From the Department of Cellular and Molecular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 and § Internal Medicine III, Erasmus University Medical School, 3015GD Rotterdam, The Netherlands
Received for publication, August 2, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Type I iodothyronine deiodinase is a ~50-kDa,
integral membrane protein that catalyzes the outer ring deiodination of
thyroxine. Despite the identification and cloning of a 27-kDa
selenoprotein with the catalytic properties of the type I enzyme, the
composition and the physical nature of the active deiodinase are
unknown. In this report, we use a molecular approach to determine
holoenzyme composition, the role of the membrane anchor on enzyme
assembly, and the contribution of individual 27-kDa subunits to
catalysis. Overexpression of an immunologically unique rat 27-kDa
protein in LLC-PK1 cells that contain abundant catalytically active
27-kDa selenoprotein decreased deiodination by ~50%, and >95% of
the LLC-PK1 derived 27-kDa selenoprotein was specifically immune
precipitated by the anti-rat enzyme antibody. The hybrid enzyme had a
molecular mass of 54 kDa and an
s20,w of ~3.5 S indicating that every
native 27-kDa selenoprotein partnered with an inert rat 27-kDa subunit
in a homodimer. Enzyme assembly did not depend on the presence of the
N-terminal membrane anchor of the 27-kDa subunit. Direct visualization
of the deiodinase dimer showed that the holoenzyme was sorted to the
basolateral plasma membrane of the renal epithelial cell.
Type I thyroxine deiodinase
(D1,1 EC 3.8.1.4) is the most
abundant member of a family of membrane-bound enzymes that catalyze the
removal of a single iodine atom from thyroid hormone. D1 generates most
of the bioactive T3 found in blood (1-4) and serves as the essential first step in the molecular mechanism of thyroid hormone action in vivo.
D1 is a selenoenzyme encoded by an ~2.1-kilobase pair mRNA and is
most abundant in liver, kidney, and thyroid (1-5). The ~27-kDa
enzyme polypeptide (p27) contains the novel amino acid, selenocysteine
(SeC) (5), and this residue is essential for full catalytic activity
(6-8). Whether p27 is catalytically active itself or requires
post-translational modification and/or assembly into a multimeric
complex remains to be established. Despite uncertainty about its
composition and the physical nature of the catalytically active enzyme,
site-directed mutagenesis, species comparisons, domain substitution,
and limited deletion analysis (9-14) have been used to characterize
some of the functional domains of p27. The p27 polypeptide contains an
uncleaved N-terminal signal/membrane anchor followed by an
uninterrupted catalytic domain facing the cell interior (10).
Iodothyronine substrate specificity is determined by amino acids
located between residues 40 and 65, as judged by site-directed
mutagenesis, analysis of species-specific differences in catalytic
properties, and amino acid composition, and by interspecies deletion-substitution analysis (9, 11). In addition to the presumed membrane anchor and the substrate specificity domain, the
active center is thought to be located in an ~15 amino acid long
region bracketing the SeC residue (7, 12).
Four D1 amino acid residues (Phe-65, Cys-124, SeC-126, and His-174)
have been identified as essential for substrate specificity and
catalysis (7, 9, 11, 12-14). The roles of Phe-65 in iodothyronine
specificity and of SeC126 in catalysis are well established (7, 9, 11),
whereas the roles of Cys-124 and His-174 in the catalytic reaction are
less understood. For example, replacement of Cys-124 with alanine led
to parallel ~2-fold decreases in the Km for
rT3 and in catalytic efficiency when assayed at a fixed
cofactor concentration (15). Determination of the limiting Michaelis
constants using two substrate reaction kinetics revealed that the
Km for rT3 was marginally affected in
the C124A mutant (14, 15), whereas the velocity of the forward reaction
decreased ~2-fold (14, 15), and the thiol cofactor requirement
increased by 6-14-fold (14, 15). The mechanism for these changes in
kinetic parameters is unclear. Traditional chemical modification
studies established that histidine(s) were essential for catalysis (16,
17), and subsequent mutagenesis and transient expression studies
identified His-174 as one of these essential residue(s) (13).
However, caution is essential when attempting to assign functional
significance to individual amino acids in an incompletely characterized
enzyme. Whereas the direct participation of any D1 residue that affects
catalysis is the first possibility often considered, it is equally
likely that individual amino acid substitutions can influence D1
activity by indirect means such as (i) abnormal post-translational
processing; (ii) altered folding of the primary translation product
that leads to rapid degradation; (iii) improper sorting of the mutant
p27 in the Golgi apparatus; and/or (iv) disturbed assembly of a
functional enzyme. Thus, the interpretation of the mechanistic
consequences of individual mutations on D1 activity in whole cell
lysates of transiently transfected cells is difficult at best and
cannot identify what molecular events are affected without an
understanding of the influence of the mutation on enzyme assembly and processing.
Much of the biochemistry of D1 was established prior to the cloning of
the enzyme using the alkylating substrate analogs, N-BrAcT4
and N-BrAcT3 (18-21). Affinity labeling studies done with liver and kidney membrane preparations identified an ~27-kDa thyroid hormone-binding protein (p27) (19-21) that was subsequently cloned by
functional expression (5). Interestingly, molecular sieve chromatography of the detergent-soluble rat kidney D1 yielded an
~50-kDa functional enzyme (22), and subsequent work showed that the
affinity labeled p27 co-migrated with catalytic activity as an
~54-kDa complex (23). These data suggested that the catalytically active D1 was composed of two similarly sized subunits. Whether the
functional enzyme is a homodimer of p27 subunits or a heterodimer formed between p27 and an unknown subunit remains to be established. The latter possibility cannot be excluded because kidney-derived cell
lines have been used for most, if not all, p27 transient expression
studies, and this organ has abundant D1 activity. Thus, it is possible
that a 25-30-kDa membrane anchor protein found in kidney cells could
partner with p27 to form a functional enzyme.
In this study, we exploited the species differences in the composition
of the C terminus of p27 to characterize the composition of
catalytically active D1. By using the native D1 found in a pig renal
epithelial cell line (LLC-PK1), we overexpressed an immunologically
unique, catalytically inert S126 mutant of rat p27 (M4), and we
examined the consequences of M4 expression on D1 subunit assembly,
enzyme activity, and subcellular trafficking. The data show that
catalytically active D1 is a dimer composed of two p27 subunits and
that the assembled enzyme is sorted to the basolateral plasma membrane
of renal epithelial cells.
Materials--
All reagents were of the highest purity
commercially available. Restriction endonucleases and DNA-modifying
enzymes were purchased from New England Biolabs (Beverly, MA). The
femtomoles of DNA sequencing and the Altered Sites Mutagenesis kits
were obtained from Promega (Madison, WI). Cell Culture--
LLC-PK1 cells were grown in
25-cm2 flasks in a humidified
atmosphere of 5% CO2 and 95% air at 37 °C as described
previously (23). Complete Growth Medium was composed of Dulbecco's
modified Eagle's medium containing 15 mM sodium
bicarbonate, 15 mM HEPES (pH 7.2), 25 mM
glucose, 1 mM sodium pyruvate, 5% (v/v) calf serum, 50 milliunits/ml penicillin, and 90 µg/ml streptomycin. Cells were
subcultured every 3-5 days by seeding 5 × 104
cells/cm2 into 25-cm2 flasks. Culture medium
was changed 3 times weekly.
Mutagenesis--
Site-directed mutagenesis was used to
inactivate the catalytic activity of rat p27 by substituting Ser for
the active center SeC. In brief, the 2.1-kilobase pair G21 cDNA was
released from pBSK using KpnI-XhoI and cloned
into the mutagenesis shuttle vector, pALTER-1 (Promega, Madison, WI). A
24-mer synthetic oligonucleotide bracketing the SeC-126 codon
(nucleotides 372-396, 5'-CAGCTGCACCAGCCCTTCATTTCTT-3') was
used to create an SeC-126
Elimination of the putative extracellular domain and the
membrane-spanning anchor of p27 was done by polymerase chain
reaction-based deletion. All upstream oligonucleotide primers contained
a KpnI site for subcloning and an in-frame Kozak consensus
translation initiation site to ensure adequate expression in mammalian
cells. The first 19 amino acids of p27 that include the putative
extracellular domain and 7 residues of the membrane-spanning region
were deleted using a 27-mer oligonucleotide upstream primer
(5'-GGGTACCATGGCCTTGGAGGTGGCTAC-3', nucleotides
65-80 of G21 underlined). The N-terminal 42 amino acids that
contain the entire membrane-spanning domain was deleted using a 29-mer
oligonucleotide upstream primer
(5'-GGGGTACCATGGCCATGGGCCAAAAGACC-3', nucleotides
131-150 of G21 underlined). The common downstream primer was a 20-mer
oligonucleotide corresponding to nucleotides 895-914 of G21
(5'-GCCTCCCGGGTAGTTATTTG-3'). Polymerase chain reaction was done using
Vent® DNA polymerase (Stratagene, La Jolla CA) for a total of 25 cycles according to the following temperature profile: 95 °C, 1.0 min; 50 °C, 1.0 min; 72 °C, 3.0 min, with a final 10-min extended
incubation at 72 °C. Polymerase chain reaction products were
digested with KpnI to generate asymmetric ends for
subcloning, and gel was isolated on 1.4% low melt agarose gels and
ligated into the KpnI-SmaI-ended pcDNA3-M4.
The N-terminal 19-residue deletion mutant, M4-19, and the N-terminal
42-residue deletion mutant, M4-42, were confirmed by DNA cycle
sequencing (see below).
Generation of M4-expressing LLC-PK1 Cells--
LLC-PK1 cells
were grown in Growth Medium to 60% confluence and transfected with 10 µg of pcDNA3 (empty vector control) or 10 µg of pcDNA3-M4,
pcDNA3-M4-19, or pcDNA3-M4-42 using cationic liposomes
(DOTAP®, Roche Molecular Biochemicals) according to the
manufacturer's instructions. Stable transfectants were selected with
800 µg/ml G418, and antibiotic-resistant cells were expanded without
clonal isolation. Cells constitutively expressing M4 (S126 cells), M4-19, and M4-42 were maintained in 200 µg/ml G418 to prevent
loss of expression of the exogenous gene product.
DNA Sequencing--
Double-stranded DNA sequencing was done by
the dideoxynucleotide method of Sanger (see Ref. 25) using cycle
sequencing and iterative primers. All sequence information was
confirmed by sequencing both strands.
Biosynthetic Labeling of Native p27wt Selenoprotein
in S126 Cells--
S126 cells were grown to 90% confluence in
25-cm2 flasks in Complete Growth medium. Cell monolayers
were washed 3 times with serum-free Dulbecco's modified Eagle's
medium, and the medium was replaced with serum-free 75Se
labeling medium composed of Dulbecco's modified Eagle's medium supplemented with 25 mM HEPES (pH 7.2), 10 mg/ml BSA, 10 µg/ml transferrin, 20 µg/ml insulin, 40 nM
75Se (specific radioactivity, 200 µCi/nmol), and
antibiotics. Cellular selenoproteins were biosynthetically radiolabeled
at 37 °C for 48 h. After labeling, cells were washed free of
labeling medium, scraped from the dish, and collected by
centrifugation. Cell pellets were stored at Production of Species-specific, Anti-p27
Antibodies--
Comparison of the p27 from fish to man revealed that
the C terminus of p27 shows species-specific variations, and the rodent p27 contains a C-terminal extension of 8-12 amino acids (26). This
unique feature was exploited to generate a rodent-specific, anti-p27
antibody. A synthetic peptide corresponding to the C-terminal 21 residues of rat p27 (NH2-YEEVRAVLEKLCIPPGHMPQF-COOH) was
coupled to mollusk hemocyanin and used to immunize rabbits. An
N-terminal tyrosine was included to allow radioiodination and to
facilitate directional coupling of the p27 peptide to carrier protein
by diaminobenzidine condensation. Where indicated, rodent-specific anti-p27 IgG was purified by affinity chromatography using a p27 peptide-Affi-Gel affinity matrix. In brief, primary antiserum was
adsorbed to the p27 peptide-Affi-Gel 10 matrix at 25 °C, and unbound
proteins were removed by repeated washes with 150 mM NaCl, 20 mM sodium phosphate buffer (pH 7.4). Anti-p27 IgG was
eluted with 100 mM Hac, and antibody elution was monitored
by absorbance at 280 nm. Fractions containing antibody were neutralized
by adding 0.1 volume of 1 M Tris (pH 8.6), pooled,
and stored frozen ( Antibody Characterization--
Anti-p27 antibodies were used for
immunoprecipitation and in a competitive binding assay. Pull-down
assays of M4 and associated proteins were done for 60 min at 4 °C in
a total volume of 100 µl. Precipitation reactions contained 50-100
units of tDOC-solubilized D1 activity prepared from purified membranes
(23), 100 mM HEPES buffer (pH 8.0), 100 mM
NaCl, 0.1 mM PMSF, 5 mM tDOC, 10 µl of immobilized rProtein ATM beads (RepliGen, Cambridge, MA),
and either preimmune rabbit antisera or anti-rat p27 antisera (1:100
final dilution). Where indicated, excess C-terminal p27 peptide (10 µg/ml, final concentration) was added to block antibody-binding
sites. Immune complexes bound to the protein A beads were removed by
centrifugation, and the deiodinase activity remaining in the clarified
supernatant was determined as described below.
M4 expression in LLC-PK1 cells was quantified by a competitive binding
assay. Cell pellets or isolated membrane preparations were solubilized
with either 0.1% Triton X-100 (v/v) or 5 mM tDOC, and the
soluble enzyme preparation was clarified by centrifugation at
14,000 × g for 30 min at 4 °C. Assays were done in
triplicate in a total of 100 µl containing 100 mM Tris
buffer (pH 8.3), 100 mM NaCl, 5 mM tDOC, 10,000 cpm 125I-labeled p27 peptide (specific radioactivity, 100 mCi/µmol), anti-p27 antisera (1:50,000 final dilution), and detergent
extracts. Incubations were done for 2 h at 4 °C, and immune
complexes were precipitated by addition of goat anti-rabbit antiserum
(1:5000 dilution). After an additional 60 min at 4 °C, 400 µl of
ice-cold PBS containing 10 mg/ml BSA was added, and the immune
precipitates were collected by centrifugation. Pellets were counted in
a well-type Construction of GFP-tagged M4 Expression Plasmids--
A green
fluorescent protein (GFP)-tagged p27 fusion construct
(M4GFP) was generated by appending GFP to the C terminus of
M4. In brief, the ~750-base pair HindIII fragment
(containing the M4 mutant of p27-coding sequence lacking the C-terminal
11 amino acids) was excised from pcDNA3-M4, gel-purified, and
ligated, in-frame, into the HindIII site of pEGFP-N1
(CLONTECH, Palo Alto, CA). The fusion construct
M4GFP cDNA was excised with BamHI and
NotI and ligated into BamHI-NotI-ended pcDNA3. The integrity of the fusion construct and expression of a
fluorescent fusion protein were confirmed by transient expression of
the pcDNA3-M4GFP in HEK293 cells and immunoblot
analysis using anti-GFP IgG (CLONTECH, CA).
Photomicroscopy--
Cells were seeded onto
poly-D-lysine (10 µg/ml)-coated glass coverslips (22 × 22 mm) and grown to ~60% confluence. Cells were then fixed with
4% paraformaldehyde and permeabilized with 0.1% Triton (v/v) in PBS.
Similarly, rat kidney slices were fixed with 4% paraformaldehyde in
PBS, embedded in paraffin, and ~4-µm sections were prepared from
the outer cortex. The distribution of p27 in the renal tubule was
determined using affinity-purified, anti-p27 IgG (0.1 µg/ml). Immune
complexes were visualized using a Texas Red-conjugated goat,
anti-rabbit IgG (1:2, 500 final dilution). The distribution of
M4GFP 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. Photomicrographs shown are representative of 20-30
independent fields.
Analytical Procedures--
D1 activity was determined in cell
lysates by measuring the release of radioiodide from 10 µM [125I]rT3 (100 cpm/pmol) at
20 mM dithiothreitol (unless otherwise indicated) in a
total volume of 100 µl. Deiodination reactions were done, in
triplicate, in 100 mM potassium phosphate buffer (pH 7.0),
1 mM EDTA, with 10-25 µg of cell protein and incubated at 37 °C for 10-20 min. Product formation was measured as described previously (24), and the data are expressed as units/mg protein where 1 unit equals 1 pmol of I
All experiments were performed at least three times. Statistical
analysis was done by Student's t test.
Characterization of the Rodent-specific Anti-p27 Antisera--
The
characterization of the anti-rat p27 antibody was done using a
competitive binding assay, immunoprecipitation, and immunocytochemistry as shown in Fig. 1. Detergent-soluble rat
kidney p27 readily displaced 125I-labeled p27 peptide from
the anti-rat p27 antisera (Fig. 1A) yielding a concentration
of p27 of 4 ± 1 pmol of p27 per mg of kidney membrane protein
(mean ± S.E., n = 3), similar to that reported
previously (19, 20). On the other hand, up to 1 mg of LLC-PK1 membrane
protein (~500 units of D1 activity) failed to displace any of the
rat-specific 125I-labeled p27 peptide indicating that the
pig p27 selenoprotein was not recognized by the anti-rat p27 antibody.
The ability of the anti-rat p27 antisera to immune precipitate
detergent-soluble D1 activity is shown in Fig. 1B.
Detergent-soluble D1 activity was prepared from rat kidney and LLC-PK1
microsomes (23), and the clarified extracts were incubated with
anti-p27 antibody in the absence or presence of excess blocking
peptide. All (>98%) of the soluble D1 activity from rat kidney
membranes was lost from the detergent extracts when the immune
complexes were removed with protein A-Sepharose, and addition of a
200-fold molar excess of p27 peptide completely blocked the
antibody-dependent loss of D1 activity. As expected from
the failure of the LLC-PK1-derived p27 to displace the rat p27 peptide
from the anti-rat p27 antibody, no soluble D1 activity was immune
precipitated from microsomes isolated from LLC-PK1 cells. Shown in Fig.
1C are representative photomicrographs of rat renal cortex
from selenium-replete and selenium-deficient rats stained with the
anti-rat p27 antisera. Selenium-dependent expression of the
abundant immunoreactive protein is found in cells lining a subset of
tubules that surround the glomerulus, consistent with earlier
subcellular localization studies (23, 27). These data establish that
the anti-rat p27 antiserum shows species-specific recognition of the
rat p27 selenoprotein by immunoprecipitation, competitive displacement
analysis, and by immunocytochemistry.
Analysis of the Effects of Constitutive Expression of M4 on Native
D1 Activity in LLC-PK1 Cells--
The abundant D1 activity in LLC-PK1
cells was used previously to characterize both the biochemistry and
physiochemical properties of the enzyme (23, 28). Gel filtration and
isopycnic density gradient centrifugation of detergent-soluble D1 from
these cells showed that catalytic activity and the
BrAcT4-labeled p27 co-migrated as a globular protein with a
Mr of ~54,000 (28), suggesting that D1 is a
dimer of two similarly sized subunits, presumably p27. Shown in Fig.
2 are the potential combinations of p27
subunits that can form in LLC-PK1 cells that constitutively express the catalytically inert M4 mutant of rat p27 (S126 cells). Combinations I
and II yield dimers with at least one selenoprotein partner. Overexpression of the M4 protein relative to the native
p27wt selenoprotein will also generate immunoreactive,
nonselenoprotein dimers of M4 (combination III), and these dimers are
favored if the rat-derived, M4 mutant cannot interact with the native
p27wt in LLC-PK1 cells. Only combination II yields a D1
dimer composed of a native p27wt selenoprotein partnered
with M4, and this hybrid D1 can be rescued with antibodies directed
against the rat p27.
Shown in Fig. 3 are the results of
pull-down assays done with anti-rat p27 antibody and soluble D1 from
control (vector) and S126 cells. Little, if any, detergent-soluble
75Se-labeled p27 was specifically bound to the protein A
beads when normal rabbit serum was used (Fig. 3A, left
bars). In contrast, ~10% of the total 75Se-labeled
protein(s) solubilized from S126 cells was specifically immune
precipitated by the anti-rat p27 antibody, whereas the quantity of
75Se-labeled proteins from the vector cells bound to the
protein A beads was identical to the nonspecific binding found when
normal rabbit serum was used (Fig. 3A). Addition of a
200-fold molar excess of blocking peptide decreased the quantity of
75Se-labeled S126 cell protein bound to the anti-rat
p27 antibody to that nonspecifically bound to the protein A beads.
Shown in Fig. 3B is the companion analysis of the effects of
anti-rat p27 antibody on soluble D1 activity prepared from microsomes isolated from vector and S126 cells. Similar to the results obtained with soluble 75Se-labeled p27 prepared from vector and S126
cells, <5% of the soluble D1 activity was lost in the pull-down assay
when normal rabbit serum was used, and the addition of excess blocking
peptide had no effect on D1 activity. Interestingly, the D1 activity in S126 cells was ~50% of that found in the vector control, LLC-PK1 cell. Just as observed with the soluble D1 activity from rat kidney and
untransfected LLC-PK1 cells (see Fig. 1B), little, if any, soluble D1 activity in vector cells was removed by immune precipitation with anti-rat p27 antisera. In contrast, >95% of the D1 activity in
detergent extracts of S126 cells was removed by the anti-rat p27
antibody, and this loss in activity was blocked by addition of excess
p27 peptide (Fig. 3B). By comparing the quantity of soluble
D1 remaining in the supernatant in the presence of excess blocking
peptide with that found when normal rabbit serum was used revealed that
Direct identification of hybrid D1 dimers composed of the
non-selenoprotein M4 and the native p27wt selenoprotein is
shown in Fig. 4. The native p27
selenoprotein in control LLC-PK1 cells and in S126 cells was
biosynthetically radiolabeled with 75Se, and detergent
extracts of the radiolabeled cells incubated with anti-rat p27 antibody
and the specific immune complexes were separated on 12.5% SDS-PAGE
gels. Shown in Fig. 4 are representative fluorograms of the
75Se-labeled proteins in starting cell lysates (lanes
1 and 3) and in immune precipitates (lanes 2 and 4) of control LLC-PK1 cells (lanes 1 and
2) and S126 cells (lanes 3 and 4).
Whereas M4 overexpression had little, if any, influence on the quantity
of 75Se-labeled p27 found in S126 cell lysates (compare
lanes 1 and 3), the native
75Se-labeled p27 co-immunoprecipitated with M4 from the
S126 cell (lane 4) but was not immune precipitated from the
parent LLC-PK1 cells (lane 2). Since only the native p27 can
incorporate 75Se into the polypeptide, these data confirm
that the native LLC-PK1-derived p27 subunit was partnered with a rat M4
polypeptide in the S126 cell. Together with the data showing that
specific M4-directed antibodies removed >97% of the D1 activity from
the S126 cell, these data show that overexpression of the inert M4
leads to the quantitative formation of a catalytically impaired, hybrid
D1 enzyme composed of pig p27wt-M4 partners.
We then measured the quantities of both the native p27wt
and M4 mutant proteins in the S126 cells by exploiting the
selenium-dependent expression of the p27wt and
the unique immunoreactivity of M4. S126 cells were grown in the absence
and presence of 40 nM 75Se for 48 h, and
cell lysates were prepared. Selenium-deficient S126 cells lost
>93% of the hybrid D1 activity when compared with control
selenium-supplemented S126 cells (Table
I). Although the 75Se-labeled
p27wt was completely lost in selenium-deficient cells, the
cellular content of M4 was unaffected by changes in selenium content.
Estimates of the p27wt and M4 content in
selenium-supplemented S126 cells (Table I) showed that there is an
~10-fold molar excess of M4 over the native p27wt, and
based on the data in Fig. 3 and in Table I this appears to be
sufficient to complex most, if not all, of the native p27wt
present in the S126 cell (Table I).
Physiochemical Properties of the Hybrid D1 Composed of
p27wt-M4 Partners in S126 Cells--
To determine whether
the catalytically active hybrid D1 composed of p27wt-M4
partners was a dimer or a higher order aggregate, detergent extracts of
75Se-labeled S126 cells were separated by isopycnic density
gradient centrifugation (28), and the sedimentation properties of the 75Se-labeled p27wt-M4 partners were determined
by immune precipitation. The distribution of 75Se-labeled
p27wt-M4 complexes peaked between the marker proteins
carbonic anhydrase and BSA as shown in Fig.
5. Assuming a frictional ratio
(f/f0) of 0.85 equal to that
determined for BrAcT4-labeled p27 (28), the sedimentation
coefficient (s20,w) for the
75Se-labeled hybrid D1 (p27wt-M4 complex) is
~3.5, yielding a calculated mass of 54 kDa in close agreement with
previous estimates of the native D1 (28). These data confirm that the
catalytically active hybrid D1 in S126 cells is a dimer composed of one
p27wt and one M4 subunit.
The Role of the Membrane Anchor in D1 Holoenzyme
Assembly--
Since the D1 holoenzyme is a dimer of two
membrane-spanning p27 subunits and assembly of the enzyme is a
post-translational event, we examined the role of the membrane anchor
in D1 holoenzyme assembly. Earlier work suggested that the N terminus
of p27 was required for either proper post-translational processing or
that specific residues located in this region were necessary for
catalysis. Two basic residues in the N terminus of p27 appear to be
required for proper folding of the p27 (10). To explore the role of the membrane anchor of p27 in D1 assembly, we prepared N-terminal deletion
mutants of M4 in which the "external" 12 amino acids, including two
basic residues at position 11 and 12, and 7 residues in the
membrane-spanning region were removed (M4-19), and/or the entire
membrane anchor was deleted (M4-42). Constitutive overexpression of
these truncation mutants in LLC-PK1 cells was done as described for the
parent M4 and yielded two new cell lines, M4-19 and M4-42.
Shown in Fig. 6, are the consequences of
expression of M4-19 and M4-42 on D1 activity. As observed with the
parent M4, overexpression of either truncation mutation decreased D1
activity by ~50%, and 90-95% of the D1 activity present in the
M4-19 and M4-42 cells was immunoprecipitated by the anti-rat p27
antibody. These data indicate that a catalytically active D1 holoenzyme
can be formed from dimers of the native p27wt and a
truncated p27 that lack the entire membrane anchor. From these data it
appears that assembly of the D1 holoenzyme does not require both p27
subunits to be membrane-associated.
Catalytic Consequences of Hybrid D1 Holoenzyme Composed of
p27wt and Inactive Subunits--
Since the D1 activity in
the S126 cell was derived from a holoenzyme composed of
p27wt-M4 subunits, and M4-M4 dimers are catalytically
inert, we used the hybrid D1 activity in S126 cells to examine the
influence of each subunit on the catalytic reaction. Hybrid D1 activity was provided by S126 cell lysates, and two-substrate reaction kinetics
were done as detailed previously (24, 29). As shown in Fig.
7, both wild type D1 activity in LLC-PK1
cells and the hybrid D1 activity derived from p27wt-M4
dimers in S126 cells showed the expected ping-pong reaction kinetics.
Secondary replots of slopes and intercepts versus the reciprocal of the thiol cofactor concentration yielded the limiting kinetic constants of the respective D1 holoenzyme (Table
II). Although the limiting
Km values for the thiol cofactor and iodothyronine
were either unchanged or marginally affected when one of the p27
subunits was catalytically inert, there was a marked 75% reduction in
the maximal forward velocity of the deiodination reaction of the hybrid
D1 when compared with that of the wild type D1.
Subcellular Distribution of M4GFP-M4GFP
Dimers and p27wt-M4GFP Dimers in S126
Cells--
Finally, we exploited the fact that the C terminus of p27
is degenerate, to generate a fluorescent p27-green fluorescent protein reporter molecule (GFP) fusion protein (M4GFP) that allows
the D1 holoenzyme to be followed in the living cell. GFP was appended
in-frame to the C terminus of M4, and constitutive expression of the
M4GFP fusion protein in LLC-PK1 cells led to the appearance
of abundant membrane-bound fluorescence (Fig.
8). D1 activity in the
M4GFP-expressing cells showed a marked decrease (45 units/mg protein in control LLC-PK1 cells versus 18 units/mg
protein in M4GFP-expressing cells) similar to that observed
when M4 alone was introduced into the LLC-PK1 cell (see Fig. 3). These
findings show that addition of the GFP reporter to the C terminus of M4 did not impair the ability of M4 to form hybrid D1 dimers with the
native p27wt subunit. To ensure that the D1 activity
present in the M4GFP-expressing cells was derived from a
hybrid D1 holoenzyme composed of native p27wt and
M4GFP subunits, anti-GFP antibodies were used in a
pull-down assay. As expected, anti-GFP IgG did not precipitate the D1
activity in control LLC-PK1 cell extracts but specifically
immunoprecipitated >95% of the D1 activity in
M4GFP-expressing cells (data not shown). These findings
show that overexpression of the inert D1 subunit, M4GFP,
results in the formation of a catalytically active D1 holoenzyme composed of a p27wt and M4GFP subunits and that
the appended GFP reporter had little, if any, influence on the
deiodination reaction of the complex.
Shown in Fig. 8 are representative photomicrographs of the distribution
of the M4GFP-derived D1 holoenzyme in LLC-PK1 cells
constitutively expressing this fusion protein (M4GFP
cells). Abundant punctate florescent signals were found between the
boundaries of adjacent cells and along the cell periphery of individual
cells. Infrequently, a more diffuse fluorescent signal is observed in
the perinuclear space. These data are consistent with earlier studies
showing that the BrAcT4-labeled native p27wt
was sorted to the basolateral membranes in the oriented LLC-PK1 cell (23, 27, 30) and indicate that the M4GFP fusion
protein, whether as a homodimer or a mixed wild type
p27:M4GFP, is also directed to the basolateral plasma membrane.
Thyroid hormone deiodination catalyzed by D1 generates most of the
bioactive T3 found in the circulation and thereby plays a
key role in the molecular mechanism of thyroid hormone action. Although
the cloning of p27 yielded a catalytically active D1 after transient
transfection (5), the subunit composition of the active enzyme, the
role of individual p27 polypeptides in the catalytic reaction, and the
physiochemical properties of the cloned enzyme have not been
determined. In this report, we describe the subunit composition of
catalytically active D1 holoenzyme, examine the role of each subunit in
the deiodination reaction, and show that the D1 dimer is sorted to the
basolateral plasma membrane of renal epithelial cells.
Prior to the cloning of p27, affinity labeling studies done with
alkylating derivatives of thyroxine were used to identify the D1
polypeptide(s). In both liver and kidney membrane preparations, a
27-kDa BrAcT4-labeled protein was identified, and this
protein showed all of the properties of the substrate binding component of D1 as judged by rate inactivation studies (23) and inhibitor competition studies (19, 20). Subsequent work showed that the p27
polypeptide and the active D1 enzyme could not be separated by gel
filtration and/or isopycnic density gradient centrifugation and that
the molecular mass of the BrAcT4-labeled D1 was 54 kDa with
an s20,w of 3.7 S (28), values similar
to those determined earlier for a detergent-soluble D1 that required
reactivation by lipid reconstitution (22). In contrast, detergent
extracts of liver D1 that contained a catalytically active proteolipid complex showed a family of active enzyme(s) with much larger molecular weights after gel filtration (31, 32). Thus, the constitution of the
membrane-bound, functional D1 appeared to differ according to the
detergent used and the tissue source.
The finding that a catalytically active D1 enzyme assembles from an
exogenous, inert p27 and a native p27 and that the hybrid enzyme had
the same physiochemical properties as the native D1 directly confirms
that D1 is a homodimer of p27 subunits. This is consistent with the
earlier work (22, 23) that followed soluble D1 activity in crude
membrane extracts. In addition, this observation shows that native and
mutant p27 subunits can combine to constitute a hybrid enzyme and
reveals that D1 assembly is a post-translational event that
presumably depends upon the local concentration of p27 in the membrane.
Since overexpression of an exogenous p27 mutant drives the native p27
subunit into hybrid D1 molecules, it appears that both the assembly and
the composition of the D1 holoenzyme can be predetermined by
experimental manipulation.
Assembly of a functional D1 holoenzyme not only requires sufficient
numbers of p27 subunits but also the correct positioning of yet to be
identified dimerization domain(s). Importantly, the orientation of the
native p27wt subunits in the D1 holoenzyme is constrained
to a head-to-head, tail-to-tail orientation by the polarity of the
membrane-bound p27 subunits and the sidedness of the catalytic activity
(23, 30). This limiting constraint imposed by the membrane-bound nature
of p27 led Larsen and co-workers (10) to conclude that residues within
the p27 membrane anchor directed either the proper folding of the p27
or participated in catalysis. Their conclusions were based, in part, on
the failure of several p27 mutants to generate D1 activity after
transient transfection when the membrane anchor of p27 was swapped with
the endoplasmic reticulum-derived membrane anchor(s) from the bovine
21-hydroxylase P450 enzyme (10). The results detailed in Fig. 7 clearly
show that truncated p27 subunits lacking the membrane anchor will bind
to the native p27 and produce a functional D1 holoenzyme in which only
one p27 subunit is anchored to the membrane. These findings indicate
that the assembly of the D1 holoenzyme is independent of the membrane anchor. Thus, the failure of the membrane anchor swapped p27s to
assemble into a functional enzyme (10) is likely due to the low
abundance of the exogenous p27 selenoprotein translation products after
transient transfection and/or to directional miscues given by the
endoplasmic reticulum resident P450-derived membrane anchor. Similar caveats apply to the failure of the p27 mutants in which the
membrane anchor derived from the deiodinase family member, type III
iodothyronine 5'-deiodinase, was substituted for the native
membrane-spanning region to generate catalytically active enzyme (10).
Since loss of the membrane anchor from one p27 subunit yields a hybrid
D1 with activity levels similar to that found when both enzyme subunits
are membrane-bound, it is likely that a catalytically active,
"soluble" D1 holoenzyme would assemble if sufficient quantities of
truncated p27 selenoprotein subunits could be produced.
Unfortunately, the limiting quantity of SeC in mammalian cells is
likely to interfere with the synthesis of sufficient quantities of
soluble p27 selenoprotein necessary to drive assembly of a
soluble D1 holoenzyme.
With the composition of the catalytically active D1 holoenzyme
established, it is now possible to revisit the role of individual amino
acids in the catalytic reaction and in enzyme assembly. For example,
whereas site-directed mutagenesis was used to identify both the SeC and
His as essential enzyme residues, these data did little to extend
earlier work using more traditional approaches of chemical modification
(16, 18, 33-37). In fact, among the histidine residues present in p27,
D1 activity was completely lost by mutations of His-158 barring further
analysis of this essential residue (13). Examination of the primary
amino acid sequence of p27 reveals that this residue is located in the
middle of an ~15-residue long stretch found in all deiodinase family members from frog to man. With the ability to predetermine the composition of D1 holoenzyme, the influence of His-158 and His-174 on
catalysis and on enzyme assembly can now be directly evaluated.
The ability to drive a native, catalytically active p27 subunit into
partnership with an inert p27 mutant allowed us to evaluate the
catalytic reaction in a D1 holoenzyme in which only one partner is
functional. By using steady-state reaction kinetics, we found that the
hybrid D1 holoenzyme had little, if any, change in substrate affinities
but showed a 75% decrease in the molar activity compared with wild
type D1 dimer. Whether this decrease in activity reflects an
interaction between the inert subunit and the native p27 that lowers
the intrinsic catalytic potential of the native p27 or indicates that
each p27 subunit in the D1 holoenzyme can serve as a catalytic center
remains to be established.
Based on the degeneracy of the C terminus of p27, we generated an inert
p27GFP fusion protein that could be followed in the living
cell. Addition of this relatively large epitope did not affect the
ability of the fusion protein (M4GFP) to partner with the wild type p27
in LLC-PK1 cells, and the fluorescent D1 holoenzyme showed the same catalytic properties as that for D1 holoenzyme composed of M4 and
p27wt subunits. Assuming the same stoichiometry of
p27GFP fusion protein and p27wt as described in
Table I, then ~10% of the florescent D1 dimers were composed of an
inert and active subunit, whereas 90% of the dimers formed were
composed of two fluorescent and inert p27s. Analysis of the subcellular
distribution of fluorescent D1 dimers revealed that the D1 dimers were
sorted to the basolateral membranes of the oriented renal epithelial
cell (23, 30).
In conclusion, the data show that catalytically active D1 is a
homodimer of p27 subunits and that hybrid enzyme with only one active
subunit can catalyze deiodination by a ping-pong reaction mechanism.
Although the membrane anchor located in the N terminus of p27
constrains the assembly of the D1 subunits into a head-to-head and
tail-to-tail orientation, p27 dimerization does not require the
membrane anchor, and enzyme assembly is a post-translational event that
appears to be driven by the local concentrations of enzyme subunits.
Once assembled the D1 holoenzyme is sorted to the plasma membrane in
both kidney and liver cells, where it can serve as an enzymic barrier
to the entry of the prohormone T4 and the source of
bioactive T3 for the cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-35S-dATP
(3000 Ci/mmol) and [
32P]dCTP (800 Ci/mmol) were
purchased from PerkinElmer Life Sciences. 75SeO32
(~200 Ci/mmol)
was purchased from the University of Missouri-Columbia Research Reactor
Facility (Columbia, MO). L-(3'- or
5'-125)I]rT3 was prepared by radioiodination
of 3,3'-T2 using methods described previously (24). The
2.1-kilobase pair p27 cDNA (G21; GenBankTM
accession number X57999) was the gift of P. R. Larsen, Harvard Medical School. Synthetic oligonucleotides were prepared in house or
purchased from Life Technologies, Inc. All iodothyronines were of the
L-configuration and were purchased from Henning Berlin GmbH. Dulbecco's modified Eagle's medium, antibiotics, Hanks' buffered salt solution, glucose, trypsin, and G418 were obtained from
Life Technologies, Inc.; supplemented bovine calf serum was from
HyClone Laboratories (Boulder, CO); acrylamide and
N,N'-methylenebisacrylamide were from U. S. Biochemical
Corp.; ammonium persulfate and TEMED were from Bio-Rad; and
dithiothreitol was from Calbiochem.
Ser substitution (underlined nucleotides) using oligonucleotide-based mutagenesis according to the
manufacturer's instructions. The integrity of the catalytically
inactivated, non-SeC containing the M4 mutant of rat p27 was confirmed
by DNA cycle sequencing of the entire coding region (nucleotides
7-780), and M4 was subcloned into the KpnI-XhoI
site of the eukaryotic expression vector, pcDNA3. Construct
orientation was confirmed by analytical restriction digestion.
70 °C until use.
70 °C) at ~1 mg/ml IgG until use.
-counter. Shown in Fig. 1A is a
representative displacement curve comparing p27 peptide to detergent
extracts containing the rodent p27 (rat kidney) to that pig p27
(LLC-PK1 cells). The minimum detection limit of rodent p27 is ~3 fmol
per assay tube (81 pg of p27/tube), and the maximum binding capacity of
the rabbit anti-rat p27 antisera is 22 nmol of p27/ml antisera as
determined by Scatchard analysis of the binding data.
released per min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Characterization of the epitope-directed
anti-rat p27 antisera. A, competitive binding analysis of
p27 peptide, detergent-soluble rat kidney membranes, and
detergent-soluble LLC-PK1 cell membranes. Cell and membrane
preparations were solubilized with 0.1% Triton X-100 or 5 mM tDOC. Assays were done as described under
"Experimental Procedures." Data are reported as the means of
closely agreeing triplicate determinations. , p27 peptide;
, rat
Mx;
, LLC-PK1 Mx. B, immune precipitation of D1 activity
from solubilized rat kidney membranes (rat Mx) and
solubilized LLC-PK1 (PK1 Mx) membranes.
Microsomes were prepared from rat kidney and LLC-PK1 cells.
Catalytically active D1 was solubilized with 5 mM tDOC, and
2 mg of soluble Mx protein (150 units of D1 activity) was incubated in
triplicate with a 1:100 dilution of anti-rat p27 antisera in the
absence (filled bars) and presence (open bars) of
10 µg/ml blocking peptide at 4 °C for 60 min. D1 activity
remaining in the supernatant after removal of specific immune complexes
by protein A beads was determined as described under "Experimental
Procedures." Data are reported as means ± S.E. of three
independent microsome preparations. C, representative
photomicrographs of immunoreactive p27 in rat kidney tubules from
selenium-supplemented and selenium-deficient rats. PCT,
proximal convoluted tubule; G, glomerulus.
Bar = 50 µm.
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Fig. 2.
Potential combinations of p27 molecules in
LLC-PK1 cells constitutively expressing the Ser-126
mutant (M4) of rat p27.
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[in a new window]
Fig. 3.
Immunoprecipitation of D1 holoenzyme composed
of pig p27wt-M4 dimers by anti-rat p27 antisera.
A, immune precipitation of 75Se-labeled
proteins. LLC-PK1 cells that constitutively express the
neor gene (vector cells) or the M4 mutant of p27
(S126 cells) were grown in 75Se-labeling media for 48 h as described under "Experimental Procedures." Cell membranes were
prepared, and 25,000 cpm 75Se-labeled protein was incubated
with protein A beads, ± anti-rat p27 IgG in the absence (open
bars) and presence (filled bars) of excess blocking
peptide as described in the legend to Fig. 1B. B, immune
precipitation of D1 activity. The ability of anti-rat-p27 antisera to
immunoprecipitate tDOC-soluble D1 from vector and S126 cell
microsomes was done as described in the legend to Fig. 1B.
Data are reported as means ± S.E., n = 3 independent preparations.
97% of the starting D1 activity in the S126 cell was protected from
antibody depletion by the blocking peptide. These findings show that
overexpression of the catalytically inert M4 protein markedly reduced
the catalytic potential of the native D1 in the host LLC-PK1 cell and
yielded a "hybrid" D1 that was specifically precipitated by
anti-rat p27 antibodies. The data also suggest that the assembly of a
catalytically active D1 is a post-translational event.
View larger version (76K):
[in a new window]
Fig. 4.
Representative fluorograms of SDS-PAGE
analysis of anti-rat p27 immune precipitates from control LLC-PK1 cells
or M4-expressing S126 cells. Immune precipitates were prepared as
described under "Experimental Procedures," denatured by heating at
100 °C for 5 min, and separated on 12.5% SDS-PAGE cells under
reducing conditions as described previously (23). Proteins were
resolved by SDS-PAGE, and the distribution of 75Se-labeled
proteins was determined by PhosphorImager. Lane 1, LLC-PK1
cell lysate; lane 2, immune precipitate from LLC-PK1 cells;
lane 3, S126 cell lysate; lane 4, immune
precipitate from S126 cells.
Summary of the D1 activity, M4, and p27wt content in S126 cells
released/min. ND, not detected
(<0.001 pmol/mg protein). Data are reported as means ± S.E.,
n = 3.
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Fig. 5.
Migration of D1 holoenzyme composed of
p27wt-M4 in 5-20% linear sucrose gradients in
H2O. D1 holoenzyme was labeled with 75Se
and solubilized from S126 cells (solid line) and control
LLC-PK1 cells (dotted line) with 5 mM tDOC as
described under "Experimental Procedures." One mg of solubilized
protein was sedimented on individual gradients as described previously
(22, 28). Carbonic anhydrase (CA) and BSA were added
internal standard proteins and identified on stained SDS-PAGE gels on
individual fractions. 75Se-labeled protein associated with
M4 was immune precipitated with anti-rat p27 antisera as described
under "Experimental Procedures" and counted in a well-type
counter. Data are representative of 3 separate gradients.
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[in a new window]
Fig. 6.
Effects of the N-terminal membrane anchor of
p27 on assembly of catalytically active D1 holoenzyme. N-terminal
truncation mutants (M4-19 and M4-42) were prepared as described under
"Experimental Procedures" and transfected into LLC-PK1 cells, and
stable transfectants were selected with 800 µg/ml G418. Cell
membranes were prepared and immunoprecipitated with anti-rat p27
antisera (1:100 final dilution) as described in the legend to Fig.
1B. Data reported as means ± S.E. n = 3 independent membrane preparations. Also shown are the putative p27
domains remaining in each truncation mutant. A, membrane
anchor; C, catalytic domain.
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Fig. 7.
Initial velocity reaction kinetics for D1
activity in control LLC-PK1 cells carrying the empty pcDNA3 vector
(D1 wild type) and in S126 cells (M4). Steady-state reaction
kinetics were done as described previously (24, 29). The hybrid D1
holoenzyme was obtained from the S126 cells detailed in Table I, and
the enzyme concentration was assumed to be equal to that of the native
p27 (~0.2 pmol/mg cell protein). , 1.0;
, 1.67;
, 2.5;
,
5;
, 20 mM dithiothreitol (DTT). Intercept
replots:
, wild type D1;
, S126 D1. Data are representative of 3 separate studies. All lines were drawn by least squares linear
regression analysis.
Limiting kinetic constants for wild type D1 and the hybrid D1 in S126
cells
1 · mol
D1 holoenzyme
1. Data reported as means ± S.E.,
n = 3 for separate determinations. DTT, dithiothreitol.
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Fig. 8.
Representative photomicrographs of S126 cells
constitutively expressing M4GFP. Phase contrast images
of the M4GFP-expressing (left panel) and
companion fluorescence images of the same field (right
panel). Sizing bar = 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cellular and
Molecular Physiology, 55 Lake Ave. N., Worcester, MA 01655. Tel.:
508-856-6687; Fax: 508-856-4572; E-mail:
jack.leonard@UMASSMED.edu.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M006973200
2 J. L. Leonard, T. J. Visser, and D. M. Leonard, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: D1, type I iodothyronine 5'-deiodinase; T4, thyroxine; T3, 3,3',5'-triiodothyronine; rT3, reverse T3; tDOC, taurodeoxycholate; p27, 27-kDa substrate-binding subunit of type I iodothyronine 5'-deiodinase; M4, Ser-126 mutant of rat p27; M4GFP, green fluorescent protein-tagged M4; BrAcT4, N-bromoacetyl-L-thyroxine; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; SeC, selenocysteine; TEMED, N,N,N',N'-tetramethylethylenediamine; PBS, phosphate-buffered saline.
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