preparation of Microsomes

Rat liver and kidney tissues and human embryonic kidney (HEK) 293 cells were homogenized in ice-cold buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol, 0.25 M sucrose, and 1 mM EDTA (homogenization buffer). Homogenates were centrifuged at 20,000 g for 20 min at 4 °C. The 20,000 g supernatants from homogenates were centrifuged at 200,000 g for 90 min at 4 °C to prepare microsomes. Microsomal pellets were resuspended in the homogenization buffer and stored at -70 °C.

Sodium Carbonate Treatment

Microsomal fractions were adjusted to pH 11.5 with 0.1 M NaCO and incubated on ice for 30 min as described previously by Fujiki et al.(17) . The suspensions were centrifuged at 200,000 g for 60 min at 4 °C. The membrane pellets were dissolved directly in SDS-sample buffer. The supernatants were neutralized with HCl and precipitated with 10% trichloroacetic acid. Then, the pellets were neutralized by addition of 0.1 M NaOH and SDS-sample buffer.

Immunoblotting

Microsomal proteins were electrophoresed on a 12.5% acrylamide (acrylamide:bis 37.5:1) SDS gel and electroblotted onto Immobilon (Millipore) in 20% methanol, 25 mM Tris-HCl, pH 8.3, 192 mM glycine at 100 mA for 16 h at 4 °C. Blots were blocked with 5% (w/v) nonfat milk in TBS-Tween (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1% Tween 20). Antisera were prepared by immunization of rabbits with a peptide corresponding to residues 45-60 of the rat D1 as described previously (18) and used at 1:100 dilution in TBS-Tween. Horseradish peroxidase-coupled goat anti-rabbit antibody was used at 1:1000 dilution. ECL detection reagents were used according to the manufacturer's instructions. Quantitation of bands on the gels was performed by scanning densitometry on a Molecular Dynamics computing densitometer.

Constructs

Vectors pUHD 10-3 (D10) and pUHD 15-1 (D15) were obtained from Dr. Manfred Gossen (19) . The rat D1 cDNA (G21) was cloned into D10 between the EcoRI and XbaI sites (18) . The codon 126 UAA (ochre, G3) and UGU (cysteine, G5) mutant deiodinase constructs have been described previously (1) . These mutant constructs were subcloned into the CDM vector (20) . The cysteine mutant (G5) was also subcloned into the T7 polymerase-dependent bacterial expression vector, pET-3 (Novagen), and expressed in the BL21 (DE3) pLysS strain of Escherichia coli as described previously (21). The PstI deletion of G5 was constructed by deleting an internal PstI fragment between position 56 in G5 (1) and the PstI site in Bluescript vector (Stratagene) and religation. For addition of a methionine and 6 histidine residues at the amino terminus of G5, we made an NdeI restriction site at the initiating methionine of G5 using the P-select in vitro mutagenesis system of Promega as described previously (1) and the synthetic oligonucleotides 5`-TATG(CAC) CATATG-3` and 5`-TATG(GTG) CA-3` were annealed and inserted into the NdeI site. The deletion mutant of G5, which starts at amino acid 26 of G5 (MEM 1), was made by PCR using the oligonucleotide, 26#; 5`-CGGAATTCACATATGGGCAAGGTGCTAATGAC-3`, which contains the nucleotides encoding amino acids 26-31 of rat D1 (underlined) and the additional sequence to create the EcoRI and NdeI restriction sites. After digestion with EcoRI and XbaI, the PCR fragment was subcloned into the D10 plasmid. The chimeric construct between bovine 21-hydroxylase P450 enzyme (P45021) (22) and G5, in which amino acids 1-20 of P45021 were substituted for amino acids 1-25 of G5 (MEM 2), was made by PCR using oligonucleotides (P450#1, 5`-CCGGAATTCCGCCACCATGGTCCTCGCAGGGCTG-3` containing the nucleotides encoding amino acids 1-6 of P45021 (underlined) and the additional Kozak consensus sequence (23) and EcoRI restriction site; P450#2, 5`-GGGAATTCCATATGCCATAGCAGGTGAGCGCC-3` containing the complementary nucleotides encoding amino acids 15-20 of P45021 (underlined) and the additional NdeI restriction site). The cDNA for P45021 was the kind gift of Dr. Michael Waterman (Vanderbilt University). The PCR fragment was digested with EcoRI and NdeI and ligated between the EcoRI and NdeI sites of the MEM 1 construct. MEM 3 construct, in which amino acid 20 of P45021, histidine and methionine of the NdeI site, and amino acids 26-32 of rat D1 were deleted from MEM 2 construct, was made by PCR mutagenesis as described previously (11) . The chimeric construct between Xeno D3 and G5, in which amino acids 1-44 of Xeno D3 (ref; GenBank/EMBL Data Bank L28111) were replaced by amino acids 1-25 of G5 (MEM 4), was made by PCR using oligonucleotides (Xeno#1, 5`-CCGGAATTCCGGCCACCATGTTGCACTGCGCGGG-3`, containing the nucleotides encoding amino acids 1-6 of Xeno D3 (underlined) and the additional Kozak sequence and EcoRI restriction site; Xeno#2, 5`-GGGAATTCCATATGCCTCCTCCTGATACACTGGAAATC-3` containing the complementary nucleotides encoding amino acids 37-44 of Xeno D3 (underlined) and the additional NdeI restriction site). The cDNA for the Xeno D3 was provided by Drs. Donald Brown and Robert Schwartzman (Carnegie Institute of Washington). The PCR fragment was digested with EcoRI and NdeI, ligated between EcoRI and NdeI sites of the MEM 1 construct. MEM 5 in which amino acids Lys-11 and Arg-12 of G21 were changed to asparagine, was made by PCR mutagenesis. MEM 6 and 7 in which amino acid Lys-27 of G21 was changed to methionine or glutamic acid, were made by PCR mutagenesis. The entire coding regions of these plasmids were sequenced to confirm that these were the only mutations.

Transfections and Deiodinase Assays

Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium + 10% fetal calf serum in the absence of tetracycline as described previously (18) . Transfections were performed by CaPO-DNA precipitation. The dishes for D10 expression vector received 10 µg of D10 plasmids, 5 µg of D15 plasmid encoding the tetracycline-repressible transactivator (18) and 4 µg of TKGH, which constitutively expresses human growth hormone to control for transfection efficiencies (24) . After 48 h of incubation, human growth hormone in the culture medium was measured and was expressed as counts/min/100 µl of medium. Two days after transfection, cells were harvested and homogenized using a Polytron. After homogenization, microsomal fractions were obtained using the same procedures as described above. For deiodinase assays, cells were sonicated in 0.25 M sucrose, 100 mM sodium phosphate, pH 6.9, 1 mM EDTA, and 10 mM dithiothreitol and incubated with 0.1-0.5 µM [I]rT3 for 30-60 min at 37 °C. Release of I was quantified as described previously (11) . For each set of transfections and deiodinase assays, negative (D10 vector alone) controls were included. Enzyme kinetics were determined from Lineweaver-Burk plots as described previously (11). All transfections were performed at least three times, and all assays were performed in duplicate. BrAc-[I]T3 Affinity Labeling and Enzyme Quantitation-BrAc-[I]T3 was synthesized from n-bromoacetylchloride and [I]T3, and affinity labeling and enzyme protein determinations were performed as described previously (11) .

In Vitro Transcription and Translation

CDM-8- or pET-based plasmids containing the relevant cDNAs were transcribed in vitro by T7 or T3 E. coli RNA polymerase. The resulting mRNA was translated in a nuclease-treated reticulocyte lysate in the presence of [S]methionine, as described previously (1) . To test for membrane translocation, rough microsomes from canine pancreas were present during the translation or added after translation, following a 10-min chase with 10 mM unlabeled methionine. Where indicated, reactions were layered over a cushion of 1 M sucrose after addition of 100 µg of carrier rat liver microsomes and centrifuged at 200,000 g for 60 min at 4 °C. Pellets were subjected to SDS-PAGE on acrylamide gels. Furthermore, some 1 M sucrose pellets were adjusted to pH 11.5 with 0.1 M NaCO, incubated on ice as described above, and centrifuged at 200,000 g for 60 min at 4 °C, and the pellets were subjected to PAGE. Gels were incubated in ENHANCE (DuPont NEN) according to the manufacturer and dried. Proteins were visualized with fluorography on film. Quantitation of bands on the gels was performed by scanning densitometry as described above. To analyze the topology of the in vitro translated D1 protein, we performed limited protease digestion using trypsin and/or chymotrypsin (100 µg/ml). Triton X-100, at 1% final concentration, was present where indicated. Reactions were incubated for 90 min on ice, followed by the addition of aprotinin (150 kallikrein-inhibiting units) to all reactions. After digestion with or without Triton X-100, some reactions were layered over a cushion of 1 M sucrose and centrifuged at 200,000 g for 60 min, and pellets were subjected to acrylamide gel electrophoresis.

Protein Structure Predictions

Hydropathy analysis and helical probabilities were predicted using the Kyte-Doolittle algorithm (window = 21) by means of the PEPPLOT program of the University of Wisconsin Genetics Computer Group (25) . Hydrophobicity and helical hydrophobic moment calculations were performed using the Moment program according to Eisenberg et al.(26) .

RESULTS

Endogenous and Transiently Expressed D1 Is an Integral Membrane Protein

Antisera against peptide 1 (amino acids 45-60 in rat D1) reacted with an approximately 28-kDa protein in euthyroid rat liver and kidney microsomes. The size of this band corresponds closely to the predicted full-length D1 protein (29.7 kDa) and is identical in size with the full-length protein translated in vitro(1) with or without microsomes (see below). This suggests that D1 is not glycosylated in vivo, nor is there post-translational cleavage of a signal peptide. To establish that D1 is an integral membrane protein, liver and kidney microsomes from euthyroid rats were prepared and exposed to 0.1 M sodium carbonate (pH 11.5) for 30 min, a treatment previously shown to remove peripheral membrane-associated proteins (17) . As shown in Fig. 3 , the pH 11.5 treatment failed to dissociate D1 from either the liver or kidney membranes. To establish that transiently expressed D1 is also incorporated into membranes and that this is true for both the wild type (SeCys 126) enzyme and the cysteine mutant (G5) protein, we transfected these constructs into human embryonic kidney (HEK) 293 cells. As shown in Fig. 4, the antisera detected a 28-kDa protein in microsomes prepared from cells transfected with either the wild type or the G5 mutant but not from cells transfected with vector alone. The sodium carbonate treatment caused no dissociation of either D1 or the G5 mutant, indicating that these transiently expressed proteins are also incorporated into the microsomal membranes as occurs for the hepatic and renal protein.


Figure 3: Sodium carbonate treatment of rat liver and kidney D1. Microsomes (80 µg) from euthyroid rat liver and kidney were incubated with 0.1 M NaCO (pH 11.5). After centrifugation, pellets and supernatants were subjected to PAGE on a 12.5% gel, transferred to membrane, and detected by immunoblotting using an antiserum to peptide 1 (residues 45-60). Lanes 1 and 4, untreated rat liver (1) and kidney (4) microsomes (80 µg); pellets (lanes 2 and 5) and supernatants (lanes 3 and 6) after NaCO treatment of microsomes from liver and kidney. Lane 7, cysteine mutant (G5) D1 protein expressed in E. coli.




Figure 4: Sodium carbonate extraction of transfected wild-type and G5 mutant D1. Microsomal fractions (120 µg) from wild-type and G5 mutant D1 transfected HEK 293 cells were treated with 0.1 M NaCO (pH 11.5). After centrifugation, pellets and supernatants were subjected to PAGE on a 12.5% gel, transferred to membrane, and detected by immunoblotting using peptide 1 antiserum. Lane 1, microsomes from vector-transfected cells; lane 2, G21 microsomes; lane 3, pellet; lane 4, supernatant of G21 microsomes after NaCO exposure; lane 5, G5 microsomes; lane 6, pellet; lane 7, supernatant of NaCO-treated G5 microsomes. The positions of full-length (28-kDa) protein are indicated by arrows.



In Vitro Translated D1 Is Incorporated into Pancreatic Microsomal Membranes

To study cotranslational membrane insertion, we employed the G5 mutant mRNA, since reticulocyte lysate systems are inefficient in the synthesis of the full-length selenoprotein (1) . Controls consisted of translation without microsomes or the addition of microsomes after a cold methionine chase. In both control situations, some S-protein appears in the 1 M sucrose pellet (Fig. 5). We interpret this as due to aggregation of this hydrophobic basic protein (pI 9.3). In the experiment shown in Fig. 5, the amount of labeled protein synthesized was lower in the presence of microsomes (lane 2 versus lanes 1and 3 in Fig. 5); however, the ratio of S-protein recovered in the microsomes (lane 8 versus lane 2) was 10-fold higher than from preparations translated either in the absence of microsomes (lane 7 versus lane 1) or if microsomes were added subsequent to the reaction (lane 9 versus lane 3) (Fig. 5), indicating that localization into the microsomes was cotranslational. The identical size of the S-proteins in lanes 1 and 2 also strongly suggests that post-translational processing does not occur.


Figure 5: In vitro translation of G5 and the PstI deletion mutant G5 in the presence or absence of microsomes. The plasmid DNAs containing the G5 or PstI deletion D1 cDNAs were transcribed, and 1.5 µg of mRNA equivalent for each lane was translated in the absence (lane 1, G5; lane 4, PstI deletion G5) or presence of nuclease-treated canine microsomes (lane 2, G5; lane 5, PstI deletion G5) or after a 10-min chase of [S]methionine with 10 mM unlabeled methionine (lane 3, G5; lane 6, PstI deletion G5) as noted under ``Experimental Procedures.'' After the translation, aliquots of each translation reaction mixture were centrifuged through 1 M sucrose, and pellets were subjected to PAGE (lanes 7-12 correspond to lanes 1-6). Aliquots of the 1 M sucrose pellets were then adjusted to pH 11 with NaCO and incubated on ice for 10 min. The pellets were then subjected to the PAGE (lanes 13-18 correspond to lanes 7-12).



To establish that the amino-terminal portion of the protein is required for membrane insertion, we used the PstI deletion G5 mutant which initiates at Met-30, producing a predicted protein of 24 kDa (Fig. 5). While the translation efficiency of this protein is lower than that of G5, presumably due to the absence of Kozak consensus sequences (23) , significant quantities of S-protein of the predicted size were synthesized (lanes 4-6). However, there was no enrichment of S-protein in the pellet when microsomes were present during the synthesis (lanes 10-12 versus lanes 4-6 in Fig. 5) due to the loss of the S-protein in the supernatant. To establish that the microsomal S-protein was integrated into the membrane, the 1 M sucrose pellets were treated with 0.1 M NaCO (lanes 13-18 in Fig. 5 ). As with the endogenous and transiently expressed proteins, pH 11 treatment had no effect on the quantity of microsomal G5 protein (lane 14 versus lane 8 in Fig. 5). The insoluble material was also unaffected by pH 11 treatment (lanes 13 and 15). Trace quantities of the 24-kDa protein also remained in the pellet during NaCO treatment (lanes 16-18). Identical results were obtained using G3, a mutant D1 terminating at codon 126 in which a 14-kDa protein was also cotranslationally inserted into the membrane (see below).

Topology of D1 in the Pancreatic Microsomal Membranes

To identify the position of the transmembrane sequence, [S]methionine-labeled D1, G5, or G3, translated in the presence of microsomes, was exposed to trypsin (T) and/or chymotrypsin (CT) in the presence or absence of Triton. Radiolabeled proteins which are contained within the membrane are protected from digestion in the absence of Triton. Since the studies shown in Fig. 5(as well as those with G3) indicated that the amino-terminal portion of the protein was required for cotranslational membrane localization and the hydropathy plots indicated that residues 1-40 constituted the only significantly hydrophobic portion of D1, the amino acid sequences of this portion of the rat and human D1 and the predicted cleavage sites for T and CT are shown in Fig. 6B. Fig. 6A shows that protection of a 4.2-4.4-kDa-labeled fragment occurred in the absence of Triton for both the G5 and G3 mutant D1 proteins (lanes 3 and 7 in Fig. 6A). No peptide is protected in the presence of Triton (lanes 4 and 8 in Fig. 6A). The results with G3 (residues 1-126) indicate that the protected protein is contained within the amino-terminal half of D1.


Figure 6: Protease digestion of mutant D1 proteins translated in the presence of microsomes. A, after translation in the presence of microsomes of G5, G3, or 6H-G5 mutant, the reaction mixtures were divided into aliquots and subjected to different reaction conditions. The symbols below the figure indicate the following treatments: T+CT, incubation with trypsin and chymotrypsin (100 µg/ml each) for 90 min on ice; Det, Triton X-100 (0.1% final concentration). After each treatment, the reaction mixtures were centrifuged through 1 M sucrose after addition of carrier rat liver microsomes, and the pellets were subjected to PAGE. B, the amino acid sequences (1-60) of rat and human D1 and predicted trypsin and chymotrypsin cleavage sites of rat D1 are shown. The symbol is above the amino-terminal residue of the predicted cleavage site.



To determine if the amino terminus per se was included in the protected fragment, we performed similar studies with a mutant G5 mRNA to which sequences encoding a methionine and 6 histidine residues were added to the amino terminus (6H-G5). The addition of these 7 amino acids did not impair the incorporation of this slightly larger protein into the membranes (lanes 9 and 10 versus 1 and 2 of Fig. 6A). After exposure to T and CT without Triton, a slightly larger (approximately 5-kDa) fragment was protected in the absence, but not in the presence of Triton (lane 11 versus 12 in Fig. 6A). This establishes that the extreme amino-terminal fragment is protected in lanes 3 and 7 of Fig. 6A. The size of this protected fragment corresponds to T cleavage between residues 36 and 37 (4.2 kDa) or 38 and 39 (4.4 kDa). To confirm this conclusion, digestion was performed with either T or CT alone. A 4.4-kDa fragment (corresponding to the same T cleavage site) and 5.5-kDa labeled fragment (corresponding to a CT cleavage site at position 56/57) were protected, respectively (Fig. 7). Thus, the potential CT cleavage site at position 33/34 is not accessible to the protease. These data indicate a type I topology for D1 in the endoplasmic reticulum (Fig. 8) with the catalytic portion of the enzyme on the cytosolic surface and an uncleaved amino-terminal signal sequence in the lumen.


Figure 7: Trypsin or chymotrypsin digestion of in vitro translated G5 mutant D1. G5 mRNA was translated in the presence of nuclease-treated canine microsomes and treated as described in Fig. 6.




Figure 8: Schematic diagram of hypothetical orientation of D1 protein in the membrane. The predicted location of the conserved positively charged amino acid residues flanking the transmembrane sequence are shown. Selenocysteine 126, the iodine acceptor (13), the essential histidine residue 174 (14), and phenylalanine 65 (human, rat) or leucine 60 (dog) (11), thought to be involved in binding of reverse T3, are also shown.



Modifications in the Amino-terminal Domain Affect D1 Synthesis and Catalytic Function

If the role of the amino-terminal portion of D1 is merely to orient the enzyme in the membrane, one would not expect those residues to have a role in catalysis. To test this hypothesis, we examined the activity of several mutant enzymes containing deletions or modifications of this portion of the protein (). Transient expression of a construct encoding a D1 protein in which amino acids 2-25 were deleted (MEM 1) resulted in the formation of two immunoreactive proteins in HEK 293 cells, one of the predicted size and one about 2 kDa smaller (Fig. 9). The smaller protein presumably initiates from the third methionine residue (amino acid 44) preceded by the nucleotide sequence CTGGCC which resembles a Kozak consensus sequence more closely than does that 5` to the first methionine (TCACAT). Neither protein was significantly retained (15%) in the microsomes after exposure to pH 11.5 (Fig. 9, compare lanes 3 and 4 to lanes 9 and 10), and there was no detectable deiodinase activity. We attribute the small quantity of residual immunoreactive protein remaining in lane 4 after alkali treatment of the microsomal pellet to insoluble aggregates of this basic protein analogous to what we observed with the in vitro translated protein (see Fig. 5).


Figure 9: Transient expression of total and alkali-resistant membrane proteins of chimeric and wild-type G5 D1. Microsomal fractions from MEM 1, 2, 4, and G5 mutant D1 transfected HEK 293 cell homogenates (120 µg) were treated with 0.1 M NaCO (pH 11.5). The 120 µg of total homogenate protein and a comparable quantity of alkali-treated membrane protein were subjected to PAGE, and D1 was detected by immunoblotting using peptide 1 antiserum as described in Fig. 4. Lane 1, homogenate; lane 2, equivalent amount of microsomal pellet protein after pH 11.5 treatment both from vector-transfected cells; similar preparations were used in lanes 3 and 4 (from MEM 1 transfected cells), lanes 5 and 6 (from MEM 2 transfected cells), lanes 7 and 8 (from MEM 4 transfected cells), and lanes 9 and 10 (from G5 cysteine mutant D1 transfected cells); lane 11 was the G5 mutant D1 expressed in E. coli.



This result indicates that amino-terminal and transmembrane domain residues are required for synthesis of an active enzyme. To determine whether transmembrane segments from other proteins could subserve this function, we prepared MEM 2 and MEM 3 in which the amino-terminal portion of the bovine 21-hydroxylase (P45021) protein, a well characterized type I membrane protein (15) , was substituted for amino acids 1-25 (MEM 2) or 1-32 (MEM 3) of the rat D1 (). An equivalent fraction (60%) of the total expressed MEM 2 protein in the homogenate was found in the membrane pellet after alkali treatment as with the control G5 (Fig. 9, compare lanes 5 and 6 with lanes 9 and 10) indicating that the amino-terminal amino acids from P45021 could function as a stop-transfer sequence when linked to the heterologous MEM 1 protein. However, even though MEM 2 was integrated into the membrane, there was no deiodinase activity with either MEM 2 or 3. This indicated that the role of the D1 amino terminus is not limited to orienting the protein in the ER membrane.

The bovine P45021 sequence we used differs from that of the D1 amino terminus in that it contains no charged amino acids (22) . Recently, St. Germain and co-workers (16, 27, 28) have reported the sequence of the X. laevis type 3 deiodinase (Xeno D3), which is also a membrane-associated selenoenzyme. A hydropathy plot of the deduced amino acid sequence of Xeno D3 is shown in Fig. 10. Analysis of this sequence predicts a single hydrophobic sequence between residues 10 and 40 centered at position 25. There are three positively charged amino acids in this region (), two of which are amino-terminal to the putative transmembrane domain (). We therefore prepared a chimeric protein containing residues 1-44 of Xeno D3 linked to residues 26 to 256 of the rat D1 protein (MEM 4). Again, a significant fraction (45%) of the homogenate protein was found in the alkali- resistant microsomal pellet (Fig. 9, lanes 7 and 8). However, despite the membrane location of this protein and the greater similarity between the putative transmembrane domains of Xeno D3 and rat D1, no catalytic activity was detected.


Figure 10: Hydropathy plot of X. laevis type 3 deiodinase protein. Hydropathy analyses of X. laevis type 3 deiodinase (GenBank/EMBL Data Bank accession number L28111) [XL D3] using the Kyte-Doolittle algorithm (window = 21) by means of the PEPPLOT program of University of Wisconsin Genomic Computer Group (25).



These results raised the possibility that specific residues of the amino-terminal domain of D1 were required for the proper synthesis or conformation of the enzyme and did not merely serve the function of tethering the protein to the lipid bilayer in the proper orientation. Since charged residues may have specific roles in the post-translational positioning of protein within the membrane or in protein folding, we prepared several mutations in the basic residues of D1 in the amino terminus and transiently expressed the mutant proteins (). Conversion of the conserved positively charged amino acids 11 and 12 to asparagine (MEM 5) had a marked effect on the transient expression of D1 in HEK 293 cells (). Despite a similar transfection efficiency of the plasmid containing the cDNA as reflected in the media human growth hormone from the cotransfected TKGH, the amount of enzyme protein covalently labeled by BrAc-[I]T3 ([E]) was reduced to 30% that of wild type (). Kinetic analysis indicated that the V of the mutant D1 was proportionally reduced and thus there was no alteration in the relative turnover number (V/[E]). The apparent Kof the transiently expressed enzyme was also normal ().

A lysine is conserved in the center of the transmembrane domain (residue 27) in rat, human, and dog D1 enzymes (Fig. 2). Mutation of this residue to methionine (MEM 6) or glutamic acid (MEM 7) () also reduced the expression of the enzyme by 75 or 40% but again had no effect on the catalytic function of the enzyme formed (). These results demonstrate that the lumenal and transmembrane charged amino acids are required for optimal synthesis, stability, or proper folding of the protein but not for its catalytic function. This can explain the absence of activity of the MEM 2-4 chimeric proteins despite their membrane location.

DISCUSSION

The present results establish that D1 is an integral membrane protein with its carboxyl-terminal portion present in the cytosol. This is consistent with the early studies of the protein in liver in which the co-fractionation of rat liver D1 activity with marker enzymes of the endoplasmic reticulum (ER) such as glucose-6-phosphatase and NADPH-cytochrome c reductase occurred (5, 6, 7) , although one early report suggested an association of deiodinase activity with liver plasma membranes (29) . A plasma membrane location for renal D1 has been suggested by its co-purification with (Na/K)-ATPase, a known plasma membrane enzyme (8, 9).

A 27-kDa protein in liver and kidney, affinity-labeled with BrAc-[I]T3, is now known to be D1 (30, 31, 32) . Schoenmakers et al.(30) reported that BrAc-[I]T3 labeling of this protein is inhibited in parallel with a decrease in deiodinase activity following treatment of rat liver microsomes with trypsin. They also showed that neither labeling of the 27-kDa protein nor deiodinase activity is affected by treatment of microsomes with 0.05% deoxycholate or with buffers of pH 8.5-9.5, conditions which are known to release lumenal proteins (30) . This is also consistent with our proposed topology (Fig. 8) since BrAc-[I]T3 interacts with the selenocysteine residue at position 126 which is found on the cytosolic surface of the ER (30) . However, Schoenmakers et al.(30) found that neither deiodinase activity nor labeling of the 27-kDa protein with BrAc-[I]T3 was detected after incubating the membranes at pH greater than 10, raising the question that it might be a peripheral membrane protein. The present studies demonstrate that both the endogenous (Fig. 3) and transiently expressed wild-type and cysteine-mutant D1 proteins (Fig. 4) are alkali-resistant and thus are integrated into the ER membrane in a type I orientation.

If a type 1 integral ER membrane protein were to be further processed and incorporated into the plasma membrane, the ER-lumenal amino-terminal portion of the protein would become extracellular with the catalytic portion in the cytosol. A renal cell plasma membrane location is consistent with all the published results (8, 9) and suggests that the post-translational trafficking of D1 differs in the liver and kidney.

The protease digestion results identify the cytoplasmic/membrane interface of D1 between residues 34 and 38. Digestion of either the full-length (G5) or the amino-terminal half of the protein (G3), translated in the presence of microsomes, results in the protection of a 4.2-4.4 kDa fragment. Inclusion of Triton eliminates this protection indicating it requires an intact lipid bilayer. Deletion of the first 29 amino acids eliminates membrane insertion ( Fig. 5and Fig. 9), consistent with a requirement for those residues for interaction with the signal recognition particle (33) . Inspection of the sequence of the three proteins (Fig. 2) shows that there is a group of positively charged and polar residues just carboxyl-terminal to the conserved proline 34: ERVKQN (rat), DRVKRN (human), and ARVKQH (dog). We hypothesize that these polar residues represent an important part of the stop-transfer portion of the signal anchor sequence and may interact with the negatively charged head group of the membrane phospholipids to prevent further transfer of D1 through the membrane. While we cannot determine which of the two trypsin cleavage sites is used (Fig. 6A and 7), the chymotrypsin site at position 33/34 is protected (Fig. 7). If this portion of the protein is helical, as suggested from the predictions of the algorithm of Eisenberg et al.(26) , and 21 to 22 residues are required to traverse the lipid bilayer, then residues 1-12 are predicted to reside in the ER lumen (Fig. 8). The two basic residues at positions 11 and 12 of the three D1 proteins would thus be excluded from the hydrophobic portion of the membrane. The putative transmembrane domains, residues 13-34, are highly hydrophobic in all three species ( Fig. 1and 2), although in both the rat and human D1 there is a single negatively charged residue, and a conserved lysine residue at position 27 is present within this domain in all three (Fig. 2). The cytosolic location of the potential glycosylation sites at 94 and 203 explains why this post-translational modification does not occur, since this process requires enzymes localized to the ER lumen. It is of interest that the addition of six histidine residues to the amino terminus (net gain of 3 positive charges) does not alter the orientation of the in vitro translated protein given the important role of such charge changes in the cytochrome P450 proteins (34) . Presumably, other information encoded in this sequence, such as the length of the hydrophobic segment (35) or the polar groups mentioned earlier, function to maintain the protein in its type I orientation.

The topology of D1 resembles that of the cytochrome P450 microsomal proteins which have similar short amino-terminal uncleaved signal recognition sequences (36) . This sequence, which incorporates topogenic functions of both signal and stop-transfer sequences, has been termed a type I signal anchor sequence (36) . Clark and Waterman (37) reported that the deletion of the hydrophobic amino terminus from bovine 17-hydroxylase (P45017), a member of the P450 enzyme group, reduced membrane targetting and insertion and abolished enzyme activity. However, replacement of the amino terminus of this P45017 with heterologous signal-anchor sequences from either 21-hydroxylase P450 enzyme (a similar sequence) or the NADPH cytochrome P450 reductase (a distinct sequence) restored activity (15) . These authors concluded that the structure and catalytic activity of P45017 is dependent on a hydrophobic amino-terminal peptide which can function as a signal-anchor sequence, but the individual amino acids composing this sequence are not critical.

We have previously observed that a PstI deletion rat D1 mutant which initiates at Met-30 has no activity (1) . Since this mutant enzyme does not have a Kozak consensus sequence, we could not eliminate the possibility that the lack of active enzyme was due to a reduced translation efficiency of the mutant D1 protein. A second mutant (MEM 1), which deletes amino acids 2-25, is also inactive, although we can detect its expression by immunoblotting in cell homogenates but not in alkali-treated microsomes (Fig. 9).

These results suggest that the amino-terminal portion of D1, like that of P45017, is required for synthesis of an active enzyme. However, unlike the result with the P450 steroidogenic enzyme, we were unable to reconstitute enzyme activity by replacing the amino terminus of D1 with either residues 1-20 of P45021 (MEM 2 and 3) or a similar putative transmembrane domain from the recently cloned X. laevis type 3 deiodinase (MEM 4) (16) . The latter protein is more similar to the D1 sequence in that it contains positively charged amino acids in the putative extramembrane domain (KLVK) and an arginine within the hydrophobic domain. While transiently expressed MEM 2, 3, and 4 are alkali-resistant membrane proteins (Fig. 9), they are inactive. Since it seems likely that the type I topologies of these proteins are preserved, these results suggest that either the proper post-translational processing of the protein or its catalytic function required specific residues in the native sequence. Thus, the function of the D1 signal-anchor sequence is not limited to membrane integration and orientation.

The results with MEM 5, 6, and 7 support this concept in that mutations of the conserved, positively charged amino acids in the amino-terminal portion of the protein reduce the synthesis or stability of active protein in a transient expression system. Since it is possible to monitor both transfection efficiency (by human growth hormone assay) and the amount of D1 synthesized during these experiments (by specific covalent labeling with BrAc-[I]T3), we can establish that there is inefficient formation of the mutant enzyme molecules. Since post-translational folding of proteins is in part a stochastic process, we speculate that the alterations in charge of MEM 5, 6, and 7 have deleterious effects on the potential of the mutant proteins to attain their native conformation, and, presumably, the aberrant folded proteins are degraded. However, those molecules which achieve a proper conformation, e.g. about 1 in 4 for MEM 5, can function normally with respect to Kand turnover number despite the absence of positive charges in the lumenal or transmembrane sequences. Presumably, the alterations induced by the global amino-terminal substitutions in mutants MEM 2, 3, and 4 are too great to permit the formation of a significant number of catalytically active molecules even though these chimeric proteins are integrated into the membrane.

Taken together, we conclude that while the topological model in Fig. 8indicates that the catalytic portion of D1 is cytosolic; specific, conserved, positively charged amino acids present in the transmembrane and lumenal domains of D1 are also essential, not for catalytic activity, but for the efficient post-translational processing of the enzyme.

  
Table: Comparison of the amino-terminal sequences of the rat G21, deletion, chimeric, and mutant D1s

MEM 1, deletion mutant of G5, which starts at amino acid 26 of G5. MEM 2, chimeric construct between bovine 21-hydroxylase P450 enzyme (P45021) and G5 in which amino acids 1-20 of P45021 and additional histidine and methionine residues replace amino acids 1-25 of G5 and MEM 3 in which amino acids 1-19 of P45021 replace amino acids 1-32 of G5. MEM 4, amino acids 1-44 of Xeno D3 (amino acids 4-10 not shown) and histidine and methionine residues replace amino acids 1-25 of G5. The putative membrane-spanning domains of the P45021 and the Xeno D3 in MEM 2, 3, and 4 are underlined. In MEM 5, amino acids Lys-11 and Arg-12 of G21 were changed to asparagine; in MEM 6 and 7, amino acid Lys-27 was changed to methionine and glutamic acid respectively.


  
Table: Quantitation of expression and analyses of the catalytic characteristics of wild-type (G21) and mutant D1 proteins after transient expression in HEK 293 cells

MEM 5, 6, and 7 are described in Table I. Transfection efficiencies were estimated by measuring human growth hormone expression from cotransfected TKGH as described under ``Experimental Procedures.'' The transfection efficiency of each construct was normalized to that of G21 (wild-type). Enzyme protein ([E]) was quantitated by saturation analysis using BrAc-[I] T3 as described under ``Experimental Procedures.'' E is expressed as picomoles of specifically bound BrAc-[I]T3/mg of sonicate protein (see ``Experimental Procedures''). Kinetic constants were derived from Lineweaver-Burk plots. Data are expressed as mean ± S.D. of at least three independent transfections.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK36256. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Thyroid Division, Dept. of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115. Tel.: 617-732-6762; Fax: 617-731-4718.

The abbreviations used are: T4, thyroxine; T3, 3,5,3`-triiodothyronine; ER, endoplasmic reticulum; rT3, 3,3`,5`-triiodothyronine; P45021, bovine 21-hydroxylase P450 enzyme; P45017, bovine 17-hydroxylase P450 enzyme; Xeno D3, X. laevis type 3 deiodinase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. Peter Arvan and Mathias Hediger for helpful discussions during the course of this work. We also thank Dr. Michael Waterman for his generosity in providing the bovine 21-hydroxylase P450 constructs.


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