From the
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.
Conversion of the prohormone thyroxine (T4)
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.
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.
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.
A 27-kDa protein in liver and kidney,
affinity-labeled with BrAc-[
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
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
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-[
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.
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.
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-[
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.
(
)
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) .
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.
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 Na
CO
, 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 EN
HANCE (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 Na
CO
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
Na
CO
exposure; lane 5, G5 microsomes;
lane 6, pellet; lane 7, supernatant of
Na
CO
-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 Na
CO
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
Na
CO
(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 Na
CO
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.
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
K
of the transiently expressed enzyme was
also normal ().
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).
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.
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.
-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.
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 K
and 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.
Table:
Comparison of the amino-terminal
sequences of the rat G21, deletion, chimeric, and mutant D1s
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
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.
-hydroxylase P450
enzyme; Xeno D3, X. laevis type 3 deiodinase; PCR, polymerase
chain reaction; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.