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INTRODUCTION |
Selenium is an essential trace element in the diet of many
organisms, including humans. It is present in the form of a
selenocysteine (Sec)1 residue
in several naturally occurring enzymes and proteins (1, 2). In
selenoenzymes with established function, such as glutathione peroxidases, thyroid hormone deiodinases, and thioredoxin reductases in
mammals, and hydrogenases and formate dehydrogenases in bacteria and
archaea, Sec is present at the enzyme active center and participates in
various redox reactions (2).
Functions of many other mammalian selenoproteins, including the 15-kDa
selenoprotein (Sep15), selenoprotein P, selenoprotein W, selenoprotein
R (also named selenoprotein X), selenoprotein T, and selenoprotein N,
have not been established. However, most of these proteins have clearly
identifiable Sec-containing redox motifs, such as the Cys-Xaa-Xaa-Sec
motif in selenoprotein W and selenoprotein T, suggesting their possible
involvement in redox processes (3).
Sep15 was recently identified in human T-cells (4). The gene for
this protein is expressed in various human tissues with highest
expression levels in the prostate and thyroid. In addition to humans,
genes encoding Sep15 were detected in mice and rats. Sep15 exhibits no
homology to previously characterized proteins, which precluded its
functional characterization. However, Sep15 has a highly conserved
motif, Cys-Gly-Sec-Lys, suggesting that this center could constitute an
active center, in which Sec and Cys form a reversible seleno-sulfide
bond. Besides this putative redox center, a previously noted feature in
the Sep15 sequence was the lack of N-terminal sequences in the isolated
human T-cell selenoprotein, which suggested the possibility of
post-translational processing of the protein. In addition, Sep15
migrated as the 15-kDa protein on SDS-PAGE gels, whereas the migration
properties of the native protein were consistent with a protein of
~160-240 kDa. The low abundance of the 15-kDa selenoprotein in human
T-cells and its lability during isolation did not permit isolation of the native protein to homogeneity to test whether the 160-kDa complex
was composed of multiple selenoprotein subunits or if other protein
components were involved in the complex (4).
The finding that the protein was expressed in the prostate at
elevated levels compared with other tissues (4, 5) provided an
opportunity to determine the oligomeric composition of Sep15 by
isolating the selenoprotein from this organ. In this report, we
describe isolation of Sep15 from rat prostate and mouse liver. In both
preparations, the native selenoprotein occurred as a complex with
UDP-glucose:glycoprotein glucosyltransferase (UGTR), an enzyme involved
in the quality control of protein folding (6). Further characterization
revealed that Sep15 was located in perinuclear cellular compartments,
consistent with the finding that UGTR is located in the endoplasmic
reticulum (ER). The observation that Sep15 was found only in a complex
with UGTR suggests that it may be involved in the regulation of protein folding.
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EXPERIMENTAL PROCEDURES |
Materials--
Rat prostate and mouse liver were purchased from
Pel-Freez. [75Se]Selenious acid (specific activity 1,000 Ci/mmol) was from the University of Missouri Research Reactor Facility
(Columbia, MO). Phenyl-TSK and DEAE-TSK HPLC columns were from
TosoHaas. The phenyl-Superose FPLC column, Q-Sepharose, and
ConA-Sepharose were from Amersham Pharmacia Biotech. LipofectAMINE was
from Life Technologies, Inc. Polyclonal anti-green fluorescent protein
(GFP) antibodies were from Invitrogen. Escherichia coli
strain NovaBlue was from Novagen. The Maxi kit for plasmid isolation
was from Qiagen, BODIPY®TR ceramide was from Molecular Probes, native
and SDS-PAGE gels and immunoblot membranes were from Novex, and
immunoblot detection systems ECL-Plus and SuperSignal were from
Amersham Pharmacia Biotech and Pierce, respectively. Other reagents
were of the highest quality available.
75Se Labeling of the 15-kDa Protein--
Rat cell
line RLE was grown on RPMI 1640 medium for 48 h in the presence of
0.5 mCi of sodium [75Se]selenite/100 ml of cell culture
medium. Cells were washed four times with phosphate-buffered saline,
collected by trypsinization, and stored at
80 °C prior to use.
75Se-Labeled mouse liver was obtained as described (7).
Briefly, 0.5 mCi of freshly neutralized [75Se]selenious
acid was injected intraperitoneally, the mouse was sacrificed 48 h
later, and 75Se-labeled tissues were collected and stored
at
80 °C prior to use.
Isolation of Sep15 from Rat Prostate--
133 g of rat prostate
was mixed with 75Se-labeled rat RLE cells and homogenized
at 4 °C in 400 ml of 20 mM Tris-HCl, pH 7.5, containing
1 mM EDTA, 1 mM AEBSF, 1 mM DTT, 5 µg/ml leupeptin, and 5 µg/ml aprotinin, and the homogenate was
further treated by sonication. Insoluble material was removed by
centrifugation, and the supernatant was applied to a 200-ml Q-Sepharose
column equilibrated with 20 mM Tris-HCl, pH 7.5, containing
1 mM EDTA and 1 mM DTT (buffer A). After
washing the column with 2 volumes of buffer A, the bound proteins were
eluted by application of a linear gradient from buffer A to 1 M NaCl in buffer A. 75Se was determined with a
-counter, and column fractions containing peaks of radioactivity
were analyzed by SDS-PAGE followed by detection of 75Se on
gels with a PhosphorImager. Fractions containing the
75Se-labeled Sep15 were identified based on migration
properties of this protein on SDS-PAGE, combined, concentrated, made in
1 M NaCl, filtered, and applied to a phenyl-HPLC column
equilibrated in 1 M NaCl in buffer A. The protein was
eluted by application of a linear gradient from 1 M NaCl in
buffer A to buffer A, and the column was washed further with a short
gradient from buffer A to water, which eluted Sep15. Fractions
containing 75Se were analyzed by SDS-PAGE followed by the
PhosphorImager analysis as described above. Fractions containing Sep15
were then applied directly to a DEAE-HPLC column equilibrated in buffer
A. Sep15 was eluted with a gradient from buffer A to 0.5 M
NaCl in buffer A. Radioactive fractions were concentrated and stored at
80 °C until ready for use.
Gel Electrophoretic Analyses of the Rat Prostate 15-kDa
Selenoprotein--
Fractions containing Sep15 were analyzed on native
and SDS-PAGE gels. To determine if the ~160-kDa protein band, which
appeared on native gels and was labeled with 75Se,
contained Sep15 and UGTR, a native PAGE gel was briefly stained with
Coomassie Blue, the band of interest cut from the gel, minced, incubated overnight in SDS-PAGE sample buffer containing 10 mM DTT, and the liquid fraction analyzed by SDS-PAGE. The
150-kDa protein band, which appeared on an SDS-PAGE gel after staining with Ponceau S, was used for the subsequent sequence analysis.
Tryptic Peptide Analysis of the 150-kDa Protein Band--
A gel
slice containing a Ponceau S-stained 150-kDa protein band was sent to
Harvard Microchem (Boston) where the protein was digested with trypsin,
and the resulting peptides were separated by reverse phase
chromatography and their masses determined by electrospray mass
spectrometry. Four tryptic peptides were sequenced by Edman degradation
comprising a total of 88 amino acid residues.
Isolation of the 15-kDa Selenoprotein from Mouse
Liver--
Initial fractionations of mouse liver Sep15 and UGTR were
carried out as described for rat prostate. Subsequent isolations took
advantage of the fact that UGTR could be isolated efficiently by
affinity chromatography on a ConA-Sepharose resin. 150 g of mouse
liver was mixed with 3 g of 75Se-labeled mouse liver
and homogenized at 4 °C in 3 volumes of buffer A containing 0.5 mM sodium orthovanadate and protease inhibitors used in the
rat prostate fractionation. The homogenate was sonicated, clarified by
centrifugation, and the clear supernatant was applied to a Q-Sepharose
column equilibrated with buffer A. The bound proteins were eluted using
a linear 0-1.5 M gradient of NaCl in buffer A. Fractions
were analyzed for the presence of 75Se-labeled proteins as
described for rat prostate. The fractions containing labeled Sep15 were
combined, concentrated, and applied to a ConA-Sepharose column that was
equilibrated with 20 mM Tris-HCl, pH 7.4, containing 0.5 M NaCl (buffer B). The column was washed with buffer B, and
proteins were eluted by the application of a step gradient of 10, 50, 100, 200, and 300 mM methyl
-D-mannopyranoside in buffer B. Fractions containing
75Se-labeled Sep15 were pooled, concentrated, and applied
to a phenyl-HPLC column as described for rat prostate. The protein was
eluted in two overlapping peaks, the first peak containing both UGTR
and Sep15, and the second peak containing only UGTR. Fractions
containing Sep15 were pooled and subsequently applied onto a DEAE-HPLC
column as described above. Proteins eluted from the DEAE column were analyzed by native and SDS-PAGE and by immunoblot assays with antibodies specific for Sep15 or UGTR.
Constructs with GFP--
N-Sep15-C-GFP denotes the following: N
is the 28-residue N-terminal signal peptide of Sep15; Sep15 is the
15-kDa selenoprotein without its signal peptide and 4 C-terminal
residues; C is the C-terminal tetrapeptide of Sep15; and GFP is the
239-residue GFP. N-Sep15-C-GFP and Sep15-C-GFP constructs were made
using the pEGFP-N1 expression vector. Human Sep15 cDNA (U93C), in
which the Sec codon, TGA, was mutated to a cysteine codon, TGC, was
amplified with primers T7 and XhoI-15-1h
(5'-CCACTCGAGGCGTTCCAACTTTTCACT-3') and designated N-Sep15-C-GFP. U93C
amplified with primers Sal-15-1h (5'-CGCAACGTCGACATGTCTGCTTTTGGGGCAGAG-3') and XhoI-15-1h was designated Sep15-C-GFP. The resulting polymerase chain reaction products were
cloned into the XhoI site of pEGFP-N1. The GFP-Sep15-C
construct was made using the pEGFP-C3 expression vector. Human Sep15
mutant cDNA, U93C, was amplified with primers Sal-15-1h and T3 and
cloned into the XhoI/Bsp120I sites of pEGFP-C3.
The fragment encoding N-terminal sequences of the 15-kDa protein was
obtained by amplification of the U93C cDNA with primers
T7-NheI, 5'-CGATGCTAGCTAATACGACTCACTATAGGG-3', and
AgeI-15-1h, 5'-CACGACCGGTGCCTCCGATGAAAACTCTGCC-3'.
N-GFP-Sep15-C and N-GFP constructs were made by cloning this fragment
into the NheI/AgeI sites of GFP-Sep15-C and
pEGFP-N1, respectively. The N-GFP-Sep15 construct was obtained by
mutagenesis of N-GFP-Sep15-C with primers 15-1h-159stopF,
5'-CCTGAGTGAAAAGTAGGAACGCATATAAATCTTGC-3' and 15-1h-159stopR,
5'-GCAAGATTTATATGCGTTCCTACTTTTCACTCAGG-3'. All constructs were
transformed into the E. coli strain NovaBlue, and the
plasmids were isolated using a Maxi kit.
Immunoblot Analyses--
Immunoblot assays with rabbit
polyclonal antibodies raised against UGTR isolated from rat liver (8)
and against the keyhole limpet hemocyanin-conjugated synthetic peptide
corresponding to the C-terminal portion of Sep15 (4) were performed
using the ECL-Plus detection system. Rabbit polyclonal antibodies
specific for GFP were used for the detection of GFP-Sep15 fusion
proteins with an ECL system.
Cell Growth, Transfection, and Dual Fluorescence Imaging Confocal
Microscopy--
Growth of monkey CV-1 cells and transfection were
carried out as described (9). 5 µg of plasmid DNA and 30 µl of
LipofectAMINE were used for transfection of each 60-mm plate. CV-1
cells transfected with the appropriate constructs were incubated for
12 h in a CO2 incubator. We used a fluorescent
BODIPY®TR ceramide as a reference marker for perinuclear structures.
This reagent has been shown to be accumulated in the ER and Golgi and
has been used to study protein trafficking (10, 11). The transfected
cells were rinsed with serum-free Dulbecco's modified Eagle's medium
containing 10 mM HEPES (DMEM-HEPES) and then incubated for
25 min at room temperature in the same medium containing 2 µM BODIPY®TR ceramide. The cells were washed twice in
serum-free DMEM-HEPES and were used immediately for image collection.
Double-labeled images of live cells were collected with a water
immersion lens using a dual excitation/emission and dual channel mode
on a Bio-Rad MRC1024ES laser-scanning microscope.
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RESULTS |
Native Rat Prostate Sep15 Is Composed of Multiple
Polypeptides--
Sep15 was previously isolated from a human T-cell
line, but small amounts of isolated proteins and protein lability
precluded molecular characterization of native Sep15 (4). Subsequent studies found that the protein is expressed at higher levels in the
prostate (4, 5). In the present study, a procedure for isolation of
Sep15 from rat prostate was developed which included fractionation of
protein extracts on conventional Q-Sepharose and phenyl-HPLC columns
followed by an HPLC procedure on a DEAE column. To allow efficient
detection of Sep15 in chromatographic fractions by tracing
-radioactivity, rat prostate homogenates were mixed with the
75Se-labeled extracts obtained from a rat cell line, and
the combined extract was used for further isolation. Immunoblot and
PhosphorImager detection of Sep15 on gradient nondenaturing and
SDS-denaturing PAGE gels in fractions from the first two columns
revealed that the native protein migrated as a large, ~165-kDa,
species. However, these same fractions migrated as the 15-kDa species
on SDS-PAGE gels (data not shown). Although native gradient PAGE does
not accurately determine the molecular weight of a protein, an 11-fold difference in masses between denaturing and nondenaturing conditions suggested that the native Sep15 was bound to another protein or proteins in the rat prostate or was composed of multiple identical selenoprotein subunits. These data were also consistent with the previous electrophoretic and gel filtration experiments of partially purified human T-cell Sep15 (4).
Native Rat Prostate Sep15 Is Associated with the 150-kDa
Protein--
Electrophoresis of the protein preparation isolated by
the three purification steps described above is shown in Fig.
1. Native gel electrophoresis revealed a
Coomassie Blue-stained band of ~165 kDa (Fig. 1A,
lane 3). This band contained Sep15 as it was labeled with
75Se and was immunoreactive with anti-Sep15 antibodies
(data not shown). SDS-PAGE analysis of the 165-kDa band showed that it
consisted of a 150-kDa protein and Sep15 (Fig. 1B,
lane 2). It was noted that the 150-kDa protein was also
present in a selenoprotein-free form (Fig. 1A, lane
2) which eluted later than the 150-kDa protein-Sep15 complex from
a phenyl-HPLC column. In contrast, we were not able to detect Sep15
that was free of the 150-kDa protein in rat prostate fractions.

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Fig. 1.
Gel electrophoretic analysis of the
UGTR·Sep15 complex. Rat prostate extracts were
fractionated sequentially using Q-Sepharose, phenyl-HPLC, and DEAE-HPLC
columns. Isolation of Sep15 was followed by immunoblot assays. In
A, Coomassie Blue staining of a native gradient PAGE gel is
shown, and the lanes contain: 1, protein standards;
2, 150-kDa band (UGTR); and 3, 165-kDa band (the
UGTR·Sep15 complex). In B, Coomassie Blue staining of an
SDS-PAGE gel is shown, and the lanes contain: 1, protein
standards (6-180-kDa range); and 2, Sep15 preparation. The
material for lane 2 was obtained by excising a protein band
similar to that shown in panel A, lane 3, from
the gel, soaking it overnight in SDS sample buffer in the presence of
10 mM DTT, and analyzing the extracted proteins by
SDS-PAGE.
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The 150-kDa Protein Is UGTR--
The 150-kDa protein was digested
with trypsin, and the sequence of four peptides consisting of a total
of 88 amino acid residues was determined (Fig.
2). Two of these peptides were not
homologous to any known sequences, but the other two showed partial
sequence homology with Drosophila melanogaster UGTR (GenBank
accession number U20554) (12). During these initial studies, no
mammalian UGTR sequences were available in GenBank. However, all four
150-kDa protein peptides were identical with internal peptides of the rat liver UGTR sequence that was deduced from the corresponding cDNA sequence (kindly provided by Dr. A. Parodi). In addition, several mammalian UGTR sequences subsequently became available in
GenBank, including rat UGTR (accession number AF200359) (13) and two
human UGTRs (accession numbers AF227905 and AF227906) (14). Sequences
of internal peptides obtained from the 150-kDa protein were identical
with the internal sequences of the rat protein and were highly
homologous with the human sequences. This further confirmed that the
150-kDa protein is UGTR. The relative locations of the four peptide
sequences within rat liver UGTR are shown in Fig. 2B. UGTR
has been shown to be responsible for the quality control of protein
folding in yeast and vertebrates (6). This protein is located in the ER
and functions by reglucosylating misfolded proteins in the ER lumen,
thus allowing them to interact with calnexin/calreticulin chaperones
(15, 16).

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Fig. 2.
Internal peptide sequences of rat prostate
UGTR. In A, amino acid sequences of four peptides
obtained by tryptic digest of isolated rat liver UGTR are shown.
Residue numbers correspond to the rat liver UGTR sequence.
In B, the relative location of the four peptides
(filled boxes) within the putative UGTR from rat liver
(long, rectangular box) is shown. Other features
shown within UGTR are the N-terminal signal peptide (diagonal
pattern) and the C-terminal ER retention signal (horizontal
pattern).
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UGTR and Sep15 were also detected by Western analysis in fractions from
a phenyl-HPLC column (Fig. 3). UGTR
eluted in two overlapping peaks, the first of which contained Sep15
(Fig. 1A, lane 3; Fig. 3, lanes 1,
3, 5, and 7), whereas the second peak did not contain the selenoprotein (Fig. 1A, lane
2; Fig. 3, lanes 2, 4, 6, and
8). The proteins shown in Fig. 3 were separated by SDS-PAGE
in the presence (Fig. 3, lanes 1 and 2, and
5 and 6) and absence (Fig. 3, lanes
2-4, and 7 and 8) of a reducing agent DTT,
which did not influence mobility of UGTR and Sep15 on SDS-PAGE. This
suggests that the isolated UGTR·Sep15 complex did not contain a significant amount of interprotein disulfide bonds.

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Fig. 3.
Immunoblot analyses of rat prostate Sep15 and
UGTR. Fractions containing hydrophobic proteins that eluted from a
phenyl-HPLC column were examined for the presence of Sep15 and UGTR by
Western blotting. Two fractions in which Sep15 and UGTR signals peaked
are shown in the figure. The first eluted fraction is shown in
lanes 1, 3, 5, and 7; and
the second fraction is in lanes 2, 4,
6, and 8. Immunoblot analysis with antibodies
specific for Sep15 is shown in lanes 1-4 and with
antibodies specific for UGTR, in lanes 5-8. Lanes
1, 2, 5, and 6 correspond to
reducing SDS-PAGE (in the presence of 10 mM DTT), and
lanes 3, 4, 7, and 8 correspond to nonreducing SDS-PAGE.
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Association between the 15-kDa Protein and UGTR in Mouse
Liver--
To determine whether the association between Sep15 and UGTR
is a general phenomenon rather than a cell type-specific event, we
fractionated mouse liver extracts and tested elution patterns of these
two proteins (Figs. 4 and
5). Initially, 75Se-labeled
liver homogenates were fractionated on DEAE and phenyl columns, and
protein fractions were analyzed for the presence of UGTR by immunoblot
assays and for the presence of Sep15 by detection of 75Se
with a PhosphorImager. These assays revealed that UGTR and Sep15
coeluted from these columns (Fig. 4). We further attempted isolation of
UGTR to near homogeneity and tested whether isolated UGTR contained
Sep15. For this purpose, we utilized a ConA column, which is an
affinity column used previously in the purification of UGTR (8) as an
additional intermediate step. The apparently homogeneous preparation of
UGTR was then analyzed by immunoblot assays for the presence of Sep15.
Analyses of five subsequent fractions from the phenyl column in the
procedure that employed the ConA column are shown in Fig. 5. Detection
of proteins on native (Fig. 5A) and SDS-PAGE (Fig.
5B) gels with antibodies specific for rat liver UGTR, as
well as the use of antibodies specific for Sep15 on native (Fig.
5C) and SDS-PAGE (Fig. 5D) gels, suggested coelution of these proteins. Overall, these data suggested that mouse
liver preparations contained Sep15 bound to UGTR. However, further
studies are required to address the quantitative relationship between
the UGTR·Sep15 complex and the remaining cellular UGTR.

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Fig. 4.
Copurification of Sep15 and UGTR in mouse
liver fractions. Elution of Sep15 and UGTR from a DEAE-HPLC column
(A) and from a phenyl-Superose column (B) is
shown. Bars represent relative 75Se
radioactivity of the 15-kDa protein determined with a
PhosphorImager, and bands represent immunoblot detection of UGTR
with anti-rat liver UGTR antibodies in the corresponding fractions. The
phenyl-Superose column did not resolve UGTR and the UGTR·Sep15
complex.
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Fig. 5.
Immunoblot analyses of UGTR and Sep15 on
native and SDS-PAGE gels. Mouse liver extracts were fractionated
on Q-Sepharose, ConA-Sepharose, phenyl-HPLC, and DEAE-HPLC columns as
described under "Experimental Procedures." Sep15 was
detected during isolation by the PhosphorImager and immunoblot assays
and UGTR, by immunoblot assays. Late eluting fractions from a phenyl
column were electrophoresed, transblotted, and blots were probed with
antibodies specific for Sep15 or UGTR. A, analysis with
antibodies specific for UGTR on a nondenaturing (native) PAGE gel.
B, analysis with antibodies specific for UGTR on a SDS-PAGE
gel. C, analysis with antibodies specific for Sep15 on a
native PAGE gel. D, analysis with antibodies specific for
Sep15 on a SDS-PAGE gel. E, Coomassie Blue staining of the
native gel.
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Intracellular Localization of the 15-kDa Protein--
Because UGTR
exhibits specific intracellular localization (ER resident protein)
(17), we determined the cellular location of Sep15. For this study, we
employed a set of constructs that encoded various fusion proteins
between Sep15 and GFP (Fig. 6). These
constructs lacked a 3'-untranslated region of Sep15 gene which contains
the Sec insertion sequence element (5). All known eukaryotic
selenoprotein genes, including Sep15, contain Sec insertion sequence
elements, which are stem-loop structures located in 3'-untranslated
regions and are necessary to dictate Sec insertion and prevent
termination of protein synthesis at in-frame UGA codons (1, 18).
Accordingly, to express a full-size polypeptide in the absence of the
Sec insertion sequence element, TGA that encodes Sec at position 93 in
Sep15 was replaced in these constructs with TGC that encodes cysteine.
These mutations were made to increase translation efficiency because
efficiency of selenoprotein synthesis from transfected constructs in
mammalian cells is low (19). The constructs were transiently
transfected into monkey CV-1 cells, and confocal microscopy was used to
localize fusion proteins by detecting the GFP green fluorescence (Fig. 7). To determine the location of the
transiently expressed proteins, we used a cell-permeable fluorescent
ceramide conjugate, which is known to label the ER and Golgi (11, 20).
In addition, expression of fusion proteins and their experimental
molecular masses was obtained by assaying transfected cellular extracts by immunoblot assays with anti-GFP antibodies (Fig.
8). Sep15 was located in the membranous
reticular structures in the perinuclear region, when it was transiently
expressed in the form fused through its C-terminal region to GFP (Fig.
7).

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Fig. 6.
Schematic representation of
selenoprotein-GFP fusion constructs. Sep15-GFP fusion constructs
were developed to determine the intracellular localization of the
15-kDa protein. Construct, Fusion protein, and
Localization in the figure show (i) the specific construct,
(ii) the organization of the fused protein, where N
designates the 28-residue N-terminal signal peptide of Sep15;
Sep15, the 15-kDa selenoprotein without its signal peptide
and its four C-terminal residues; C, the 4 C-terminal
residues of Sep15; and GFP, a 239-residue GFP; and (iii)
polypeptides localized in the ER, respectively. For details, see
"Results."
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Fig. 7.
Confocal microscopy. Confocal
images of CV-1 cells expressing various GFP-tagged Sep15 and control
proteins are shown. A set of three images is shown for each construct.
Left panels show green fluorescence corresponding to
transiently expressed fusion proteins. Center panels show
fluorescence of the ER/Golgi marker. Right panels show
images obtained by merging left and center
panels. The scale bar is 100 µm. The GFP fusion
constructs used in this experiment are shown on the
left.
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Fig. 8.
Immunoblot detection of the GFP-Sep15 fusion
proteins. CV-1 cells were transiently transfected with plasmids
encoding GFP (lane 1), N-Sep15-C-GFP (lanes 2 and
4; two independent transfection experiments are shown),
Sep15-C-GFP (lane 3), GFP-Sep15-C (lane 5),
N-GFP-Sep15-C (lane 6), N-GFP-Sep15 (lane 7), and
N-GFP (lane 8) as described under "Experimental
Procedures." Samples were probed with antibodies specific
for GFP.
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The N-terminal Signal Peptide of Sep15 Is Necessary for ER
Localization--
Known ER resident proteins contain N-terminal signal
peptides that are required for translocation of proteins into the ER. The signal peptide may be subsequently cleaved from translocated proteins. As expected, Sep15 contained a highly hydrophobic N-terminal signal peptide. We observed previously that Sep15 isolated from a human
T-cell line lacked the N-terminal sequence (4), which is consistent
with it being an ER luminal protein.
To test a possible role of the N-terminal peptide, we developed a
construct that encoded Sep15 located downstream of GFP (the GFP-Sep15-C
construct). This fusion protein, transiently expressed in CV-1 cells,
was distributed in a non-ER-specific manner, including localization to
the cytosol (Fig. 7). Similar patterns were also observed in cells
expressing a fusion protein lacking the N-terminal peptide
(Sep15-C-GFP) (Fig. 7). Thus, removal of N-terminal sequences from the
selenoprotein resulted in a failure to direct Sep15 to the ER/Golgi
domain, suggesting an essential role of the N-terminal peptide in
selenoprotein translocation.
Sep15 Lacks an ER Retention Signal--
ER resident proteins
generally contain a common C-terminal tetrapeptide, KDEL, or very
similar analogs. This tetrapeptide is necessary to prevent ER resident
proteins from exiting the ER cellular compartment. However, the Sep15
sequence lacked such a signal and instead terminated with LERI. To
determine if the last four residues of Sep15 constitute a novel ER
retention signal or if this selenoprotein is retained in the ER by
another mechanism, we developed a construct in which the GFP gene was
inserted between the N-terminal signal peptide and the rest of Sep15
(the N-GFP-Sep15-C construct). Such a design allowed the GFP-Sep15
fusion protein to terminate on the natural C-terminal sequence of Sep15
while containing the N-terminal signal peptide for ER translocation. In
addition, a construct was made which differed from the N-GFP-Sep15-C construct in that it lacked the last four residues (N-GFP-Sep15; Fig.
6). Upon transient expression in CV-1 cells, no differences were
observed between fusion proteins with and without the C-terminal peptide, and both proteins were found residing in the ER/Golgi structures (Fig. 7). Thus, the C-terminal sequence was not important for intracellular trafficking and retention of Sep15.
To test further if the N-terminal signal peptide of Sep15 was alone
responsible for ER localization of the selenoprotein, we expressed a
GFP form containing N-terminal signal peptide of Sep15 (N-GFP). This
protein was detected in the ER and other cellular compartments. Thus,
the N-terminal signal was not sufficient for exclusive ER localization
of the protein. These data suggested that the internal (without the
N-terminal peptide and the C-terminal tetrapeptide) selenoprotein
sequence was responsible for retention of the protein in the ER. It is
likely that the ER-translocated Sep15 is kept in this cellular
compartment through its tight interaction with UGTR, which does have
the C-terminal ER retention signal and the N-terminal signal peptide.
To test whether the N-terminal single peptide was cleaved from the
fusion proteins upon translocation into the ER, we calculated molecular
masses for seven fusion products and for their predicted forms
obtained by cleavage of the N-terminal signal peptide (Table I). These values were compared with
experimental masses obtained from immunoblot assays (Fig. 8). This
experiment revealed that the signal peptide of Sep15 was cleaved in
every case in which it was present as an N-terminal sequence in a
fusion protein, i.e. upstream of either Sep15 or GFP.
Indeed, immunoblot assays indicated similar mobility of N-Sep15-GFP-C
(Fig. 8, lanes 2 and 4) and Sep15-GFP-C fusion
proteins (Fig. 8, lane 3) on SDS-PAGE gels, as well as
similar mobility of GFP (Fig. 8, lane 1) and N-GFP (Fig. 8,
lane 8). It should be noted that the proteins being compared
should have differed by 2.7 kDa if the N-terminal signal peptide was
retained. The difference of 2.7 kDa should be sufficient to be resolved
by our SDS-PAGE analysis because a difference of 1.8 kDa was clearly
seen when Sep15-C-GFP and GFP-Sep15-C fusion proteins were compared
(see Fig. 8, lanes 3 and 5, respectively).
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Table I
Characteristics of Sep15-GFP constructs
The design of the constructs is described under "Experimental
Procedures."
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DISCUSSION |
In this report, we described the association between Sep15 and
UGTR in the ER of mammalian cells. The data show that Sep15 is tightly
bound to UGTR, which suggests that this selenoprotein may be linked to
the quality control of protein folding.
Sep15 had previously been isolated only from a human T-cell line and
only under denaturing conditions (4). Thus, a possibility remained that
Sep15 was a component of a multiprotein complex or a homomultimer.
Isolation of Sep15 from mammalian tissues and cell lines was proven to
be difficult because of its extreme lability and low abundance.
However, taking advantage of the finding that Sep15 exhibits high
expression levels in prostate (4), we isolated the protein from rat
prostate. A procedure for isolation of Sep15 was developed which
combined conventional chromatography and HPLC. This procedure allowed
rapid isolation of the protein and minimized losses through denaturation.
The isolated selenoprotein was found to occur in a complex with a
150-kDa protein. Comparison of sequenced peptides from the 150-kDa
protein with the deduced sequence of rat liver UGTR revealed 100%
identity in four peptide sequences. Western blot analyses of the
150-kDa protein with the anti-UGTR antibodies, as well as analyses of
fractionated mouse liver extracts, further supported the conclusion
that Sep15 was purified in a complex with UGTR. This finding was
unexpected for the following reasons. (i) UGTR is known for its role in
the quality control of protein folding (6). This enzyme recognizes
misfolded protein domains in the ER lumen of eukaryotic cells and
specifically glucosylates these proteins, which retains misfolded
proteins in the ER for the next cycle of folding by the
calnexin/calreticulin glycoprotein folding system (15, 16, 21).
Previously characterized eukaryotic selenoproteins were involved in
redox processes (2), and redox function has been anticipated for Sep15,
but the quality control of protein folding has not been linked to a
redox process. (ii) UGTR is located in the ER (17), and no
selenoprotein has yet been found to occur in this cellular compartment.
In addition to Sep15, two other known mammalian selenoproteins,
glutathione peroxidase 3 and selenoprotein P, contain N-terminal signal
peptides. These proteins are secreted and are the major selenoproteins
in the plasma of mammals (22). Most other known mammalian
selenoproteins are cytosolic, nuclear, or mitochondrial proteins.
To determine the intracellular localization of Sep15, we made a series
of constructs that encoded fusion proteins between Sep15 and GFP. In
addition, we tested the relevance of the N-terminal portion of Sep15
for the ER translocation and of the C-terminal portion of the protein
for ER retention. Expression patterns of the GFP fusion constructs
containing Sep15 sequences in CV-1 cells were examined and compared
with location of a marker by live-cell imaging confocal microscopy.
Although the marker that was used in the present study is known to
label both ER and Golgi, the fact that UGTR is the ER resident protein
strongly suggests that Sep15 colocalized with UGTR in the ER. The data
from imaging analyses in combination with other biochemical evidence
demonstrated that the N-terminal signal peptide of Sep15 was necessary
for ER localization. In contrast, the C-terminal tetrapeptide of the
selenoprotein lacked a typical ER retention signal, and this sequence
was not necessary to keep the protein in the ER. It appears that the
selenoprotein sequence itself was responsible for retaining Sep15 in
the ER and preventing its secretion. The data thus suggested that Sep15 was maintained in the ER because of its interaction with UGTR.
The binding between Sep15 and UGTR appeared to be very strong because
these proteins copurified at each isolation step. Moreover, Sep15 was
found exclusively in the UGTR-bound form. The lack of the UGTR-free
selenoprotein may also be consistent with the idea that Sep15 and UGTR
are subunits of a two-subunit protein. It is possible that the presence
of the selenoprotein subunit in UGTR preparations was unnoticed
previously because of the small size of Sep15, which made it difficult
to visualize the selenoprotein by protein staining on SDS-PAGE gels. In
addition, low percentage SDS-PAGE gels have been used previously for
homogeneity assessment of isolated UGTR (8). In these gels,
selenoprotein would migrate in the dye front.
In contrast to the exclusive binding of Sep15 to UGTR, the latter
protein was detected in both selenoprotein-bound and selenoprotein-free forms. It remains to be determined if the selenoprotein-free form arose
by the release of the denatured selenoprotein during protein isolation,
if it was a natural UGTR form or if UGTR also occurred in a complex
with other proteins and/or selenoproteins.
UGTR was shown previously, by immunoprecipitation, to associate with
misfolded proteins, such as
1-antitrypsin (23), and with
other ER resident proteins, such as protein disulfide isomerases, carboxylesterase, and the glucose-regulated protein (24). However, these proteins do not copurify with UGTR, and only a small fraction of
them was associated with this enzyme. Sep15, on the other hand, was
found exclusively in the UGTR-bound form in rat prostate and mouse liver.
UGTR is the only known quality control protein that recognizes
misfolded proteins in the ER, and its mechanism has been characterized in great detail. Interestingly, UGTR is able to glucosylate misfolded domains specifically while not reacting with properly folded domains within a protein composed of identical folded and misfolded domains (16).
The possible role of redox processes in the ER-based protein folding
has received much attention recently. In particular, protein disulfide
isomerase was found to remove electrons, through the disulfide bond
formation, from folding proteins and to transfer reducing equivalents
further to the ER membrane protein Ero1 (25, 26). The formation of
disulfide bonds in nascent polypeptides is believed to be associated
with folding by the calnexin/calreticulin chaperones. Although properly
folded proteins may proceed further to secretory pathways, misfolded
polypeptides including those containing disulfide bonds are
glucosylated by UGTR to retain them for the next cycle of folding.
Sensing or reduction of disulfides within misfolded proteins prior to
folding appears to be required. Whether Sep15 is involved in such redox
reactions is a direction for further research.