Expression of recombinant membrane-bound type I
iodothyronine deiodinase in yeast
George G J M Kuiper, Willem Klootwijk and Theo J Visser
Department of Internal Medicine, Room Ee 502, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands
(Requests for offprints should be addressed to T J Visser; Email: firstname.lastname@example.org)
The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone
thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD)
to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3
(rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very
difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression
system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed
a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1
Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with
dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be
detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in Km(2 µM) for rT3 as compared
with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as
compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125I]T3
affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and
32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity.
Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique
myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a
combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal
sequence and the membrane-spanning domain (amino acid residue 2–35), did not result in the production of a soluble
D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence
of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast
cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further
Journal of Molecular Endocrinology (2005) 34, 865–878
The type I iodothyronine deiodinase (D1) selenoprotein
is found in mammals predominantly in the microsomal
fractions of liver, kidney and thyroid gland (Bianco et al.
2002, Köhrle 2002, Kuiper et al. 2005). This enzyme is
responsible for a large part of the peripheral production
of 3,3?,5-triiodothyronine (T3) from thyroxine (T4) in
euthyroid animals (Doorn et al. 1983, Nguyen et al. 1998,
Bianco et al. 2002). Remarkably, D1 is capable of both
deiodination (IRD) of T4, and shows preference for
reverse T3 (rT3; 3,3?,5?-triiodothyronine) as the sub-
strate (Berry et al. 1991a, Moreno et al. 1994). D1 activity
in vitro is stimulated by thiol compounds such as
dithiothreitol (DTT) and is uncompetitively inhibited by
6-propyl-2-thiouracil (Visser 1980).
(ORD)and inner ring
N-bromoacetyl-[125I]T3 has proven to be a useful
affinity label of D1, allowing the specific labeling of the
enzyme in rat liver microsomal fractions (Schoenmakers
et al. 1992), despite the fact that it only constitutes about
0·01% of protein content (Mol et al. 1988). Progress in
hampered by the very low expression level. Due to the
low expression level only small amounts of purified D1
protein (approximately 50% pure) were obtained by
affinity chromatography from detergent-solublized mi-
crosomal fractions of rat liver (Mol et al. 1988).
Molecular sieve chromatography of the detergent
solubilized D1 from rat liver or kidney cells yielded a ?
50 kDa active enzyme preparation, suggesting that the
D1 protein is composed of a homodimer of 27 kDa
subunits (Mol et al. 1988, Köhrle et al. 1990, Leonard
et al. 2001). After the D1 cDNA cloning (Berry et al.
Journal of Molecular Endocrinology (2005) 34, 865–878
0952–5041/05/034–865 © 2005 Society for EndocrinologyPrinted in Great Britain
Online version via http://www.endocrinology-journals.org
1991a), site-directed mutagenesis and deletion analysis
have identified several functional regions of the D1
protein (Berry et al. 1991b, 1992, Toyoda et al. 1995a,b,
1997, Leonard et al. 2001, Kuiper et al. 2003). The rat
D1 protein contains an uncleaved endoplasmic reticu-
lum (ER) transfer sequence and transmembrane domain
(amino acid residue 2–35), while iodothyronine substrate
selectivity is influenced by the domain between amino
acid residues 40 and 70. The D1 core active center
consists of a region of about 20 amino acid residues long,
surrounding the single essential selenocysteine (SeC)
At the same time it should be realized that analyzing
the effects of individual mutations in impure D1
enzymes only reveals that the changed amino acid
residue directly or indirectly affects activity, but does not
necessarily deliver reliable information on the molecular
mechanism(s) involved. In order to obtain pure D1
protein for more detailed structure–function analysis it is
necessary to develop a heterologous (over)-expression
system. The fact that D1 is an integral membrane
protein limits the choice of expression systems to
eukaryotic cells, for instance yeast or insect cells, since
expression in bacteria of D1 protein results in the
production of inactive D1 that accumulates in inclusion
bodies (G G J M Kuiper, W Klootwijk & T J Visser,
unpublished observations). A heterologous expression
system that combines several advantages of both
prokaryotic and eukaryotic expression systems are yeasts
(Guengerich et al. 1991, Bill 2001, Griffith et al. 2003).
modifications, and contain identical secretory pathways
as higher eukaryotic cells. Yeast cells can be grown
inexpensively in large quantities and several well-
characterized inducible promoter systems that allow
regulated expression of specific genes have been
established. Several cytochrome P450 enzymes and
short-chain dehydrogenase/reductase enzymes, inserted
in membranes in much the same manner as deiodinases,
(Guengerich et al. 1991, Blum et al. 2001, Hult et al.
The D1 protein contains a single SeC residue in the
catalytic center, encoded by a UGA stop codon.
Successful insertion of SeC at UGA codons in
eukaryotes requires the presence of a specific stem-loop
structure in the 3?-untranslated region of the mRNA
(Berry et al. 1993). Although yeast cells are eukaryotic
cells they are unable to synthesize selenoproteins. In fact,
the yeast homologs of several higher eukaryotic proteins
(glutathione peroxidase, SelX) contains cysteine residues
instead of SeC residues (Lescure et al. 1999). In order to
overcome this potential problem, we have made D1
expression vectors in which the UGA stop codon
encoding SeC incorporation was replaced by UGC (Cys)
overexpressed in yeast
or UCA (Ser). In the present paper we describe the
heterologous expression and characterization of type I
deiodinase activity in Saccharomyces cerevisiae.
Materials and methods
Nonradioactive iodothyronines were obtained from
Henning Berlin R&D (Berlin, Germany) and [3?,5?-
125I]rT3 was prepared by radioiodination of 3,3?-
diiodothyronine (T2) as described (Visser et al. 1978).
[3?-125I]T3 (1500–2000 mCi/µmol) was obtained from
Amersham Biotech (Little Chalfont, Bucks, UK). Un-
labeled N-bromoacetyl-T3 (BrAcT3) and BrAc[125I]T3
were synthesized from N-bromoacetylchloride and T3 or
[3?-125I]T3 as described (Mol et al. 1984). HPLC analysis
demonstrated that the purity of BrAc[125I]T3 was at
Iodoacetate (IAc) and 6-propyl-2-thiouracil (PTU) were
obtained from Sigma (St Louis, MO, USA). Rat liver
microsomes were prepared as previously described
(Fekkes et al. 1979).
The yeast expression vector pYES6/CT and the
diploid Saccharomyces cerevisiae strain INVSc1 (genotype
his3n1; leu2; trp1–289; ura3–52) were obtained from
blasticidin S was also obtained from Invitrogen.
Peptone, bacto-yeast extract and the yeast nitrogen base
without amino acids were from DIFCO Laboratories
(Detroit, MI, USA). Yeast lytic enzyme (80 000 U/g)
was obtained from ICN Biochemicals (Costa Mesa,
CA, USA). Tunicamycin was obtained from Sigma
Chemicals. N-glycosidase F was obtained from New
England BioLabs (Beverly, MA, USA). Pfu thermostable
DNA-polymerase and DpnI restriction endonuclease
were obtained from Promega (Madison, WI, USA).
XL-10 competent Escherichia coli cells were obtained from
Stratagene (La Jolla, CA, USA).
The rat D1 antiserum 3049/JL (Leonard et al. 2001),
directed against the C-terminus (epitope YEEVRAVLE-
KLCIPPGHMPQF) was a kind gift of Drs Jack Leonard
and Alan Farwell (University of Massachusetts Medical
School, Worcester, MA, USA).
Construction of yeast expression vectors
In order to construct the D1 Cys (SeC126 Cys) and D1
Ser(SeC126 Ser) expression
mutagenesis was used to substitute the SeC (TGA
codon) of rat D1 cDNA with Ser (AGC) or Cys (TGC)
codons as described (Leonard et al. 2001). The
oligonucleotide primers G21A 5?-GCGGGATCCATGG
GGCTGTCCCAGCTA and G21B 5?-GCGGGATCC
CTAGAACTGAGGCATGTGTCC (BamHI sites in
italics and start/stop codons underlined) were used to
PCR amplify the D1 cDNA. The PCR products were
G G J M KUIPER and others· Recombinant D1 expression in yeast866
www.endocrinology-journals.org Journal of Molecular Endocrinology (2005) 34, 865–878
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Received 1 March 2005
Accepted 7 March 2005
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