Molecular Biology of the Cell
Vol. 14, 4114–4125, October 2003
The Retrieval Function of the KDEL Receptor
Requires PKA Phosphorylation of Its C-Terminus
Margarita Cabrera,* Manuel Mun ˜iz,* Josefina Hidalgo, Lucia Vega,
Marı ´a Esther Martı ´n, and Angel Velasco†
*Department of Cell Biology, Faculty of Biology, University of Seville, 41012 Seville, Spain
Submitted April 2, 2003; Revised May 26, 2003; Accepted June 9, 2003
Monitoring Editor: Benjamin Glick
The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval
of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by
COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has
been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this
truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and
GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas
the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-
binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic
acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into
the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant
protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of
the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We
conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the
Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.
In recent years, different retrograde transport routes have
been described to be operative in the early secretory path-
way. Together, these fulfills several important functions
such as the retrieval of escaped endoplasmic reticulum (ER)
proteins (Pelham, 1988; Dean and Pelham, 1990), retention of
misfolded proteins (Hammond and Helenius, 1994; Vashist
et al., 2001), recycling of Golgi glycosyltransferases (Storrie et
al., 1998), the internalization of bacterial and plant toxins
(Lord and Roberts, 1998), and the disassembly of the Golgi
complex during mitosis (Zaal et al., 1999). Among these, the
recycling of ER residents has been particularly well studied.
During normal anterograde flow a certain number of endog-
enous ER proteins continuously leave the organelle and
reach downstream compartments in the secretory pathway
where they are recognized and returned back to their orig-
inal location (Pelham, 1991). Soluble ER proteins such as
chaperones and components of the control quality machin-
ery contain a C-terminal KDEL (HDEL in yeast) sequence
that is responsible for their recognition and retrieval from
post-ER compartments (Munro and Pelham, 1987; Pelham et
al., 1988). The evolutionary extent of this pathway is illus-
trated by the fact that some bacterial toxins such as cholera
toxin and Pseudomonas exotoxin A also contain a C-terminal
KDEL sequence that allows them to reach the ER by retro-
grade transport after their uptake by endocytosis (Majoul et
al., 1996; Jackson et al., 1999). Throughout their association
with molecular chaperones containing the KDEL signal mis-
folded proteins are also efficiently recovered from post-ER
compartments and retained in the ER (Yamamoto et al.,
2001). Many ER transmembrane proteins, on the other hand,
contain a dilysine (KKXX) motif at their C-terminus cyto-
plasmic tail. This is also a retrieval signal that allows recog-
nition and subsequent retrograde transport (Nilsson et al.,
1989; Jackson et al., 1990, 1993).
In addition to KDEL and KKXX sorting signals displayed
by ER residents, retrieval of these proteins depends on re-
ceptors that recognize the appropriate signals. ERD2, the
KDEL receptor, is an integral membrane protein located at
the Golgi complex and the ER-Golgi intermediate compart-
ment (Lewis and Pelham, 1990; Semenza et al., 1990; Griffiths
et al., 1994). At these locations the receptor specifically binds
KDEL-bearing proteins with high affinity and mediates their
uptake into transport intermediates (Lewis and Pelham,
1992). These ferry the ligand-receptor complexes to the ER
where dissociation occurs. Ligands are thus released within
the ER and the receptor is recycled back to the Golgi for
further rounds of transport. pH differences between the ER
and the Golgi have been proposed to account for the differ-
ent affinities exhibited by the receptor toward ligands at
both locations (Wilson et al., 1993).
COPI-coated transport intermediates, either in the form of
round vesicles or as tubular processes, mediate retrograde
traffic followed by both the KDEL receptor-ligand com-
plexes and membrane proteins containing a dilysine re-
trieval motif (Cosson and Letourneur, 1994; Letourneur et
al., 1994; Orci et al., 1997; Presley et al., 1998). Formation of
these carriers depends on a highly conserved transport ma-
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E03–04–0194. Article and publication date are at www.
†Corresponding author. E-mail address: firstname.lastname@example.org.
* These authors contributed equally to this work.
4114 © 2003 by The American Society for Cell Biology
chinery (Wieland and Harter, 1999). An essential component
of this machinery is coatomer, a heptameric protein complex
that is recruited from cytosol to the membrane before bud-
ding. Coatomer recruitment, in turn, requires previous as-
sociation of ARF1, a ras-like GTPase that in its GTP-bound
form initiates COPI coat assembly (Barlowe, 2000; Donald-
son and Lippincott-Schwartz, 2000). Thus, ARF1-GTP binds
to the Golgi/intermediate compartment membranes and re-
cruits coatomer. ARF1 activation consists in the exchange of
GDP for GTP catalyzed by an ARF1-specific guanine nucle-
otide exchange factor (GEF; Jackson and Casanova, 2000). By
contrast, hydrolysis of GTP by ARF1 gives rise to its deac-
tivation. This reaction is regulated by a Golgi-associated
GTPase-activating protein (ARF-GAP; Cukierman et al.,
1995), and recent studies indicate that this activity is also
required for cargo sorting and budding (Lanoix et al., 2001;
Yang et al., 2002). Additional constituents of the COPI-
coated transport intermediates are the p24 proteins, which
are type I transmembrane proteins that have been proposed
to function in both cargo selection and coat recruitment
Although the KDEL recycling pathway is well established,
several important questions remain unanswered. In partic-
ular, how the occupied KDEL receptor is sorted into COPI-
coated transport intermediates is largely unknown. Recent
studies indicate that upon ligand binding the receptor oli-
gomerizes and interacts with components of the transport
machinery such as ARF-GAP and ARF1 (Aoe et al., 1997,
1998; Majoul et al., 2001). This most likely contributes to the
formation at the donor membrane of prebudding complexes
that should facilitate evagination. However, it does not ex-
plain the mechanism that allows the occupied receptor to be
sorted, whereas the unoccupied receptor would be ex-
cluded. In principle, sorting would take place throughout
the interaction of COPI coat proteins with the cytoplasmic
domains of the KDEL receptor (Bremser et al., 1999; Wieland
and Harter, 1999). The latter should bear some kind of ER
retrieval motif for that. However, no such a signal has been
characterized for the KDEL receptor so far, and therefore the
mechanism of sorting of this protein remains unresolved. In
this study, we have analyzed the functional role played by
the C-terminal cytoplasmic domain of the KDEL receptor.
The results indicate that this protein region is phosphory-
lated by cAMP-dependent protein kinase A (PKA). This
phosphorylation event allows the interaction of the KDEL
receptor with ARF-GAP and coatomer proteins, which in
turn determines both the Golgi-ER retrograde transport fol-
lowed by the receptor and the retrieval of ligands containing
the KDEL signal.
MATERIALS AND METHODS
Expression in bacteria and purification of His-tagged recombinant proteins
(murine RII? and YFP-Sar1dn) was performed as described (Martı ´n et al.,
1999). Tissue culture media and antibiotics were from Life Technologies
(Paisley, Scotland, United Kingdom) and restriction endonucleases from
Roche Diagnostics (Mannheim, Germany). C? and H89 were purchased from
Calbiochem (La Jolla, CA). Protein G-Sepharose, ATP, GTP, DTT, and pro-
tease inhibitors were from Sigma-Aldrich (St. Louis, MO). Streptolysin O
(SLO) was purchased from Dr. H. G. Meyer (University of Mainz, Germany).
The following antibodies were provided by other investigators: F6.26.1 mouse
mAb against hen lysozyme (M.M. Riottot, Institut Pasteur, Paris, France;
Goldbaum et al., 1999), 12G5 mouse mAb against CXCR4 (Dr. A. Caruz,
University of Jaen, Spain; Amara et al., 1997), and rabbit polyclonal against
GMAP-210 (Dr. R. Rios, University of Seville, Spain; Infante et al., 1999).
Mouse monoclonal against the C-terminal domain of the native bovine KDEL
receptor was from Stressgen Biotechnologies (Victoria, BC, Canada), 9E10
mouse monoclonal against c-myc from Roche Diagnostics, goat polyclonal to
ARF-GAP1 from Abcam (Cambridgeshire, United Kingdom), FITC- and
HRP-conjugated secondary antibodies from Biosource (Camarillo, CA), and
Texas Red–labeled secondary antibodies from Molecular Probes (Eugene,
OR). Rabbit polyclonals against coatomer proteins were purchased from Dr.
F. Wieland (BZH, Heidelberg, Germany).
DNA Construction and Production of Recombinant
Plasmid HE24M coding a version of the human KDEL receptor containing a
c-myc epitope inserted between the last transmembrane domain and the
C-terminal cytoplasmic domain was generated by site-directed mutagenesis
according to the overlapping extension technique (Ho et al., 1989). An EcoRV-
BamHI fragment of the coding sequence present in plasmid HE24 (provided
by Dr. H.R.B. Pelham, MRC, UK; Lewis and Pelham, 1990) was subcloned into
pBluescript II SK (Stratagene, La Jolla, CA) and used as template. Single
amino acid changes and deletion of the C-terminal domain were also carried
out by this method. For expression, modified sequences were returned to the
original vector by replacement. Simultaneous expression of both lysozyme-
KDEL and c-myc–tagged versions of the KDEL receptor was achieved by
inserting a XhoI-NcoI fragment of HE24M into HYKE4 plasmid (provided by
Dr. L.M. Roberts, Warwick University, UK; Jackson et al., 1999). To generate
KDEL receptor fluorescent constructs, variants of the green fluorescent pro-
tein, namely enhanced cyan fluorescent protein (CFP) and enhanced yellow
protein (YFP), were fused to the C-terminus of the receptor. Sequences coding
different KDEL receptor variants were subcloned into pECFPN1 and pEY-
FPN1 vectors (Clontech, BD Biosciences Clontech, Palo Alto, CA). The over-
lapping extension procedure was also used to replace the C-terminal domain
of CXCR4 with that of the native KDEL receptor. Templates in this case were
an expression plasmid coding wild-type CXCR4 (provided by Dr. A. Caruz;
Amara et al., 1997) and the above mentioned pBluescript vector containing a
C terminal fragment of the KDEL receptor. Oligonucleotides coding amino
acids 307–311 of CXCR4 and amino acids 200–205 of the KDEL receptor were
used as primers. The resulting chimera was subcloned into the original
CXCR4 expression vector. All constructs were verified by DNA sequencing.
To generate recombinant baculoviruses, EcoRI and XhoI restriction sites were
inserted at the N and C ends, respectively, of the coding sequence of HE24M.
This fragment was subcloned into pFastBacI (Life Technologies). Baculovi-
ruses were obtained in bacmid-transfected Sf9 insect cells according to the
instructions provided by the manufacturer. The vector pEYFPC2-Sar1pdn
coding a YFP-tagged version of the dominant-negative mutant form of Sar1
(Sar1[H79G], Sar1dn) was provided by Dr. R. Pepperkok (EMBL, Heidelberg,
Germany). For expression in bacteria a NcoI-BamHI fragment was subloned
into pRSETB (Invitrogen, Carlsbad, CA).
Membrane and Cytosol Preparations
Cytosol was prepared from either bovine brain or rat liver as described
(Hidalgo et al., 1995; Martı ´n et al., 2000). Total microsomes were prepared
from Sf9 insect cells infected with recombinant baculoviruses. Cells (5–7 ?
108) were harvested 50–60 h postinfection by centrifugation at 400 ? g for 10
min. They were rinsed with cold PBS and resuspended in 12–15 ml of 0.25 M
sucrose in 25 mM HEPES, pH 7.2, 5 mM MgCl2containing protease inhibitors
(1 mM PMSF, 5 mM benzamidine, 100 ?g/ml soybean trypsin inhibitor, 20
?g/ml aprotinin, and 10 ?g/ml each pepstatin A, leupeptin, antipain). Ho-
mogenization was performed in a ball-bearing homogenizer. The homogenate
was centrifuged at 12,000 ? g for 10 min at 4°C to remove nuclei, mitochon-
dria, and unbroken cells. The supernatant was centrifuged at 100,000 ? g for
1 h at 4°C. Membranes were incubated on ice with 3 M KCl for 30 min. They
were recovered by centrifugation as above on a 2 M sucrose cushion. Micro-
somes were resuspended in 25 mM HEPES-KOH, pH 7.2, 25 mM KCl, and 2.5
mM MgCl2at 5–9 mg/ml protein concentration, snap-frozen in liquid nitro-
gen, and stored at ?80°C.
Mammalian Cell Culture, Transient Transfection,
Microinjection, and Immunofluorescence
Vero and COS-7 cells were grown in MEM and DMEM, respectively, and
supplemented with 10% (vol/vol) FCS, 2 mM l-glutamine, 50 U/ml penicil-
lin, and 50 ?g/ml streptomycin. Cells were transfected by electroporation.
Briefly, 1–2 ? 106cells were resuspended in 0.2 ml of hypoosmolar electro-
poration buffer (Eppendorf, Hamburg, Germany) containing 12 ?g of pure
plasmid DNA and 14 ?g sperm DNA. The cell suspension was transferred
into a 4-mm gap sterile cuvette. Electroporation was carried out in Multipo-
rator (Eppendorf) at 600 v, ?: 100 ?s. Cells were diluted in complete culture
medium containing 15 mM HEPES and recovered by centrifugation at 400 ?
g for 10 min. They were used 24–48 h posttransfection. Cells grown on glass
coverslips for 1 d were microinjected into the cytoplasm with 2 mg/ml
YFP-tagged Sar1dnusing an Automated Microinjection System (Eppendorf).
For indirect immunofluorescence, cells were fixed for 5 min in cold methanol
or, alternatively, for 20 min in 3% (wt/vol) paraformaldehyde in PBS. They
were rinsed several times with plain PBS and PBS/0.5%(wt/vol) BSA/
PKA Phosphorylation of the KDEL Receptor
Vol. 14, October 2003 4115
0.05%(wt/vol) saponin. Incubation with antibodies diluted in PBS/BSA/
saponin was performed at 37°C for 30 min. Cells were rinsed with PBS and
mounted with Fluoromont G (Southern Biotechnology, Birmingham, AL).
Golgi-ER Redistribution Assay
Cells cultured on glass coverslips were rinsed with ice-cold buffer (20 mM
HEPES, pH 7.2, 2 mM magnesium acetate, 90 mM potassium acetate, 1 mM
DTT). They were incubated on ice for 20 min with 1 ?g/ml SLO. Coverslips
were rinsed thoroughly with cold buffer and then incubated at 37°C with 0.3
ml buffer containing 1 mM of both ATP and GTP in a 17-mm well of a 24-well
After transfection, 0.5 ? 106cells were plated on each 35-mm well of a six-well
dish. One day after, the cells were depleted by incubation with methionine-
and cysteine-free medium for 30 min and then radiolabeled at 37°C for 10 min
with 1 ml of the same medium containing 25 mCi Tran35S-label (1000 Ci/
mmol). They were rinsed with ice-cold PBS and chased in 0.5 ml of complete
medium containing 1.5 mg/ml both unlabeled methionine and cysteine. At
each time point, the medium was collected and the cells were washed with
cold PBS and lysed with 0.4 ml lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1 mM EDTA, 1% [vol/vol] Triton X-100) containing protease inhibitors.
Samples were cleared by centrifugation at 12,000 ? g for 20 min at 4°C, and
supernatants were transferred to new tubes. Media samples were mixed with
0.5 ml of 2? lysis buffer. Incubation with antibody against hen lysozyme was
carried out overnight at 4°C followed by 1-h treatment with protein G-
Sepharose. Immunoprecipitates were rinsed with the above buffer supple-
mented with 1% (wt/vol) sodium deoxycholate and 0.1% (wt/vol) SDS (RIPA
buffer) and 10 mM Tris-HCl, pH 7.8, before electrophoresis.
Synthetic peptides were coupled to thiopropyl Sepharose 4B (Amersham,
Piscataway, NJ) according to the manufacturer’s instructions. Coupled pep-
tides (2–3 nmol) were incubated at room temperature for 5 min with 200 ?g
bovine brain cytosol in 0.5 ml coupling buffer (50 mM Tris-HCl, pH 7.3, 0.1–1
M NaCl). Beads were rinsed several times with coupling buffer before pro-
cessing for electrophoresis.
High salt–washed microsomal membranes (35 ?g) were incubated at 30°C for
10 min with 0.1 ?Ci [?-32P]ATP (3000 Ci/mmol) and 1 U PKA catalytic
subunit (C?) in phosphorylation buffer (20 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 1 mM DTT). Alternatively, 100 ?g crude rat liver cytosol and phos-
phatase inhibitors (10 mM sodium pyrophosphate, 20 mM NaF) were added
to the incubation medium instead of C?. Final volume was, in all cases, 50 ?l.
The reaction was stopped by transferring the tubes to ice and adding 1 ml of
ice-cold phosphorylation-arresting buffer (20 mM Tris-HCl, pH 7.5, 100 mM
ATP, 100 mM EDTA). Membranes were pelleted at 12,000 ? g for 20 min at
4°C, rinsed with phosphorylation-arresting buffer, and lysed in 0.5 ml RIPA
buffer. The KDEL receptor constructs were immunoprecipitated with anti-
myc antibody and protein G-Sepharose.
Electrophoresis and Immunoblot Analysis
Beads with bound proteins were resuspended in electrophoresis sample
buffer, reduced with 10 mM DTT, and boiled for 5 min. SDS-PAGE, 15%, gels
were used to resolve both lysozyme and KDEL receptor molecules, whereas
coatomer proteins were separated on 10% SDS-PAGE gels. Proteins were
transferred onto either nitrocellulose or polyvinylidene difluoride (Immo-
bilon-P, Millipore, Bedford, MA) membranes. Blots were blocked overnight at
4°C with 5% (wt/vol) nonfat milk in TBS containing 1% (vol/vol) Tween 20.
They were incubated with primary antibody for 1 h at room temperature
followed by treatment with the corresponding HRP-conjugated secondary
antibody. Signals were detected by ECL (SuperSignal; Pierce Chemical Co.,
Rockford, IL) and quantitated by scanning densitometry. Alternatively,35S-
and32P-labeled proteins were visualized in PhosphorImager (FUJIXBas 1000;
Fuji, Tokyo, Japan) using PC-BAS 2.08 software.
Role of the C-terminal Domain in Subcellular
Localization of the KDEL Receptor and Retrieval of
The KDEL receptor is believed to adopt a seven-transmem-
brane-domain topology with a short, 12–13-amino acid tail
at the C terminus projecting out of the membrane toward the
cytosol (Townsley et al., 1993; Scheel and Pelham, 1998). To
analyze the functional role of this domain, we inserted a
c-myc epitope between the last transmembrane domain and
the putative cytoplasmic tail, which is between amino acids
199–200 of the native protein. When expressed in COS cells
this tagged version of the receptor localized at the Golgi
complex as evidenced by double immunofluorescence stain-
ing with an antibody specific for GMAP-210, a protein as-
sociated to the cis-Golgi network (Infante et al., 1999; Figure
1A). This indicated that the presence of the intercalated myc
sequence did not affect the normal, steady state localization
of the receptor. Deletion of the last 12 amino acids of our
construct leaves the myc epitope only covered by a threo-
nine residue at the C terminus. This truncated form of the
receptor was still localized at the Golgi complex (Figure 1A),
suggesting the absence of targeting signals within the C-
terminal domain. To test this we investigated the conse-
receptors. (A) COS cells were transfected with plasmid HE24M
coding a version of the KDEL receptor containing a myc epitope
inserted between the last transmembrane domain and the C-termi-
nal cytoplasmic domain. Cells expressing either the intact, wild-
type receptor (WT) or a truncated form lacking the last 12 amino
acids (?C) were double-stained with anti-myc and anti-GMAP-210
antibodies. (B) COS cells were transfected with a plasmid coding
either wild-type CXCR4 (WT) or a chimera resulting from the re-
placement of the last 41 amino acids of this protein with the C-
terminal domain of the KDEL receptor. They were single-stained
with anti-CXCR4 antibody. Bars, 20 ?m.
Immunofluorescence localization of KDEL and CXCR4
M. Cabrera et al.
Molecular Biology of the Cell 4116
quences of replacing the C-terminal domain of a plasma
membrane protein with that of the native KDEL receptor.
CXCR4, a chemokine receptor, was chosen because of its
structural similarity with the KDEL receptor (Amara et al.,
1997). Both the chimera and the wild-type protein were able
to travel throughout the exocytic pathway to the plasma
membrane when expressed in COS cells (Figure 1B). There-
fore, according to these results the C-terminal domain does
not confer Golgi retention to a reporter protein.
We examined the role of this domain in the retrieval from
the Golgi to the ER of proteins bearing the KDEL signal at
their C terminus. Cells expressing lysozyme-KDEL were
pulse-labeled and chased to monitor secretion of this protein
to the medium. As shown in Figure 2A, a significant amount
(21%) of the initially labeled lysozyme-KDEL was secreted
to the medium during a 3-h chase period, indicating satura-
tion of the endogenous retrieval system. By contrast, secre-
tion of lysozyme-KDEL did not occur in cells simultaneously
expressing the wild-type, myc-tagged version of the KDEL
receptor (Figure 2B). In these cells, lysozyme-KDEL was
localized in the ER by immunofluorescence, whereas the
receptor, detected with anti-myc antibody, was mostly con-
centrated in the perinuclear Golgi region (our unpublished
results). This indicated that the efficiency of retrieval was
restored after expression of the ectopic receptor that was,
therefore, fully functional in terms of ligand recognition and
recovery at the Golgi complex. By contrast, during a 4-h
chase period 44–47% of the initially labeled lysozyme mol-
ecules were secreted to the medium by cells expressing a
truncated form of the KDEL receptor lacking the C-terminal
domain (Figure 2B). This suggests a severe defect in the
cellular mechanisms that are responsible for the normal
retention of escaped ER proteins.
The C-terminal Domain Is Required for the Transport of
KDEL Receptor from the Golgi Complex to the ER
To gain insight on the role played by the C-terminal domain
in retrograde transport we designed a morphological assay
aimed to monitor the redistribution into the ER of the KDEL
receptor. SLO-permeabilized cells were incubated at 37°C in
the presence of both ATP and GTP. Under these conditions
the endogenous receptor left the Golgi complex into elon-
gated tubular processes that with time fragmented and
fused with the ER (Figure 3). In addition to ATP and GTP,
this process seemed to require residual cytosolic factors
remaining inside the cells. Thus, permeabilized cells that
were rinsed with high-salt buffer before incubation could
not drive tubule formation. Addition of exogenous cytosol
did not restore the cytosolic factors lost during the high salt
wash (our unpublished results).
Cells expressing fluorescently tagged versions of the
KDEL receptor were permeabilized and used in the redis-
tribution assay. The wild-type receptor readily redistributed
into the ER after incubation with both ATP and GTP
whereas it remained localized in the Golgi in their absence
(Figure 4). In contrast, a truncated form of the receptor
lacking the C-terminal domain did not redistribute either in
the presence or in the absence of ATP and GTP. This sug-
gested the existence of differences in the ability of both
molecular forms to participate in the Golgi-ER retrograde
pathway. To reveal such differences, we used immunofluo-
rescence staining with an antibody against the C-terminal
domain. Cells expressing the truncated form of the receptor
and incubated for 30–35 min with ATP and GTP showed the
endogenous receptor, recognized by the antibody, redistrib-
uted into the ER, whereas the ectopic fluorescent form lack-
ing the C-terminal domain remained retained in the Golgi
(Figure 4). This clearly indicated that transport from the
Golgi complex to the ER of the KDEL receptor depends on
its C terminus.
Role of the C-terminal Domain in the Interaction of the
KDEL Receptor with Coatomer and ARF-GAP
In the Golgi-ER retrograde pathway, sorting of membrane
proteins occurs throughout the interaction of coat proteins
with their cytoplasmic domains. The latter typically bear
some kind of ER retrieval motif such as a dilysine (KKXX)
motif at their C-terminus. Although apparently the KDEL
receptor lacks such a signal, it is well established that it is
transported from the Golgi complex to the ER in COPI-
coated transport intermediates (Orci et al., 1997; Girod et al.,
1999). Therefore, in order to be sorted into this kind of
carriers the KDEL receptor must interact with components
of the COP I coat. To examine this, we carried out pull-down
experiments of cytosolic coatomer proteins with synthetic
fected with a plasmid coding hen lysozyme containing the KDEL
signal at the C terminus. (B) Alternatively, they were transfected
with a plasmid coding both lysozyme-KDEL and a particular myc-
tagged version of the KDEL receptor, either the intact, wild-type
receptor (WT) or a truncated form lacking the last 12 amino acids
(?C). Cells were pulse-labeled for 10 min with [35S]methionine and
-cysteine and chased for the indicated time periods. At each time
point, lysozyme-KDEL was immunoprecipitated from both medium
(M) and cell pellet (C) and resolved by SDS-PAGE.
Secretion of lysozyme-KDEL. (A) COS cells were trans-
PKA Phosphorylation of the KDEL Receptor
Vol. 14, October 20034117
peptides corresponding to the C-terminal domain of the
KDEL receptor coupled to beads (Figure 5, A and B). On
incubation with cytosol the beads were rinsed and processed
by SDS-PAGE before protein detection by immunoblotting.
Recruitment was dependent on peptide because control
beads with no peptide coupled did not bind any coatomer
protein. At 0.1 M NaCl a 21-amino acid peptide covering the
complete C-terminal domain of the KDEL receptor did not
recruit coatomer. However, a small amount of some
coatomer proteins such as ??-COP and ?-COP could be
bound at higher, 0.5–1 M, NaCl concentration (Figure 5A).
Interestingly, this peptide sequence contains at positions ?6
and ?7 from the C terminus two consecutive lysine resi-
dues. Although located far away from the C terminus, these
residues could be part of a hidden ER retrieval motif that
under certain circumstances would be exposed for interac-
tion with coatomer. To investigate this possibility a similar
peptide lacking the last three amino acids was also assayed.
Despite the presence of a potential retrieval dilysine motif at
the C terminus coatomer proteins did not interact either
with this peptide sequence (Figure 5A). Therefore, other
factors different from the classic dilysine retrieval motif
would be responsible for the interaction of the C-terminal
domain of the KDEL receptor with coatomer proteins. Thus,
a serine residue located at position 209 in the native protein
was shown to be critical for interaction. Even at high salt
concentration no coatomer protein bound to a peptide se-
quence in which this particular amino acid was omitted. We
reasoned that serine 209 could be a potential phosphoryla-
tion target. Accordingly, serine was replaced by either ala-
nine, which cannot be phosphorylated, or alternatively, by
aspartic acid, which mimics a phosphorylated residue. The
latter peptide sequence was able to recruit coatomer under
all conditions assayed. By contrast, alanine replacement
abolished coatomer binding (Figure 5A). These results sug-
gested that phosphorylation of serine 209 would determine
the interaction of the C-terminal domain of the KDEL recep-
tor with cytosolic coatomer proteins.
Although phosphorylation of serine 209 could be a rele-
vant event, additional determinants might be also involved
in coatomer binding. As mentioned above, a putative dil-
ysine motif is present within the C-terminal domain of the
KDEL receptor. In principle, this motif should not be func-
tional in retrieval because it is located far away from the C
terminus. Figure 5B shows that peptides in which this motif
was altered by alanine replacement did not bind coatomer
proteins. Apparently, this situation could not be overcome
by phosphorylation of serine 209. Thus, a peptide lacking an
intact dilysine motif and simultaneously containing an as-
partic acid residue at a position equivalent to 209 in the
native protein could not bind coatomer (Figure 5B). There-
fore, in addition to phosphorylation of serine 209, a func-
tional dilysine retrieval motif must be present within the
C-terminal domain of the KDEL receptor for coatomer in-
teraction to occur.
The KDEL receptor, on the other hand, has been shown
to interact with ARF-GAP in a ligand-stimulated process
(Aoe et al., 1997, 1998). This interaction is thought to
promote ARF-GAP recruitment from cytosol to the mem-
brane where ARF-GAP has been described to participate
in cargo sorting and formation of COPI-coated transport
carriers (Yang et al., 2002). We asked whether the amino
acid determinants involved in coatomer recruitment also
affected the interaction of the KDEL receptor with cyto-
solic ARF-GAP (Figure 5C). In our assay, ARF-GAP did
not bind to a peptide corresponding to the complete C-
terminal domain of the KDEL receptor unless serine 209
was replaced by aspartic acid. Again, the internal dilysine
motif had a dominant effect because no binding occurred
when it was altered by alanine replacement either in the
presence or in the absence of aspartic acid at a position
equivalent to 209 in the native protein (Figure 5C). Taken
together, these results suggest that phosphorylation of
serine 209 promotes the interaction of the KDEL receptor
with both coatomer proteins and ARF-GAP.
at 37°C for the indicated time periods (min) with 1 mM of both ATP and GTP. Cells were fixed and processed for indirect immunofluores-
cence with an antibody against the C-terminal domain of the KDEL receptor. Bars, 20 ?m
Golgi-ER redistribution of the native KDEL receptor. Vero cells were permeabilized with SLO, rinsed with buffer, and incubated
M. Cabrera et al.
Molecular Biology of the Cell 4118
Role of Serine 209 in Golgi-ER Transport and Retrieval of
The above data suggested that within the C-terminal do-
main serine 209 could play a critical role in the interaction of
the KDEL receptor with COPI coat proteins and hence in the
Golgi-ER retrograde transport followed by the former. To
investigate this further, we analyzed the redistribution of a
mutated form of the receptor in which this particular amino
acid was replaced by alanine. Cells expressing fluorescently
tagged forms of both the wild-type and the S209A mutated
receptor were permeabilized and used in the redistribution
assay. During a 25–30-min incubation period a significant
amount of the wild-type receptor redistributed to the ER in
an ATP- and GTP-dependent manner. In contrast, the S209A
mutated receptor lagged behind in the Golgi (Figure 6A)
and only after prolonged (40–45 min) incubation periods
started to appear in the ER network. This is consistent with
the fact that most of the Golgi residents redistributed to the
ER after long incubations (our unpublished results). These
results revealed striking differences between both molecular
forms in their ability to be transported from the Golgi com-
plex to the ER within the same living cell. According to these
CFP-tagged version of either the wild-type KDEL receptor (WT) or a truncated form lacking the last 12 amino acids (?C). They were
permeabilized with SLO and incubated at 37°C for 35 min either in the presence (?) or in the absence (?) of both ATP and GTP. Cells were
fixed and processed for indirect immunofluorescence with an antibody against the C-terminal domain of the KDEL receptor. Bars, 16 ?m
Golgi-ER redistribution of different fluorescent KDEL receptor constructs. COS cells were transfected with plasmid coding a
PKA Phosphorylation of the KDEL Receptor
Vol. 14, October 20034119
data the S209A receptor does not travel from the Golgi to the
ER at normal speed and most likely this could affect the
retrieval of ligands containing the KDEL sequence. We
therefore examined the effect of this amino acid replacement
on the intracellular retention of lysozyme-KDEL (Figure 6B).
Cells expressing simultaneously lysozyme-KDEL and either
the wild-type or the S209A mutated receptor were pulse-
labeled and chased for 3 h. During this time period 18.2% of
the initially labeled lysozyme-KDEL was secreted to the
culture medium by cells expressing the S209A mutant form.
Because lysozyme-KDEL was not secreted to the medium by
cells expressing the wild-type receptor (Figure 6B), this re-
sult indicated that expression of the mutated receptor de-
creased the efficiency of the retrieval system.
PKA Phosphorylation of the KDEL Receptor
The above data indicated that serine 209 is a key residue for
the normal functioning of the KDEL receptor. The sequence
context in which Serine 209 is located (KKLSL) is a potential
consensus site for PKA phosphorylation (K/R-K/R-X-S-X).
We therefore examined the possibility that this kinase might
be involved in phosphorylation of the C-terminal domain of
the KDEL receptor (Figure 7). High-salt–washed microsomal
membranes from insect cells expressing different versions of
the myc-tagged KDEL receptor were incubated with both
[?-32P]ATP and pure PKA catalytic subunits, C?. Mem-
branes were then rinsed with buffer and lysed. The KDEL
receptor constructs were immunoprecipitated with anti-myc
antibody, subjected to SDS-PAGE, and transferred to West-
ern blots. Protein bands labeled with32P were identified by
immunoblotting with anti-myc antibody. The wild-type
KDEL receptor was efficiently phosphorylated in a C?-de-
pendent reaction. By contrast, the S209A mutant form was
not phosphorylated either in the absence or in the presence
of C? in the incubation medium (Figure 7A). Phosphoryla-
tion of the wild-type KDEL receptor could also be achieved
by incubation with crude cytosol (Figure 7B). An endoge-
nous kinase activity catalyzed receptor phosphorylation in
this case. We identified such kinase activity as PKA by using
cytosol preincubated with pure RII? regulatory subunits.
These are expected to complex and inactivate endogenous
C? subunits present in the cytosol preparation. As shown in
Figure 7B, addition of 50–100 nM RII? abolished KDEL
receptor phosphorylation induced by cytosol.
These data implied that PKA could potentially phosphor-
ylate the C-terminal domain of the KDEL receptor at serine
209. The question that arose was the functional relevance of
such modification. To address this, we performed an exper-
iment aimed to evaluate the role of PKA phosphorylation in
the dynamic cycling of the endogenous KDEL receptor. A
GTP-restricted form of Sar1 (Sar1[H79G], Sar1dn) was used
as a specific reagent to arrest the anterograde ER-Golgi
transport while leaving the retrograde Golgi-ER pathway
unaffected. Nontransfected cells were microinjected with
recombinant Sar1dn-tagged with YFP. In the presence of this
protein the KDEL receptor redistributed to the ER where it
became trapped. This effect was observed in most (66%, n ?
115) of the microinjected cells and seems to be the result of
a situation of continuous retrograde transport in the absence
of anterograde flow. By contrast, noninjected cells showed
the typical Golgi/intermediate compartment localization
pattern (Figure 8). We next used H89 as a selective inhibitor
to reveal the involvement of PKA in the transport of the
native receptor from the Golgi to the ER. Used at 5–10 ?M,
this reagent specifically inhibits PKA activity, whereas much
higher concentrations (mM range) are required to inhibit
other serine/threonine kinases. In cells incubated with 5 ?M
H89 the KDEL receptor was seen concentrated in the Golgi
area either in the presence or in the absence of Sar1dn(Figure
8). Because redistribution did not occur in ?70% of the cells
microinjected with Sar1dn, this result indicated that the
with peptides. Synthetic peptides were covalently coupled to
thiopropyl Sepharose beads. They were incubated with crude
bovine brain cytosol for 5 min at room temperature, rinsed with
buffer, and processed by SDS-PAGE and immunoblotting. The
same Western blot membrane was reused to detect several
coatomer proteins. Incubation with cytosol and rinses with buffer
were carried out at the indicated NaCl concentrations (A) or,
alternatively, at 0.1 M NaCl (B and C). Peptide . . . GKKLSLPA
corresponds to the entire C-terminal domain of the native KDEL
receptor. Changes on this sequence are indicated as underlined
residues and the caret indicates deletion). As a negative control,
beads with no peptide coupled were similarly processed (con-
trol). Also, 20 ?g of crude cytosol was processed and loaded on
gels as a positive control (cytosol).
Interaction of native coatomer proteins and ARF-GAP
M. Cabrera et al.
Molecular Biology of the Cell4120
transport of the native receptor from the Golgi to the ER is
indeed PKA modulated.
We took advantage of the inhibitory effect of H89 on
ER-Golgi anterograde transport (Mun ˜iz et al., 1996; Aridor
and Balch, 2000; Lee and Linstedt, 2000) to demonstrate the
redistribution from the Golgi to the ER of the S209D mutant
receptor. We reasoned that if this form would mimic a
phosphorylated receptor, it would travel from the Golgi to
the ER in a H89 insensitive way. Then it would be retained
in the ER because of blocking of the anterograde transport.
Cells expressing fluorescently tagged versions of both the
wild-type KDEL receptor and the S209D mutated form were
therefore subjected to H89 treatment. Both molecular forms
colocalized at the Golgi complex in control, untreated cells
(Figure 9). As expected from the above data, H89 inhibited
ER redistribution of the wild-type KDEL receptor that re-
mained localized at the Golgi after 1-h incubation with a
high dose, 20–30 ?M, of this inhibitor. By contrast, under
these conditions the mutant S209D receptor expressed by the
same cell redistributed efficiently from the Golgi to the ER
(Figure 9). These results emphasized the relevance of PKA
phosphorylation of serine 209 to promote the retrograde
traffic of the native KDEL receptor from the Golgi complex
to the ER.
Retrieval of endogenous ER proteins tagged with the KDEL-
(HDEL) sequence at their C-terminus is an essential, con-
served process in eukaryotic cells that contributes to the
quality control in the secretory pathway. The wild-type
KDEL receptor does not contain a classic dilysine ER re-
trieval motif. Despite of this, it is firmly established that,
once loaded with cargo, the KDEL receptor travels from the
Golgi complex to the ER in COPI-coated transport carriers
(Orci et al., 1997; Girod et al., 1999). Therefore the mechanism
that allows the receptor-ligand complexes to be sorted into
these containers is a major unresolved problem.
In this study we have analyzed the functional role of the
C-terminal cytoplasmic domain of the KDEL receptor. Ac-
cording to our results, this part of the protein does not
contain dominant targeting information that could direct the
KDEL receptor or any other membrane protein to the Golgi
complex. By contrast, the data indicate that it is necessary for
receptor sorting into COPI-coated transport intermediates
and, therefore, is required for the retrograde transport of the
KDEL receptor from the Golgi region to the ER as well as for
the retrieval of KDEL ligands. Thus, in a Golgi-ER redistri-
bution assay that makes use of SLO-permeabilized cells a
receptor redistribution and secretion of ly-
sozyme-KDEL. (A) COS cells were cotrans-
fected with two plasmids, one coding a YFP-
receptor and the other coding a CFP-tagged
version of a mutant form in which S209 was
changed to A (S209A). Cells were permeabil-
ized and incubated at 37°C for 25 min either
in the presence (?) or in the absence (?) of
both ATP and GTP before fixation. Bars, 16
?m. (B) COS cells were transfected with a
plasmid coding both lysozyme-KDEL and the
myc-tagged version of the KDEL receptor.
Cells expressing either the wild-type receptor
(WT) or the S209A mutant form were pulse-
labeled for 10 min and chased for 3 h. Ly-
sozyme-KDEL was immunoprecipitated from
both medium (M) and cell pellet (C) and re-
solved by SDS-PAGE.
Effects of S209A replacement on
PKA Phosphorylation of the KDEL Receptor
Vol. 14, October 20034121
truncated form of the receptor lacking the C-terminal do-
main remained retained in the Golgi complex, whereas the
native receptor present in the same cell efficiently redistrib-
uted to the ER. Accordingly, a significant amount of ly-
sozyme-KDEL was secreted to the extracellular medium by
cells expressing this truncated form of the KDEL receptor. In
contrast, those expressing the wild-type form retained ly-
sozyme-KDEL intracellularly. Together, these results indi-
cate that the short C-terminal domain plays an essential role
for the functioning of the KDEL receptor. A relevant role of
the C-terminal domain in retrograde transport is also sup-
ported by studies showing that the arrival of KDEL-bearing
bacterial toxins to the ER is inhibited in cells microinjected
with antibodies directed against this region of the receptor
(Majoul et al., 1998; Jackson et al., 1999). Alternatively, it is
possible that tail-less forms of the KDEL receptor would
become aggregated in the Golgi complex where they would
be unable to participate in retrograde transport.
Within the C-terminal domain serine 209 is an important
residue for the function of the KDEL receptor. Replacement
by alanine (S209A) affected the ability of the receptor to be
transported from the Golgi complex to the ER as judged by
results obtained with the redistribution assay. At least in this
case formation of molecular aggregates that could not be
transported along the retrograde pathway seems unlikely.
The S209A mutant form remained arrested in the Golgi
complex, whereas the wild-type receptor expressed by the
same cell redistributed to ER at normal speed. Additionally,
intact cells expressing the S209A mutant form did not retain
lysozyme-KDEL properly. In contrast to these findings,
Townsley et al. (1993) reported that point mutations at the
different predicted cytoplasmic domains of the KDEL recep-
tor including the C-terminal tail did not affect its retrograde
traffic from the Golgi to the ER. This was evaluated by
receptor redistribution to the ER during coexpression with
lysozyme-KDEL. In particular, alanine replacement of serine
209 did not prevent receptor relocation to the ER induced by
overexpression of lysozyme-KDEL. Instead, using the same
criteria an aspartic acid residue located in the seventh trans-
membrane domain was judged to be critical for retrograde
transport (Townsley et al., 1993). The molecular versions of
the KDEL receptor here analyzed, including the S209A and
S209D mutant forms and also a truncated form lacking the
entire C-terminal domain, were all concentrated at the Golgi
complex during transient expression. Still they exhibited
different capabilities to be transported from the Golgi to the
membranes containing myc-tagged versions of the KDEL receptor
were prepared from baculovirus-infected insect cells, washed with
high-salt buffer, and subjected to PKA-mediated phosphorylation.
(A) Membranes containing either the wild-type KDEL receptor
(WT) or the S209A mutant form were incubated at 30°C for 10 min
with [?-32P]ATP either in the presence or in the absence (?) of 1 U
C?. (B) Membranes containing the wild-type KDEL receptor were
similarly incubated with both [?-32P]ATP and 100 ?g rat liver cy-
tosol. The latter was preincubated for 10 min with both phosphatase
inhibitors and the indicated concentrations of recombinant RII?.
Membranes were rinsed with buffer and lysed with detergents
before immunoprecipitation of the KDEL receptor with anti-myc
antibody. Immunoprecipitates were resolved by SDS-PAGE and
either directly visualized in PhosphorImager (B) or previously
transferred to Western blot membranes (A). Radioactive (32P) bands
in A were identified by immunoblotting with anti-myc antibody. In
this case, protein G-Sepharose beads with anti-myc antibody co-
valently cross-linked were used during immunoprecipitation to
avoid interference of immunoglobulin light chains during immuno-
PKA phosphorylation of the KDEL receptor. Microsomal
the native KDEL receptor. Vero cells were preincubated (?) or not
(?) for 10–15 min with 5 ?M H89 in FCS-free medium supple-
mented with 25 mM HEPES. They were microinjected in the same
medium with recombinant YFP-tagged Sar1dnand incubation con-
tinued at 37°C for 1 h. Cells were fixed and processed for indirect
immunofluorescence with an antibody against the KDEL receptor.
Bars, 16 ?m
Effect of H89 treatment on the Golgi-ER redistribution of
M. Cabrera et al.
Molecular Biology of the Cell4122
ER as shown by their relative contribution to the intracellu-
lar retention of lysozyme-KDEL. This indicates that receptor
localization pattern is determined by several factors (i.e.,
ER-Golgi anterograde transport) and is not necessarily an
indication of its functionality.
Phosphorylation of serine 209 seems be a key event in
controlling the dynamic behavior of the KDEL receptor. The
available evidences indicate that PKA is involved. Thus, the
receptor can be phosphorylated in vitro by both pure, re-
combinant PKA catalytic subunit and by a cytosolic activity
inhibited by PKA regulatory subunits. Involvement of PKA
in Golgi-ER retrograde transport is supported by experi-
ments with H89. Cells incubated with low concentrations of
this inhibitor did not show redistribution of the native KDEL
receptor from the Golgi region to the ER after a block in the
ER-Golgi anterograde flow induced by Sar1dn. This suggests
that in order to be transported from the Golgi complex to the
ER the KDEL receptor must be first phosphorylated by PKA.
Conversely, in the absence of PKA phosphorylation the
native receptor would not be recruited into the Golgi-ER
retrograde pathway and proteins bearing the KDEL signal
would be secreted to the extracellular medium. In addition,
because of its inhibitory effect on ER to Golgi anterograde
transport (Mun ˜iz et al., 1996; Aridor and Balch, 2000; Lee and
Linstedt, 2000) H89 was used to evaluate the consequences
of the S209D mutation. In this case, treatment with H89 does
not interfere with the Golgi-ER retrograde traffic of the
S209D mutant form but inhibits exit from the ER and this
gives rise to retention at this location. Presence of an aspartic
acid at position 209 would mimic a phosphorylated residue.
This could have the effect of making the KDEL receptor to be
permanently activated for retrograde transport. However,
the Golgi localization pattern of this form in untreated cells
suggests that it is also rapidly moving out of the ER to the
Our observations indicate that the C-terminal domain is
involved in the interaction of the KDEL receptor with com-
ponents of the COPI coat such as coatomer proteins and also
with the ARF1 regulator ARF-GAP. We have been able to
study such interaction in assays using synthetic peptides
that mimic the cytoplasmic tail of the KDEL receptor. From
these experiments we conclude that binding of both
coatomer proteins and ARF-GAP apparently depends on
two factors. First, a hidden dilysine motif is required. This
corroborates a recent study showing that mutation of these
residues abolishes the interaction of the cytoplasmic domain
with purified coatomer and recombinant ARF-GAP (Yang et
al., 2002). On the other hand, serine 209 was shown to be
critical for both coatomer and ARF-GAP recruitment. Bind-
ing in both cases was maximal after replacement of this
residue by aspartic acid, whereas it was abolished after
alanine replacement. This result places PKA phosphoryla-
cotransfected with two plasmids, one coding a YFP-tagged version of the wild-type KDEL receptor and the other coding a CFP-tagged
version of a mutant form in which S209 was changed to D (S209D). Twenty-four hours after transfection they were incubated or not (?) at
37°C for 1 h with 20 ?M H89 in FCS-free medium before fixation. Bars, 16 ?m
Effect of H89 treatment on the Golgi-ER redistribution of the S209D mutant form of the KDEL receptor. Vero cells were
PKA Phosphorylation of the KDEL Receptor
Vol. 14, October 20034123
tion in the center of the regulation of the interaction of the
KDEL receptor with the retrograde transport machinery. A
possibility would be that PKA phosphorylation of serine 209
could trigger a conformational change in the C-terminal
domain of the KDEL receptor. This would result in the
cryptic dilysine motif becoming exposed and available for
interaction with ARF-GAP and coatomer. Although this
mechanism might occur in mammalian cells, it certainly
does not apply to other cell systems. Thus, neither the inter-
nal dilysine motif nor the critical serine residue are strictly
conserved in evolution. For instance, the C-terminal domain
of the yeast HDEL receptor have only one lysine residue,
which is not sufficient to constitute a retrieval motif. Fur-
thermore, no serine or other phospho-acceptor residue is
present in such domain (Semenza et al., 1990). Interestingly,
the two determinants here discussed are present in the C-
terminal domain of yeast Rer1, which also functions in ret-
rograde transport as a receptor for the COPI-dependent
retrieval of certain type II transmembrane proteins (Nish-
ikawa and Nakano, 1993; Boehm et al., 1997). Therefore, it is
possible that PKA activity regulates the interaction with the
COPI transport machinery of several membrane receptors in
We postulate the existence of a signal-transduction path-
way across the membrane of the Golgi complex. This could
become activated once the KDEL receptor interacts with
ligands. The predicted topology of the KDEL receptor, struc-
turally similar to G-protein–coupled receptors, supports the
existence of such a pathway (Townsley et al., 1993; Scheel
and Pelham, 1998). PKA, on the other hand, is associated to
the cytoplasmic side of the Golgi membranes (Martı ´n et al.,
1999), and it could also be activated upon ligand binding.
Activated PKA phosphorylates serine 209 at the C-terminus
of the KDEL receptor and this would trigger its recruitment
into the Golgi-ER retrograde pathway by promoting ARF-
GAP and coatomer association and sorting into COPI-coated
We thank Drs. A. Caruz, H. R. B. Pelham, R. Pepperkok, R. Rios, M. M.
Riottot, and L. M. Roberts for providing reagents used in this study. This
work was supported by Grants PB98–1119 and BMC2002–00295 from Min-
isterio de Ciencia y Tecnologı ´a (to A.V.) and Grant 00/1065 from Ministerio
de Sanidad y Consumo (to J.H.). M.C. and L.V. were supported by predoc-
toral fellowships from Ministerio de Educacio ´n and Junta de Andalucı ´a,
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