Department of Molecular Animal Physiology, Nijmegen Center for Molecular Life Sciences (NCMLS), University of Nijmegen, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands
* Author for correspondence (e-mail: gmartens{at}ncmls.kun.nl )
Accepted 28 November 2001
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Summary |
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Key words: p24 proteins, Protein transport, Prohormone-producing cell, Pituitary, Xenopus laevis
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Introduction |
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Cargo proteins can leave the ER without prior concentration
(Martínez-Menárguez
et al., 1999; Warren and
Mellman, 1999
), but several studies have demonstrated that the
cell has mechanisms for concentration of cargo in ER-derived vesicles and for
accelerated transport out of the ER (Kuehn
et al., 1998
; Mizuno and
Singer, 1993
; Nishimura and
Balch, 1997
), suggesting a selective mechanism of cargo transport,
presumably via cargo receptors. Thus far, three evolutionarily conserved
families of integral membrane proteins have been proposed to facilitate
ER-to-Golgi transport. Representatives of these protein families are fairly
abundant and binding sites for COPI and/or COPII coat subunits are found in
the cytoplasmic tail of these proteins; these binding sites enable the
proteins to cycle constantly within the early secretory pathway. The BAP
family seems to regulate trafficking of certain membrane proteins out of the
ER (Adachi et al., 1996
;
Kim et al., 1994
;
Terashima et al., 1994
).
BAP31, a representative of this family, has been shown to bind with high
specificity to the endosomal membrane protein cellubrevin and to control its
export out of the ER (Annaert et al.,
1997
). ERGIC-53/p58, a mannose-specific membrane lectin, belongs
to another class of receptors involved in the transport of a number of
glycoproteins from the ER to the ERGIC
(Hauri et al., 2000
). The
third group of putative cargo receptors is a family of structurally related
24-kDa type I transmembrane proteins, collectively termed p24 proteins. Based
on their amino acid sequences, these proteins have been classified into four
main subfamilies, designated p24
, -ß, -
and -
(Dominguez et al., 1998
).
Members of the various p24 subfamilies exhibit only a low degree of amino acid
sequence identity (17-30%) but all p24 proteins have certain structural
characteristics in common, such as a relatively large lumenal domain with two
conserved cysteine residues forming a disulfide bridge, a C-terminally located
transmembrane stretch and a short cytoplasmic tail with sequence motifs known
to specify interactions with vesicle coat proteins. Experimental evidence
indicates that members of the various subfamilies can interact and tetrameric
complexes are formed containing one representative of each subfamily
(Belden and Barlowe, 1996
;
Füllekrug et
al., 1999
; Marzioch et al.,
1999
). Consistent with this view, the stability of other family
members is compromised in yeast mutants and knockout mice deficient in the
expression of a single p24 member (Denzel
et al., 2000
; Marzioch et al.,
1999
). A function for p24 proteins in cargo transport has been
proposed on the basis of the observation that in yeast, deletion of certain
p24 members slows ER export of a set of secretory proteins, whereas the export
rate of a number of other cargo proteins is normal
(Schimmöller
et al., 1995
). More recently, it was shown that two of these yeast
p24 members, Emp24 (yp24ß) or Erv25p (yp24
), which coexist in a
heteromeric complex, can be directly crosslinked to the lumenal cargo protein
Gas1p in ER-derived vesicles. Efficient packaging of Gas1p was reduced when
vesicles were generated from membranes lacking Emp24p activity
(Muñiz et
al., 2000
). Furthermore, genetic experiments in yeast and
Caenorhabditis elegans indicated that loss of p24 protein activity
affects the fidelity of ER sorting
(Elrod-Erickson and Kaiser,
1996
; Wen and Greenwald,
1999
). Although in yeast, p24 proteins are not essential for
vesicular transport (Springer et al.,
2000
), deleting a single p24 member (p23) leads to early embryonic
death in mice (Denzel et al.,
2000
).
In this study, we identified the members of the p24 family that are
expressed in the intermediate pituitary of the South African clawed toad
Xenopus laevis. The intermediate pituitary consists of a homogenous
population of melanotrope cells that are involved in the process of background
adaptation of the animal. The central function of the melanotrope cells is the
production of proopiomelanocortin (POMC) and in an active cell this prohormone
constitutes over 80% of all newly synthesized proteins
(Holthuis et al., 1995a). The
processing of POMC yields a number of bioactive peptides of which the
-melanophore stimulating hormone (
-MSH) stimulates the
dispersion of the black pigment melanin in skin melanophores, causing
darkening of the animal (Jenks et al.,
1977
). In the melanotrope cells, the expression levels of POMC can
be manipulated in a physiological way simply by changing the background color
of the animal. On a black background, the POMC gene is highly active, whereas
on a white background the gene is virtually inactive. The high levels of POMC
production in black-adapted animals cause an enormous increase in cargo
transport in the melanotrope cells, reflected by an extremely well-developed
biosynthetic and secretory pathway, and a melanotrope cell size about twice as
large as that in white-adapted animals (reviewed by
Roubos, 1997
). One would thus
expect that the p24 proteins which have a presumed function in POMC transport
are coordinately expressed with this prohormone. We could demonstrate that the
expression of a selective set of p24 proteins is induced in the melanotrope
cells of black-adapted animals, whereas others are not or only slightly
induced. The coordinate expression of Xp24
3,
-ß1, -
3 and -
2 with POMC
suggests that these p24 proteins assemble into a tetrameric complex involved
in the ER-to-Golgi transport of the prohormone.
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Materials and Methods |
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Library screening and DNA sequencing
The nonredundant GenBank/EMBL/DDBJ database at NCBI was searched for p24
sequences from mouse with the BLAST program. Expressed sequence tags (ESTs)
containing the entire ORFs of mouse p243,
-
1, -
2, -
3 and
-
4 were identified and received from the IMAGE Consortium
(Zehetner and Lehrach, 1994
).
PCR products, constituting parts of the ORFs, were generated using the mouse
EST clones as templates. Degenerated oligonucleotides encoding conserved
sequences in vertebrate p24ß proteins were used to amplify a 313-bp
fragment of Xp24ß1 (nucleotides 286-599 in
Xp24ß1 ORF) from cDNA obtained through standard
reverse-transcriptase reactions (Sambrook
et al., 1989
) on RNA isolated from Xenopus brain by the
Trizol isolation method (Life Technologies-BRL). Fragments were gel purified,
labelled with [32P]dATP by random primer extension
(Ausubel et al., 1989
), and
unincorporated nucleotides were removed using NucTrap Probe purification
columns (Stratagene, Cedar Creek, TX). The probes were used to screen an oligo
dT-primed cDNA library of neurointermediate lobes of the pituitary gland of
black-adapted X. laevis (Kuiper
et al., 2000
). In addition, ZapII-cDNA libraries made from whole
Xenopus embryos (stage 42; Michael King, Indiana University,
Bloomington, IN) or embryo heads (stage 28-30; Richard Harland, University of
California, Berkeley, CA) were used. Plaques (density 400/cm2) were
replicated on duplicate nylon membrane filters by standard procedures
(Sambrook et al., 1989
).
Filters were prehybridized for at least 1 hour at 50°C in hybridization
solution (10% dextran sulfate, 1% SDS, 1 M NaCl, 0.1% sodiumpyrophosphate,
0.2% bovine serum albumin, 0.2% polyvinylpyrollidone K90, 0.2% Ficoll 400, 50
mM Tris pH 7.5) and hybridized under conditions of low stringency (at
50°C) with a labelled 524 bp probe for mp24
3
(nucleotides 150-674 in accession number AA109932), a 313-bp probe for
Xp24ß1 (nucleotides 286-599 in Xp24ß1 ORF), a
537-bp probe for mp24
1 (nucleotides 107-644 in accession
number W08294), a 462-bp probe for mp24
2 (nucleotides
248-710 in accession number W58982), a 301 bp probe for
mp24
3 (nucleotides 5-306 in accession number AA020489), and
a 453 bp probe for mp24
4 (nucleotides 274-727 in accession
number AA060892). Filters were washed twice for 40 minutes at 50°C in
2xSSPE/0.1% SDS (where 1xSSPE is 0.15 M NaCl, 10 mM sodium
phosphate, 1 mM EDTA; pH 7.4) and exposed to X-ray films between two
intensifying screens at 70°C. Positive plaques were purified, in
vivo excised and analyzed by DNA sequencing using the ABI-PRISM DNA sequencing
kit and the ABI-PRISM automatic sequencer (Perkin Elmer-Cetus Applied
Biosystems, Foster City, CA). The cDNA libraries were screened under
high-stringency hybridization conditions with the 3'-untranslated
sequences of isolated cDNA clones encoding the various Xenopus p24
proteins. These sequences were amplified in a PCR reaction yielding for
Xp24
3 a 0.43 kb fragment, for Xp24ß1
1.8 kb, for Xp24
2
0.6 kb and for
Xp24
3
1.7 kb. The high-stringency hybridizations were
performed at 63°C and filters were washed to a final stringency of
0.1xSSPE/0.1% SDS at 63°C. Standard procedures, such as PCR and
single clone in vivo excision of
-phage, were performed as described
(Ausubel et al., 1989
).
RNA isolation and Northern blot analysis
Total RNA was isolated with RNAgents isolation system according to the
instructions of the manufacturer (Promega, Madison, WI) and quantified by
spectrophotometry. Aliquots of 5 µg per lane were separated by
electrophoresis on 2.2 M formaldehyde-containing 1.2% agarose gels in MOPS
buffer (Sambrook et al., 1989)
and blotted onto nylon membranes using downward capillary transfer.
Hybridizations were performed for 18 hours at 42°C in ULTRAhyb
hybridization solution (Ambion, Austin, TX) with probes comprising the entire
ORFs of Xp24
3, -
1, -
2,
and -
3, a 364-bp fragment of Xp24ß1
(nucleotides 77 to 441 in Xp24ß1 ORF) or a 230 bp fragment of
XGAPDH (nucleotides 266 to 496 in XGAPDH ORF), as a control for RNA loading
and integrity. Blots were washed at 60°C to a final stringency of
0.1xSSPE/0.1% SDS (twice for 30 minutes) and exposures were taken using
a PhosphorImager (BioRad Personal FX).
Antibodies
For antibody production, a region in the lumen of Xp243
(residues 31-128) was cloned into the expression vector pQE30 (Qiagen,
Chatsworth, CA), the recombinant protein was produced in E. coli and
purified by Ni2+-NTA agarose affinity chromatography. The lumenal
domains of Xp24
1 (residues 18-194) and
Xp24
2 (residues 32-191) were cloned as GST fusion proteins
into the bacterial expression vector pGEX-2T (Pharmacia Biotech Benelux, NL).
The expressed GST fusions were largely insoluble. Therefore, the aggregated
fusion proteins were isolated as inclusion bodies from E. coli
(Nagai and Thogersen, 1987
). A
synthetic peptide against the cytoplasmic tail sequence of
Xp24
3 (C-FSDKRTTTTRVGS) was coupled to keyhole limpet
hemocyanin (Pierce, Rockford, IL). All antigens described were injected into
rabbits and the immunization was done as described
(Kuiper et al., 2000
).
Polyclonal antibodies against amino acid sequences in the lumenal part of
Xp24
1 (C-FDSKLPAGAGRVP; anti-
1) and
-
2 (residues 72-150; anti-
2), as well as
the C-terminally directed p24
antibody (anti-
C) have been
described previously (Kuiper et al.,
2000
). The p24ß1 and -
3 peptide
antibodies (anti-ß1L and anti-
3) recognize
orthologs in human and Xenopus, and were kindly provided by T.
Nilsson (EMBL, Heidelberg, Germany)
(Dominguez et al., 1998
).
Affinity purifications of antisera using antigen-sepharose 4B columns were
performed according to standard protocols
(Harlow and Lane, 1988
).
Immunological characterization
Extraction of proteins from different tissues of X. laevis,
SDS-PAGE gel electrophoresis, Western blotting, antibody detection and
immunocytochemistry were performed as described
(Kuiper et al., 2000).
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Results |
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The p24 subfamily
Within the p24 subfamily two branches of p24 proteins can be
distinguished, namely, the
1-branch and the
2/
3-branch
(Dominguez et al., 1998
)
(Fig. 1B). Thus far, the only
isolated representative of the
1-branch is from dog
pancreatic microsomes (Wada et al.,
1991
). Two closely related representatives of the
2/
3-branch are expressed in mouse, with
mouse p24
2 (mp24
2; GMP25/mp25) being 80.8%
identical to mp24
3 (GMP25iso)
(Dominguez et al., 1998
). In
our screening for p24
subfamily members, we used a 524 bp fragment of a
mouse EST p24
3 clone as a probe, yielding 12
hybridization-positive plaques from 2x105 plaques of the
Xenopus NIL cDNA library screened. Nucleotide sequence analysis
revealed that the positive clones all contained overlapping cDNAs. The insert
size of two full-length clones was approximately 1.2 kb with a 687-bp open
reading frame (ORF). Since the deduced Xenopus p24
protein
(excluding the signal peptide region) was more closely related to
mp24
3 (92.4% identity) than to mp24
2
(81.3% identity), the protein was named Xp24
3
(Fig. 1). The low degree of
amino acid sequence conservation between the
1- and the
2/
3-branch (identity <60%) does not
allow the isolation of a p24
1-related protein using mouse
Xenopus p24
3 cDNA as a probe. However, the degree
of homology within the
2/
3-branch
(identity >80%) should be high enough for the identification of a
p24
2 ortholog in Xenopus. We therefore performed a
low-stringency hybridization using the coding region of
Xp24
3 cDNA as a probe. From a total number of
4.2x105 plaques, 51 hybridization-positive plaques were
obtained. All clones positive in this screening were also recognized by the
0.43-kb 3'-untranslated region of Xp24
3 cDNA on a
duplicate filter under stringent hybridization conditions, indicating that
only p24
3, and not p24
2, is expressed in
the Xenopus intermediate pituitary. Nevertheless,
p24
2 does exist in X. laevis, because recently
performed database searches revealed two Xenopus EST clones isolated
from embryo and liver cDNA libraries, of which the deduced amino acid
sequences were more similar to mp24
2 than to
mp24
3 (Fig.
1).
|
The p24ß subfamily
Only one member of the p24ß subfamily exists in higher vertebrates and
its high degree of sequence conservation enabled us to amplify a 313-bp
fragment of Xp24ß1 in a PCR reaction using Xenopus
brain-derived cDNA and degenerate primers corresponding to two conserved
domains in vertebrate p24ß1 proteins. This fragment was used
to screen the Xenopus NIL cDNA library, and two full-length clones of
Xp24ß1 with an insert size of 2.4 kb were isolated. A
comparison of the deduced primary sequence of Xp24ß1 with
mouse p24ß1 revealed that, when the signal peptide sequence is
excluded, the two proteins share a sequence identity of 99.4%
(Fig. 1). To assess whether
other representatives of the p24ß subfamily exist and are expressed in
the library, duplicate filters were prepared, of which one filter was
hybridized under low-stringency conditions with a probe comprising the entire
ORF of Xp24ß1, whereas the second filter was hybridized under
high stringency conditions with a probe directed against the 1.8 kb
3'-untranslated sequence of Xp24ß1. All 345
hybridization-positive plaques from 6.7x105 plaques screened
were recognized by both probes, indicating that Xenopus melanotrope
cells express only one member of the p24ß subfamily.
The p24 subfamily
The p24 subfamily is more diverse than the other p24 subfamilies,
and database searches revealed four members in mouse. An evolutionary tree
showed that two groups of p24
proteins can be distinguished, one
represented by p24
1 and -
2, and the other
by p24
3 and -
4
(Fig. 1B). Low-stringency
hybridization of the Xenopus NIL cDNA library
(3.4x105 plaques) with a 462-bp mouse p24
2
fragment allowed the identification of 9 hybridization-positive cDNA clones,
which carried an insert that coded for a p24
2 ortholog in
X. laevis. The deduced protein sequence of Xp24
2
displays 72.4% identity with mouse p24
2 and 52.9% with mouse
p24
1 (excluding signal peptides)
(Fig. 1). Out of
7.3x105 NIL cDNA plaques screened under stringent conditions,
30 plaques were recognized by a
0.6 kb probe corresponding to the
3'-untranslated region of Xp24
2. A subsequent
low-stringency screening of the same library filters with the coding sequence
of Xp24
2 yielded no additional hybridizing plaques. Thus,
under the conditions used, no cross hybridization with other Xp24
sequences was found.
A low-stringency hybridization of the NIL cDNA library detected no positive
clones when a mouse p241 fragment was used. However,
screening of two Xenopus embryo cDNA libraries with the mouse
p24
1 probe resulted in the identification of several
overlapping cDNA clones, encoding the Xp24
1 protein
(Fig. 1). Therefore,
Xp24
1 does not appear to be expressed in the
Xenopus intermediate pituitary, but is expressed in other
Xenopus tissues. In its mature form (without signal peptide), the
Xp24
1 protein shares an overall sequence identity of 68.5%
with mouse p24
1, 56.4% with mouse p24
2 and
56.2% with Xp24
2. Moreover, nucleotide sequence analysis
revealed that 2 out of the 17 Xp24
1 clones isolated from the
embryo head library showed an insertion of 30 nucleotides at position +442 in
Xp24
1 cDNA. Consequently, the protein encoded by the two
cDNAs contained an in-frame insertion of 10 amino acid residues following
amino acid 148, with the amino acid sequence VRFCPLTFEE (single-letter code).
This finding suggests that in a low incident of cases the transcripts of
Xp24
1 are subject to alternative splicing, giving rise to
two structurally distinct Xp24
1 proteins that differ in size
by
1.1 kDa. The in-frame insertion was not found in other known
p24
1 proteins and so far alternative splicing has not been
described for other p24 proteins. In conclusion, since under identical
hybridization conditions Xp24
1 could be isolated from an
embryo cDNA library but not from a NIL cDNA library, only
Xp24
2 but not -
1 is expressed in the
melanotrope cells of the Xenopus intermediate pituitary.
For the isolation of Xenopus orthologs of p243
and p24
4, the NIL cDNA library was screened with a 453 bp
fragment derived from a mouse p24
4 cDNA. Out of the
4x105 plaques screened, 25 were positive on a duplicate set
of filters. Nucleotide sequence analysis of 10 of these clones revealed a
single ORF and the corresponding amino acid sequence (without signal peptide)
was 93.2% identical to mp24
3 and 69.1% identical to
mp24
4. Thus, the cDNA clones isolated with the
mp24
4 probe encode a Xenopus ortholog of
p24
3 (Fig.
1). The NIL cDNA library was re-screened and 175
hybridization-positive plaques were observed when the filters were first
hybridized with a 3'-untranslated probe of Xp24
3 under
stringent hybridization conditions (6x105 plaques screened).
A subsequent low-stringency hybridization with the coding sequence of
Xp24
3 revealed no differences in the hybridization pattern,
suggesting that only Xp24
3 and not Xp24
4
is expressed in Xenopus melanotrope cells. Besides the NIL cDNA
library we also screened the whole-embryo cDNA library with a 306-bp
mp24
3 probe under conditions of low-stringency. This led to
the identification of 66 positive plaques (4x105 plaques
screened), which also remained positive after a more stringent wash
(0.1xSSC/0.1% SDS; 50°C). Together with the fact that from 20 of
these clones a specific Xp24
3 fragment was amplified in a
PCR reaction, the hybridization-positive clones most likely contain an
Xp24
3 cDNA insert. From these experiments we conclude that a
p24
4 ortholog is not expressed in the Xenopus
intermediate pituitary and embryos.
The p24 subfamily
In Xenopus, the p24 subfamily has already been
characterized in our laboratory, leading to the identification of two
p24
proteins, Xp24
1 and -
2,
expressed in the melanotrope cells (Kuiper
et al., 2000
). Up to then, only one representative of the
p24
subfamily had been described in vertebrates, with mouse p24
being more related to Xp24
1 than to Xp24
2
(amino acid sequence identities of 82.2% and 70%, respectively).
Analysis of p24 sequences
A multiple amino acid sequence alignment of all known Xenopus p24
proteins revealed a low degree of overall amino acid sequence identity between
the members of the different subfamilies
(Fig. 1A). However, the
topology common to all p24 proteins is also preserved in the Xenopus
proteins (an N-terminal signal sequence, a large lumenal domain followed by a
transmembrane stretch, and a cytoplasmically exposed C-terminal region of
10-16 residues), as are conserved amino acid residues such as the two cysteine
residues that form a disulfide bridge in the N-terminal region, a glutamine
residue within the transmembrane domain and a phenylalanine residue that
constitutes part of a COPII-binding motif
(Dominguez et al., 1998).
Furthermore, heptad repeats of aliphatic amino acids are found in the membrane
proximal parts of the lumenal domains of Xp24
2,
-
3, -ß1, -
1,
-
1 and -
2 (but not in
Xp24
2 and -
3) that have a medium to high
propensity to form coiled-coil structures (>0.4 by coils algorithm, version
2.2) (Lupas, 1996
).
Coiled-coil interactions between members of different p24 subfamilies are
involved in the formation of hetero-oligomeric complexes
(Ciufo and Boyd, 2000
). The
alternatively spliced form of Xp24
1 has in this particular
region an insertion of ten amino acids (following residue +148), which reduces
its propensity to form coiled-coil structures. The cytoplasmic tail sequences
are highly conserved between the two Xenopus p24
subfamily
members, as they are in the p24
subfamily and the
p24
1/
2 subgroup. A classical ER
retrieval/COPI-binding motif in the C-terminal region (K(X)KXX) is present
only in the Xenopus p24
proteins, whereas members of the other
subfamilies show variations of this motif. Binding studies with the
cytoplasmic domains of human p24 proteins have revealed efficient COPI binding
for hp24
2 and also for hp24
1 (despite an
imperfect COPI-binding motif), while all human p24 proteins analyzed
(
2, ß1,
3,
4,
1) have been found to interact with
COPII (Dominguez et al.,
1998
). Since the critical amino acid residues in the cytoplasmic
tails are conserved between the human and Xenopus p24 proteins,
similar binding properties are to be expected for the Xenopus p24
proteins.
During evolution, the genome of Xenopus laevis underwent a genome
duplication event, causing this species to be tetraploid
(Graf and Kobel, 1991). As a
consequence, two highly conserved genes (paralogs) are usually found. This was
also the case for every novel p24 protein isolated in this study. The
nucleotide sequence identities over the entire ORFs were found to be in the
order of 94 to 95.5%, and the nucleotide substitutions were either neutral or
led to conservative amino acid substitutions in the deduced primary sequences
of the respective p24 proteins. For clarity, the primary sequence of only one
paralog is shown in Fig. 1.
In summary, the melanotrope cells in the intermediate pituitary of
Xenopus express one member of the p24 (
3),
one of the p24ß (ß1), two of the p24
(
2,
3) and two of the p24
(
1,
2) subfamily. Two members,
Xp24
2 and -
1, show a tissue-specific
distribution in that they were expressed in Xenopus embryos but not
in the intermediate pituitary.
Expression of the various Xenopus p24 proteins in the
pituitary gland
To study the expression of the various Xenopus p24 proteins,
polyclonal antisera directed against sequences in the lumenal domains of these
p24 proteins were generated. Western blot analysis revealed that the various
antisera recognize specifically the corresponding Xp24 proteins of the
melanotrope cells (Kuiper et al.,
2000) (data not shown), except for the anti-
C antiserum,
which recognizes both Xp24
proteins (
1 and
2). To study the expression of the p24 proteins in the
pituitary, lobes from black- and white-adapted Xenopus were manually
dissected into the NIL and the anterior lobe (AL). Together with tissue
extracts from brain and hypothalamus, NIL and AL lysates were separated with
SDS-PAGE and analyzed by immunoblotting (three times more protein was loaded
for brain and hypothalamus). In NIL lysates of black-adapted animals, the
antibodies directed against epitopes in the lumenal domains of
Xp24
3, -ß1, -
2,
-
3, -
1, and -
2
recognized proteins with a molecular mass of
25, 21, 24, 25.5, 19 and 21
kDa, respectively (Fig. 2, data
not shown for anti-
1 and anti-
2). With an
antibody against Xp24
1 (residues 18-194), a 22 kDa protein
was identified in tissue lysates from brain and hypothalamus, while no protein
was detected in the NIL (Fig.
2), in line with the finding that a p24
1 cDNA
could be isolated only from a Xenopus embryo but not from a NIL cDNA
library. At present it is not clear if the 23 kDa product in the anterior lobe
of the pituitary detected with the anti-
1L antiserum
corresponds to the alternatively spliced form of Xp24
1, or
is due to nonspecific binding to the similarly sized and highly expressed
anterior pituitary hormones prolactin/growth hormone (judged by staining of
the western blot with Ponceau S). Interestingly, immunoblot analysis of
cellular extracts showed that the protein levels of Xp24
3,
-ß1, -
3, and -
2 were
highly upregulated in NILs of black-adapted Xenopus when compared
with that in white-adapted animals (
20-30-fold). By contrast, the level
of Xp24
1 expression was only slightly induced
(
threefold) in NIL lysates of black-adapted animals, and no change was
observed for Xp24
2 (Fig.
2). As previously demonstrated for POMC
(Holthuis et al., 1995a
), the
physiologically induced changes in the expression levels of
Xp24
3, -ß1, -
3 and
-
2 were strictly confined to the melanotrope cells of the
NIL and did not occur in cells from the AL of the pituitary
(Fig. 2). Semiquantitative
reverse transcription (RT)-PCR revealed a three- to fivefold increase in the
levels of Xp24
3, -ß1, -
3
and -
2 mRNA in the melanotropes of black-adapted animals,
whereas the levels of Xp24
1 and -
2 mRNA
were similar (data not shown).
|
Next, we tried to confirm our results using immunocytochemistry. The p24
proteins Xp243, -ß1, -
3
and -
2, shown by western blot analysis to be differentially
regulated in the NIL, displayed intense staining in the melanotrope cells of
the intermediate pituitary of a black-adapted animal, whereas their expression
in a white-adapted animal was low (Fig.
3). Conversely, for Xp24
2 and
-
1, a low degree of expression was observed in the
melanotrope cells, independent of background adaptation. The differential
regulation of the various p24 proteins was again confined to the melanotrope
cells, because no differences in the expression patterns were observed in the
anterior lobes of black- and white-adapted animals. Furthermore, the
homogenous distribution of the p24 proteins throughout the intermediate lobe
suggests that they are expressed in all melanotrope cells. Antisera directed
against Xp24
1 showed no immunoreactive staining in the
melanotrope cells, whereas in the anterior pituitary some immunoreactive
material was detected, again likely due to nonspecific cross-reactivity as was
observed during Western blot analysis. In conclusion, we find that during
background adaptation only a subset of the p24 proteins
(Xp24
3, -ß1, -
3, and
-
2) expressed in the melanotrope cells of the intermediate
pituitary is coordinately expressed with the prohormone POMC.
|
p24 expression in Xenopus tissues
The distributions of the various Xp24 proteins in tissues other than the
pituitary was studied at the level of RNA (Northern blot analysis) and at the
protein level (Western blotting). For Northern blot analysis, total RNA was
isolated from a number of Xenopus tissues and hybridized with
[32P]-labelled probes covering the entire ORF of
Xp243, -
1, -
2,
-
3 or with a 364-bp fragment of Xp24ß1
(nucleotides +77 to +364). The applied method was not sensitive enough for the
detection of Xp24
1 and -
2 transcripts,
whereas more than one transcript was found for Xp24
3,
-ß1 and -
3
(Fig. 4A).
Xp24
3, -ß1 and -
3 mRNAs
were found to be expressed in brain, liver, kidney, spleen, heart and lung, as
was previously found for Xp24
1 and -
2
mRNAs (Holthuis et al., 1995b
)
(data not shown). Owing to the tetraploid nature of the Xenopus
genome, a pair of closely related genes (paralogs) is expressed that often
gives rise to transcripts with different sizes. Xp24
3 is
represented by two transcripts of about 1.2 and 2.5 kb, ubiquitously expressed
in all tissues examined. The size of the 1.2 kb transcript corresponds to the
insert sizes of two full-length cDNA clones encoding paralog A of
Xp24
3. The transcript length expected for paralog B of
Xp24
3 is not known and may be represented by the 2.5 kb
transcript. With an Xp24ß1 probe, a predominant mRNA
transcript of 2.5 kb and a weak one of 1.2 kb were detected. The
Xp24ß1-encoding cDNAs for the two paralog genes were derived
from a 2.5-kb transcript and thus the 1.2-kb transcript may arise from
alternative splicing of nuclear RNA or from the use of an alternative
polyadenylation signal. Three transcripts (2.3, 2.5 and >4 kb) with similar
intensities were observed for Xp24
3 mRNAs. Since the sizes
of two transcripts (2.3 and 2.5 kb) correspond to full-length
Xp24
3 cDNA clones, the presence of the >4 kb transcript
indicates that Xp24
3 gene expression may also include
alternative splicing and/or alternative usage of polyadenylation signals.
|
The ubiquitous tissue expression of Xp24 proteins observed at the mRNA
level was also seen at the protein level
(Fig. 4B). With the
anti-3L antibody, expression of Xp24
3 was
found to be high in ovarium and liver, intermediate in hypothalamus, brain,
kidney and spleen, and low in heart and lung. This antibody recognized a
second protein band in liver and lung that was slightly larger in size and
represents the only protein detected in heart tissue. The two protein bands
(
25 and
26 kDa) may reflect different glycosylation states of
Xp24
3, which is N-linked glycosylated at a single site (data
not shown). Tissue-dependent variations in the glycosylation state have also
been described for hp24
2
(Füllekrug
et al., 1999
). Alternatively, the anti-
3L
antibody may recognize with low affinity the highly related
Xp24
2 protein, which at least in liver is known to be
expressed. The overall tissue distributions of Xp24ß1,
-
3, and -
2 were similar to that obtained
for Xp24
3 and the four proteins are predominantly expressed
in liver and ovarium. Except for the intermediate pituitary, the
Xp24
1 protein was expressed in all tissues examined
(Fig. 4). When compared to the
levels obtained for Xp24
3, -ß1,
-
3 and -
2, the relatively low levels of
Xp24
1 expression in ovarium and liver are remarkable. The
tissue distribution of Xp24
1 is similar to those of
Xp24
2 and -
1 (the two proteins that are
not differentially regulated in the intermediate pituitary), although both
Xp24
2 and -
1 show higher levels of protein
expression in ovarium. Taken together, these experiments indicate that the
members of the p24 family are widely expressed but they may display a
cell-type specific expression, as was found for Xp24
2 and
-
1 in the melanotrope cells of the intermediate
pituitary.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the physiologically manipulated Xenopus melanotrope cells, we
demonstrated that during black background adaptation the protein levels of
Xp243, -ß1, -
3 and
-
2 were increased 20 to 30 times, whereas the expression of
the Xp24
2 and -
1 proteins remained
unchanged or increased by only three times, respectively. In yeast and
mammals, p24 proteins form tetrameric complexes with a defined complex
composition, in which one member from each subfamily is present
(Füllekrug
et al., 1999
; Marzioch et al.,
1999
). Furthermore, the steady-state protein level and the
intracellular localization of a p24 protein is dependent on the presence or
absence of other p24 members that participate in the oligomeric complex
(Denzel et al., 2000
;
Emery et al., 2000
;
Füllekrug et
al., 1999
; Marzioch et al.,
1999
). Since Xp24
3, -ß1,
-
3 and -
2 show similar dynamics in protein
expression in the melanotrope cells and also in other tissues, they may well
form a tetrameric p24 complex, whereas Xp24
2 and
-
1, which are not differentially regulated in the
melanotrope cells, could be constituents of other p24 complexes.
Interestingly, the apparent composition of the main tetrameric melanotrope p24
complex (Xp24
3, -ß1, -
3,
and -
2) is different from the one previously identified in
HeLa cells, where a
p24
2/ß1/
3/
1
p24 complex exists (GMP25/p24/gp27/p23)
(Füllekrug
et al., 1999
). Variations in p24 complex formation are likely to
occur, for example, because of the observed cell-type-specific expression of
the various Xenopus p24 proteins. Furthermore, in yeast an
Erp1p(yp24
)/Erp2p(yp24
)/Emp24p(yp24ß)/Erv25p(yp24
)
complex is present, in which Erp1p can be substituted by another p24
subfamily member (Erp5p and/or Erp6p) if Erp1p is not expressed
(Marzioch et al., 1999
). Also,
hp24
4 has been found to be excluded from the HeLa cell
2/ß1/
3/
1
p24 complex
(Füllekrug
et al., 1999
), while mp24
4 may well participate
in a p24 complex (Denzel et al.,
2000
). Therefore, the composition of a tetrameric p24 complex
appears to be cell-type specific.
The exact role of the p24 proteins is still elusive, but p24 proteins have
been implicated in a number of functions that are all linked to vesicular and
protein transport, such as regulation of cargo inclusion in ER vesicles
(Muñiz et
al., 2000;
Schimmöller
et al., 1995
), quality control mechanisms in the ER
(Belden and Barlowe, 1996
;
Wen and Greenwald, 1999
),
recruitment and regulation of COPI/II vesicle coat assembly
(Bremser et al., 1999
;
Kaiser, 2000
;
Kuehn et al., 1998
), and
generation of vesicular tubular clusters
(Lavoie et al., 1999
;
Rojo et al., 2000
). In yeast,
an interaction between the cargo protein Gas1p and p24 proteins has been
demonstrated, and loss of function of certain p24 proteins reduces the
kinetics of ER-to-Golgi transport of a subset of secretory proteins, whereas
resident ER proteins (Kar2p and Pdi1p) are less efficiently retained in the ER
(Elrod-Erickson and Kaiser,
1996
; Marzioch et al.,
1999
;
Muñiz et
al., 2000
;
Schimmöller
et al., 1995
). In C. elegans, reducing the activity of
certain p24 proteins restores at least partially the ER transport block of a
mutant protein to the plasma membrane (Wen
and Greenwald, 1999
). Proper p24 function may thus facilitate the
transport of certain cargo molecules and restrict the entry of ER proteins and
incorrectly folded proteins into COPII vesicles. Along this line, in the
activated Xenopus melanotrope cells a
Xp24
3/ß1/
3/
2
complex could be involved in the inclusion of POMC into transport vesicles, as
the four p24 members are coordinately expressed with this prohormone.
Unfortunately, extensive crosslinking and co-immunoprecipitation studies using
the Xenopus intermediate pituitary cells have not allowed us to
establish a direct physical interaction between the Xenopus p24
proteins or between p24 and POMC.
In conclusion, we isolated and characterized the set of p24 proteins expressed in a single cell type (the Xenopus intermediate pituitary melanotrope cell), and revealed that their expression is cell-type specific and can be selectively induced. In the melanotropes, four of the six p24 members are coexpressed and these representatives of the four subfamilies may form a complex that is involved in the efficient ER to Golgi transport of its major cargo protein POMC. Together, our results thus point to an involvement of p24 proteins in the process of selective protein transport within the early secretory pathway.
![]() |
Acknowledgments |
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