1 Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer
Feld 328, 69120 Heidelberg, Germany
2 Max-Planck-Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, D-01307 Dresden, Germany
3 Central Spectroscopy Department, German Cancer Research Center, Im Neuenheimer
Feld 280, 69120 Heidelberg, Germany
4 Max-Planck Institut für Biochemie, Martinsried,
Germany
* Author for correspondence (e-mail: helms{at}uni-hd.de )
Accepted 5 November
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Summary |
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By mass spectrometry, GAPR-1 was shown to be myristoylated. Immunoprecipitation of GAPR- 1 from Golgi membranes resulted in the coimmunoprecipitation of caveolin-1, indicating a direct interaction between these two proteins. Myristoylation, together with protein-protein or electrostatic interactions at physiological pH owing to the highly basic pI of GAPR-1 (pI 9.4) could explain the strong membrane association of GAPR-1.
Tissue screening revealed that GAPR-1 is not detectably expressed in liver, heart or adrenal glands. High expression was found in monocytes, leukocytes, lung, spleen and embryonic tissue. Consistent with the involvement of PR-1 proteins in the plant immune system, these data could indicate that GAPR-1 is involved in the immune system.
Key words: Golgi, Microdomains, Rafts, Plant pathogenesis-related protein, PR-1, Myristoylation, Caveolin
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Introduction |
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Since the identification of plant PR-1 proteins, secretory proteins with a
significant sequence homology to PR-1 have been identified in various other
organisms. Examples of PR-1-related proteins include fruiting body proteins in
fungi that are expressed during infection
(Schuren et al., 1993), insect
allergens (Lu et al., 1993
;
Schreiber et al., 1997
),
mammalian CRISP proteins, which may be involved in sperm maturation or
sperm-egg fusion (Kjeldsen et al.,
1996
; Kratzschmar et al.,
1996
), human GliPR/RTVP-1, which is specifically expressed in
glial tumors (Murphy et al.,
1995
; Rich et al.,
1996
) and snake or lizard venoms, which are reported to block
ryanodine receptors or cyclic nucleotide-gated ion channels
(Brown et al., 1999
;
Morrissette et al., 1995
).
Together with plant PR-1 proteins, these proteins constitute a large PR-1
protein superfamily.
Here we describe a novel member of this family that was identified as a
component of lipid-enriched microdomains at the Golgi complex. The lipid
scaffold for this type of microdomain is built mainly by sphingomyelin (SM)
and cholesterol (Brown and London,
1998b; Simons and Ikonen,
1997
). By differential participation of proteins and lipids,
microdomains are believed to play an important role in signal transduction and
intracellular membrane transport events such as protein sorting
(Anderson, 1998
;
Brown and London, 2000
;
Simons and Toomre, 2000
).
Although the in vivo existence of microdomains in biological membranes has
been difficult to prove, evidence is accumulating in favour of their presence.
Important details such as their size and dynamics, however, are still
intensely discussed (Brown and London,
1998a
; Brown and London,
1998b
; Brown and London,
2000
; Simons and Toomre,
2000
). A widely used approach to study microdomains has been their
isolation as low-density detergent-insoluble complexes. We used this method
for the isolation of lipid-enriched microdomains from isolated Golgi membranes
(Gkantiragas et al., 2001
). To
determine the architecture and function of microdomains at the Golgi complex,
we initiated a systematic identification of their proteins by microsequencing.
Two peptide sequences derived from a protein with an apparent molecular weight
of 17 kDa did not match any of the proteins identified to date. Here we report
the cloning and biochemical characterization of this protein. On the basis of
sequence homology, this novel protein belongs to the superfamily of plant
pathogenesis-related proteins. Biochemical characterisation revealed some
unique properties of this protein that have not yet been reported for other
protein family members.
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Materials and Methods |
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Subcellular fractions from CHO cells
CHO Golgi membranes and COPI-coated vesicles were isolated as described
previously (Balch et al., 1984;
Malhotra et al., 1989
;
Serafini et al., 1991
). CHO
cytosol was prepared by centrifugation of a CHO homogenate (prepared as
described for the isolation of CHO Golgi membranes) for 1 hour at 100,000
g at 4°C. The supernatant was stored at -80°C before
use.
Isolation of low-density detergent-insoluble fractions from Golgi
membranes and from total cell lysates
For the identification of GAPR-1, low-density detergent-insoluble complexes
were isolated from CHO Golgi membranes. This method is described in detail by
Gkantiragas et al. (Gkantiragas et al.,
2001). In short, 5 mg (protein) of CHO Golgi membranes (isolated
as described above) were pelleted and resuspended in 2 ml of PEN buffer (25 mM
Pipes, pH 6.5, 2 mM EDTA, 150 mM NaCl) containing 1% Triton X-100. The
suspension was incubated for 30 minutes on ice, mixed with 2 ml of 80% (w/v)
sucrose in PEN buffer and transferred to a SW41 rotor tube (Beckman). The 40%
sucrose fraction was overlayed with subsequently 1.3 ml of each 30%, 25%, 20%,
15%, 10% and 5% sucrose in PEN buffer. The samples were centrifuged for 22
hours at 190,000 g at 4°C. After centrifugation, the
Golgi-drived detergent-insoluble complexes (GICs) migrating as an opalescent
band at the 10-15% sucrose interface was collected.
Low-density detergent-insoluble complexes from total-cell lysates (total DRM) were isolated according to the same procedure by solubilization of a subconfluent cell monolayer of NRK cells (5x175 cm2 plates) in 2 ml of PEN buffer+1% TX-100.
Cloning, sequencing and sequence analysis of GAPR-1
Two peptide sequences, obtained from microsequening of p17 from CHO GICs,
were used to search DNA databases. Several ESTs containing both peptide
sequences in the same reading frame were identified (GenBank accession numbers
T78145, aa339686, aa428123). EST44799 (obtained from ATCC (Rockville, USA))
was sequenced by TopLab, Martinsried, Germany, before being cloned into
pBluescript SK- (GenBank accession number aa339686). The sequence revealed a
complete open reading frame with 462 bases by use of vector primer M13 and
primer walking. The corresponding sequence is accessible at the EMBL/GenBank
database (NP_071738). Homologues of GAPR-1 were identified by searching the
Swissprot database using the BLASTP2 search engine (EMBL, Heidelberg,
Germany). The predicted coil-coil sequence (amino acid 29-49) was identified
by the software described by Lupas et al.
(Lupas et al., 1991)
(www.ch.embnet.org/software/COILS_form.html).
In-gel tryptic digest
The protein band obtained after SDS-PAGE was excised, cut into small
pieces, destained with 30% acetonitrile 0.1 M NH4HCO3,
washed with water, dried in a centrifugal evaporator (vacuum concentrator,
Bachofer, Reutlingen, Germany) and rehydrated with 10 µ1 of the digest
solution. This solution contained 50 µg/ml trypsin in 0.1 M
NH4HCO3. After incubation at 37°C over night, the
tryptic fragments were extracted with 2x10 µl of 5% formic acid. The
combined extracts were desalted using C-18 ZipTips (Millipore, Bedford, MA).
The peptides were eluted in 3 µl 50% acetonitrile, 2% formic acid and
transferred into a nanoESI capillary.
Mass spectrometry
Mass spectra were recorded using a quadrupole time-of-flight instrument
Q-TOF 2 (Micromass, Manchester, UK) equipped with a nanoESI source and
operated with a resolution of about 8000 (FWHM). Spray capillaries were
prepared in-house using a micropipette puller P-87 (Sutter Instruments,
Novato, CA), and subsequently surface coated with a semitransparent film of
gold using a gold sputter unit. The spray voltage used was about 1200 V, and
argon was used as a collision gas.
Immunoprecipitation
10 µl of polyclonal serum against the C-terminus of GAPR-1 (#1852) was
incubated with 50 µl of protein A Sepharose (Fast Flow, Amersham Pharmacia
Biotech, Freiburg, Germany) and 50 µl of PBS containing 0.5% milk for 90
minutes at RT. The beads were washed twice with PBS and twice with IP buffer
(PEN+1% TX-100) before use. Golgi membranes (500 µg) were centrifuged
(100,000 g for 30 minutes at 4°C), and the pellet was
resuspended in 100 µl 1%SDS and incubated for 5 minutes at 95°C. The
sample containing denatured proteins was diluted with 900 µl of PEN+1%TX-
100 and used for immunoprecipitation of GAPR-1 by incubation overnight at
4°C with the protein A beads. The beads were subsequently washed twice in
PEN+1%TX-100 and four times in PEN before analysis. For western blot analysis,
a protein A-HRP conjugate (Biorad Laboratories GmbH,
München, Germany) was used that does not
recognize the denatured IgGs on the blot that were eluted from the
immunoprecipitation beads.
Immunofluorescence
Vero cells were cultured in Dulbecco's minimal essential medium (DMEM)
supplemented with 10% FCS to subconfluency and prepared for indirect
immunofluorescence according to standard procedures. Cells were fixed either
in MeOH (1-2 minutes at -20°C) or fixed in 3% (w/v) paraformaldehyde for
20 minutes and permeabilized for 5 minutes in 0.1% saponin. After incubation
of first and secondary antibodies, cells were washed and embedded in
Fluoromount G (biozol, Eching, Germany). Images were taken using the Leica
TCS-NT confocal laser scan microscope (Leica Lasertechnique, Heidelberg,
Germany). Confocal images of double labelling experiments were obtained
simultaneously to exclude any artefacts from sequential acquisition. Both
channels were adjusted to ensure that the maximum fluorescence intensity was
still in the recording range. Only one focal plane was analysed. Stainings
shifted against each other were confirmed by series of z sections and repeated
simultaneous scans. Micrographs were arranged with Adobe Photoshop and
Illustrator.
Viral infection
Vero cells were maintained and infected with tsO45 vesicular stomatitis
virus (VSV; Indiana Serotype) as described previously
(Kreis, 1986). Incubation of
cells at 15°C was performed under conditions as described in Fullekrug et
al. (Fullekrug et al., 1999
).
In short, cell culture medium was replaced by DMEM containing 20 mM Hepes, pH
7.2 and 200 µM cyclohexamide. After incubation, the cells were fixed with
methanol for 4 minutes at -20°C. A mouse mAb that recognizes an exoplasmic
epitope of VSV-G (Pepperkok et al.,
1993
) was used to detect the presence of tsO45-G protein, followed
by Cy3-labelled antimouse secondary antibody (Alexis Corporation, CA). Cells
were mounted in Fluoromount G (Biozol) and analysed using a Zeiss Axiovert 35
microscope equipped with the appropriate filters for Cy3-derived
fluorescence.
Protease digestion of Golgi membranes
Isolated CHO Golgi membranes (10 µg) were incubated with Trypsin (2.5
µg) in 25 mM Hepes/KOH, pH 7.2, 20 mM KCl, 2.5 mM MgOAc for 30 minutes at
30°C. Where indicated, Trypsin inhibitor (25 µg) or Triton X-100 (1%
final concentration) were added to the assay. The Golgi membranes were
pelleted through a sucrose cushion (15% sucrose w/v) at 14,000
g for 30 minutes. The proteins were analysed by SDS-PAGE and
subsequent western blot analysis (ECL) using a GAPR-1-specific antibody
(#1852) and a p23-specific antibody raised against the N-terminus of the
protein (KAI2/3 (Sohn et al.,
1996)).
Preparation of a total membrane fraction from various tissues
Isolated tissues (heart, brain, testis, lung, liver, spleen, muscle,
kidney, adrenal gland and pancreas) from male rats were homogenized in 0.2 M
sucrose in PEN buffer (tissue:buffer 1:4 (w/w)) in the presence of protease
inhibitors. The homogenate was centrifuged for 10 minutes at 650
g at 4°C to obtain a postnuclear supernatant (PNS). A
membrane fraction was isolated from the PNS by centrifugation for 1 hour at
100,000 g at 4°C. Leukocytes were prepared from rat blood
by extensive washing of 4 ml of rat blood in lysis buffer (0.15 M
NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2) until all
erythrocytes were lysed. The white cell pellet is enriched in leukocytes and a
total membrane fraction was obtained as described above.
The embryo, uterus and placenta from rats were kindly provided by Celina Cziploch (ATV, Heidelberg). Monocytes (from human blood) were kindly provided by Anne Große Wilde (DKFZ, Heidelberg).
Miscellaneous
Protein-determination was performed according to Lowry
(Lowry et al., 1951).
Quantitation of phosphatidylcholine in CHO Golgi membranes and COPI-coated
vesicles was performed by nano-electrospray ionization tandem mass
spectrometry
(Brügger et
al., 1997
; Helms et al.,
1998
).
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Results |
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Sequence analysis revealed the presence of a consensus sequence for
myristoylation (Met-Gly-X-X-X-Ser/Thr)
(Resh, 1999) at the N-terminus
of GAPR-1 and a coiled-coil region (Lupas,
1996
) between amino acids 29-49, which might be involved in
protein-protein interactions. A caveolin-binding motive
(Okamoto et al., 1998
)
(-X-XXXX-, where - is aromatic amino acid Trp, Phe or Tyr) is found between
amino acids 90-97.
GAPR-1 belongs to a superfamily of proteins
No homologous proteins have been identified to date, although several
expressed sequence tags from various mammalian sources (including bovine, pig,
mouse, and zebrafish) have been reported to contain fragments with identical
sequences. GAPR-1 belongs to a superfamily of proteins that include human
GliPR (Murphy et al., 1995),
mammalian cysteine-rich secretory proteins
(Kratzschmar et al., 1996
),
plant pathogenesis-related proteins group 1 (plant PR-1 proteins)
(Bol et al., 1990
), allergens
of insect venoms (Lu et al.,
1993
) and snake or lizard venoms
(Morrissette et al., 1995
).
The members within these subfamilies that share the highest homology to GAPR-1
are shown in Fig. 1C. The
identity of GAPR-1 to these proteins varies between 30% (TPX1) and 50%
(Drosophila CG2337), and the homology varies between 46% (AG5) and
63% (Drosophila CG2337). In yeast there are three hypothetical
proteins (PRY1-3) that have a domain with a similar level of identity (35-41%)
and homology (51-53%) to GAPR-1. The functions of this protein superfamily are
presently unknown. GAPR-1 contains two histidine (His54 and His103) and two
glutamate residues (Glu65 and Glu86) that are highly conserved throughout the
protein family. In the NMR structure of p14a, a plant PR-1 protein, these four
amino-acid residues are located in a cleft that might represent an active site
of the protein (Fernandez et al.,
1997
; Szyperski et al.,
1998
).
GAPR-1 is a Golgi localized peripheral membrane protein
GAPR-1 was originally identified in a low-density detergent-insoluble
fraction from a Golgi-enriched fraction named GICs
(Gkantiragas et al., 2001). To
confirm the identity of the cloned protein, enrichment of GAPR-1 in Golgi
membranes was determined by use of a peptide antibody that was generated from
the sequence of the cloned protein. As shown in
Fig. 2A, GAPR-1 is enriched
(30-50 fold) in an isolated Golgi fraction, which corresponds well with the
enrichment of Golgi markers in this fraction
(Brügger et
al., 2000
). In addition, GAPR-1 is highly enriched in a GIC
fraction (
65 fold compared to Golgi membranes, as evaluated by
quantitation of Fig. 2A, lanes
3 and 4). Quantitative analysis showed that GAPR-1 represents
0.1% of
total Golgi proteins (data not shown). GAPR-1 could not be detected in the
cytosolic fraction (see also Fig.
4).
|
|
The topological orientation of GAPR-1 was determined by trypsin treatment
of Golgi membranes. As shown in Fig.
2B, GAPR-1 was completely digested by trypsin in the absence of
detergent. As a control, only the C-terminal fragment (1 kDa) was removed
from p23, a Golgi-localized type I membrane protein with a short cytoplasmic
tail, showing that its large luminal domain was not accessible to trypsin and
thus the membranes are sealed under these conditions. In a control experiment
it was shown that the luminal domain of p23 can in principle be digested by
trypsin in the presence of detergent (Fig.
2B, lane 4). Thus, with intact membranes, GAPR-1 is accessible to
trypsin and therefore has a cytosolic orientation.
As GAPR-1 is localized to Golgi membranes, we determined whether GAPR-1 is
also present in COPI vesicles, which mediate transport of proteins between
different Golgi cisternae. COPI-coated vesicles were isolated from a
large-scale cell-free incubation of Golgi membranes in the presence of
GTPS. The vesicles were quantified on the basis of their
phosphatidylcholine (PC) composition. This lipid is present in relatively high
amounts in membranes and is not expected to be sorted within the Golgi Stack
(Brügger et
al., 2000
; van Meer,
1989
). Fig. 2C
shows a comparison of equal amounts of donor Golgi membranes and COPI-coated
vesicles, on the basis of their phosphatidylcholine content. They are
characterized by p23, a Golgi-localized protein that is enriched in
COPI-coated vesicles, in agreement with previous observations
(Sohn et al., 1996
). In
addition, ß'-COP, a subunit of the coatomer complex, is enriched in
the vesicle fraction, as expected. Antibodies against GAPR-1 were then used to
identify the presence of GAPR-1 on the vesicles. Whereas GAPR-1 is present on
Golgi membranes, GAPR-1 could not be detected in the purified COPI-vesicle
fraction derived from the donor Golgi membranes. This is in agreement with
previous observations, where we showed that some of the GIC proteins were
found to be excluded from COPI vesicles
(Gkantiragas et al.,
2001
).
N-myristoylation of GAPR-1
To determine whether the consensus sequence for N-myristoylation results in
in vivo myristoylation of GAPR-1, proteins from a large-scale preparation of
Golgi-derived detergent-insoluble complexes were separated by SDS-PAGE. The
protein band at 17 kDa was excised from the gel and digested with trypsin. The
resulting peptides were analysed by electrospray mass spectrometry
(Fig. 3A, top panel). If native
GAPR-1 is myristoylated, trypsinization should generate the myristoylated
dipeptide myrGK with an [M+H]+ signal at m/z 414.33. The survey
spectrum of the tryptic digest of GAPR-1 showed a signal at m/z 414.327
(Fig. 3A, lower panel), which
is consistent with the calculated m/z value for the myristoylated T1-fragment
(within the accuracy of our mass determination). The ion at m/z 414.327 was
further analysed by ESI tandem mass spectrometry, and the product obtained
from the ion spectrum is displayed in Fig.
3B. All major fragment ions in this spectrum can be assigned to
the myrGK sequence, as indicated in Fig.
3B. In particular the fragment ion triplet at 211, 240 and 268 is
indicative of the myrG structure. In summary, the mass spectroscopic data show
that native GAPR-1 is N-terminally myristoylated. It remains to be established
whether GAPR-1 is completely or only partially myristoylated.
|
Association of GAPR-1 with Golgi membranes
GAPR-1 is tightly associated with Golgi membranes, which is reflected in
the absence of GAPR-1 from the cytosolic fraction
(Fig. 2). In agreement with
this, GAPR-1 could not be detected by immunoprecipitation from large amounts
of cytosol (Fig. 4A). GAPR-1
could, however, be efficiently immunoprecipitated from Golgi membranes and the
total membrane fraction. This indicates that GAPR-1 is completely absent from
the cytosol and that GAPR-1 is not likely to cycle on and off Golgi membranes
like other myristoylated Golgi-localized proteins such as ARFs. The
characteristics of this tight membrane association of GAPR-1 were further
investigated by incubation of isolated Golgi membranes under various
conditions. As shown in Fig.
4B, treatment of Golgi membranes with 1 M KCl does not strip
GAPR-1 off the membranes, whereas NSF, a peripheral Golgi membrane protein
(Block et al., 1988), and
exogenous GAPR-1 (see below) are affected by this treatment. Alkaline
treatment of the membranes causes the dissociation of most peripheral membrane
proteins, including NSF and GAPR-1 (Fig.
4B). In contrast, p23, a type I transmembrane protein of the Golgi
complex (Sohn et al., 1996
),
remains present in salt or alkaline-treated membranes
(Fig. 4B). To determine whether
the myristoyl moiety of GAPR-1 could contribute to the saltresistant membrane
binding, purified non-myristoylated GAPR-1 was bound to isolated Golgi
membranes (Fig. 4C). Upon salt
treatment of the membranes, most of the non-myristoylated GAPR-1 is stripped
from the membranes. These data indicate that native GAPR-1 is bound to Golgi
membranes not only by ionic interactions but also through the myristoyl
moiety, which might affect the membrane anchoring of the protein.
GAPR-1 contains a caveolin-binding motive, which might contribute to its
strong membrane-binding characteristics. A direct interaction between
caveolin-1 and GAPR-1 was investigated by coimmunoprecipitation studies. Under
native conditions using various detergents, caveolin-1 could not be
coimmunoprecipitated with GAPR-1 (data not shown). Therefore,
coimmunoprecipitation studies were performed after chemical crosslinking of
Golgi membranes. As shown in Fig.
4D (lane 1 and 2), crosslinking with
NHydroxylsulfosuccinimidyl-4-azidobenzoate results in irradiation-dependent
crosslink products of caveolin-1 at 45-50 kDa
(Fig. 4D, X-link 45-50 kDa) and
at high molecular weights (Fig.
4D, X-links HMW). At 45-50 kDa, a similar crosslink product was
observed for GAPR-1 (data not shown). When GAPR-1 was immunoprecipitated from
these incubations, caveolin-1 was found to coimmunoprecipitate with GAPR-1 in
a crosslink product of 45-50 kDa (Fig.
4D, lane 3). These results indicate a direct interaction of GAPR-1
with caveolin-1. The crosslink products at high molecular weights did not
coimmunoprecipitate with GAPR-1. These crosslink products thus probably
reflect an interaction of caveolin-1 with other proteins or with other
caveolin molecules, as caveolins are known to form stable high molecular
weight oligomers (Anderson,
1998).
GAPR-1 localizes to early Golgi compartments
The Golgi localization of GAPR-1 was confirmed by immunofluorescence. In
CHO cells, NRK cells and Hela cells, GAPR-1 colocalizes with established Golgi
markers such as coatomer and TGN38 (data not shown). As shown in
Fig. 5, GAPR-1 also colocalizes
with the KDEL receptor in Vero cells, which cycles through the early secretory
pathway but has a predominant Golgi localization under steady-state conditions
(Tang et al., 1993). In the
presence of brefeldin A (BFA), the Golgi structure is disrupted, and Golgi
enzymes are transferred to the endoplasmic reticulum (reviewed in
(Klausner et al., 1992
)).
However, certain cycling membrane proteins of the early secretory pathway such
as ERGIC-53, KDEL receptor and p24 family proteins accumulate instead in
tubulo-vesicular clusters scattered in the cytoplasm
(Fullekrug et al., 1999
;
Lippincott-Schwartz et al.,
1990
; Tang et al.,
1995
; Tang et al.,
1993
). As shown in Fig.
5, GAPR-1 also has a BFA-sensitive Golgi localization and
colocalizes with the KDEL receptor in a punctuate pattern scattered throughout
the cytosol. Interestingly, the kinetics by which the Golgi localization of
GAPR-1 is disrupted is different from that of the KDEL receptor. The
localization of the KDEL receptor is already affected by a 5 minute treatment
of the cells with BFA. In contrast to the KDEL receptor, the Golgi
localization of GAPR-1 is affected only after a 10 minute incubation of the
cells with BFA. After prolonged incubation with BFA, GAPR-1 and KDEL receptor
show an increased colocalization to tubulo-vesicular clusters. These data
indicate that GAPR-1 is localized to the early secretory pathway and behaves
like other cycling proteins. During the BFA-induced redistribution of GAPR-1
from Golgi membranes to tubulo-vesicular structures, GAPR-1 remains associated
with membranes. GAPR-1 could not be detected in the cytosol after
BFA-treatment of the cells (Fig.
4A).
|
To further corroborate this finding, we analysed the localization of GAPR-1
relative to the anterograde cargo accumulating at 15°C in the intermediate
compartment between the ER and the cis Golgi
(Saraste and Kuismanen, 1984;
Schweizer et al., 1990
). A
temperature-sensitive mutant of the membrane glycoprotein VSV-G is misfolded
at the restricted temperature of 39.5°C and accumulates in the ER
(Fig. 6). At 15°C, VSV-G
accumulates in punctuate structures that are scattered throughout the
cytoplasm and probably represent the tubulo-vesicular structures of the
intermediate compartment. Double immunofluorescence of GAPR-1 and VSV-G
protein at the restricted temperature shows an ER-localization pattern for
VSV-G, whereas GAPR-1 is localized to the perinuclear region
(Fig. 6). After incubation at
15°C in the presence of the protein synthesis inhibitor cyclohexamide,
VSV-G accumulates in punctuate structures. Under these conditions, some GAPR-1
colocalizes to these structures, resulting in a partial colocalization of
GAPR-1 with VSV-G. The colocalization of GAPR-1 with VSV-G is not as
pronounced as other cycling proteins such as the KDEL receptor
(Tang et al., 1993
) and VIP36
(Fullekrug et al., 1999
). This
indicates that the cycling of GAPR-1 through the early secretory might be
slow, in accordance with the slow redistribution induced by BFA as compared to
the cycling protein KDEL receptor.
|
As the early compartments of the secretory pathway contain strikingly lower
levels of sphingomyelin and cholesterol than the Golgi
(van Meer, 1998), we
determined whether the characteristics of detergent insolubility of GAPR-1
would change when GAPR-1 is present in the tubulo-vesicular structures. To
this end, cells were treated with BFA, and low-density detergent-insoluble
complexes were isolated from these cells by isopycnic sucrose density
centrifugation. As shown in Fig.
7, GAPR-1 remains in a low-density detergent-insoluble fraction,
indicating that once these microdomains have formed, they are stable enough to
survive in an altered membrane environment such as tubulo-vesicular
structures. This is consistent with the behaviour of other GIC proteins
(Gkantiragas et al.,
2001
).
|
Differential expression of GAPR-1
During the course of these studies, we found that in contrast to isolated
Golgi membranes from CHO cells, Golgi membranes from other sources such as
from rat and rabbit liver did not contain any GAPR-1
(Fig. 8A). This prompted us to
determine the expression levels of GAPR-1 in various rat tissues. As shown in
Fig. 8B, GAPR-1 is
differentially expressed and could not be detected in liver, heart and adrenal
glands. High expression levels were found in monocytes, the lung, spleen,
kidney lymphocytes, uterus and embryonic tissue. Thus, high expression levels
are found in immunocompetent cells and organs
(Klein and Horejs'i, 1999) and
might indicate a function for GAPR-1 in the immune system.
|
![]() |
Discussion |
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The mechanisms of microdomain localization of GAPR-1 remain to be
determined. We previously showed that several proteins that localize to
microdomains in the Golgi complex (GICs), including the B subunit of the
v-ATPase, flotillin-1, caveolin-1 and p17 (GAPR-1), interact with each other,
even after disruption of the microdomain scaffold
(Gkantiragas et al., 2001).
This indicates that the lipid scaffold is not absolutely required for
interactions between these proteins. We have now extended these findings by
the demonstration of a direct interaction between GAPR-1 and caveolin-1. The
interaction of GAPR-1 with caveolin-1 and/or other GIC proteins might provide
a means to localize GAPR-1 to microdomains.
Cycling of GAPR-1 through the early secretory pathway
As shown in Fig. 5, GAPR-1
redistributes into tubulo-vesicular clusters that are scattered in the
cytoplasm upon treatment of cells with BFA. BFA inhibits anterograde but not
retrograde transport of proteins, causing disassembly of the Golgi complex.
Under these conditions, Golgi-resident proteins such as mannosidase II are
transported back to the endoplasmic reticulum. Other proteins that cycle
through the early secretory pathway, such as ERGIC-53, KDEL receptor and p24
family proteins, have been reported to localize to tubulo-vesicular clusters
scattered in the cytoplasm (Fullekrug et
al., 1999; Hendricks et al.,
1992
; Lippincott-Schwartz et
al., 1990
; Tang et al.,
1993
). Not much is known about the nature of these dispersed
tubulo-vesicular clusters, also termed Golgi remnants. Recently, Warren and
co-workers suggested that Golgi remnants may be the substrates for the
synthesis of new Golgi cisternae (Seemann
et al., 2000
). In contrast to, for example, the KDEL receptor and
p24 family proteins, GAPR-1 is unlikely to be transported by COPI-coated
vesicles, as it is excluded from these carriers
(Fig. 2C). In support of this,
we find that the kinetics of BFA-induced redistribution of GAPR-1 are
different from proteins that are transported by COPI-coated vesicles such as
the KDEL-receptor. COPI-independent pathways through the early secretory
pathway have been described (Girod et al.,
1999
; White et al.,
1999
). It remains to be established, however, whether the slower
kinetics observed are due to an alternative pathway or due to an effect on
sorting (e.g. due to the presence of GAPR-1 in microdomains).
GAPR-1 is a new member of the protein superfamily of PR proteins
As shown in Fig. 1, GAPR-1
shares significant amino-acid sequence homology and identity with a large
variety of proteins derived from the three kingdoms of animals, plants and
fungi. These proteins share several distinctive features unique to this
superfamily and present in most family members: (i) two highly conserved
histidines (AA 54 and 103 in GAPR-1) and glutamates (amino acids 65 and 86),
which might represent an active center in a cleft, as observed in the NMR
structure of family member p14a (Fernandez
et al., 1997; Szyperski et
al., 1998
); and (ii) two motifs, GENL(A) and gHyTQvVW, are
conserved in all family members (corresponding to amino acid 64-68 and 102-109
in GAPR-1).
Despite the diversity within this superfamily, very little is known about
the function of the individual members. The first identified and most
intensely studied family members are the plant pathogenesis-related (PR)
proteins (Bol et al., 1990).
There are five groups of PR proteins (PR-1 to PR-5) that do not share
structural or functional similarities except for the induction of expression
of PR proteins after infection of the plants by bacteria, fungi and viruses.
More recently, the induction of PR proteins is used as an early marker for
systemic acquired resistance, a long lasting defence mechanism of the plants
against pathogens (Klessig et al.,
2000
; Maleck et al.,
2000
; Zhang et al.,
1999
). Of all the mammalian homologues identified to date, GAPR-1
has the highest level of identity and homology to PR-1 proteins. In addition,
the molecular mass of GAPR-1 (17 kDa) is very similar to that of PR-1 proteins
(16-19 kDa). The size of all other family members ranges between 23 and 89
kDa, reflecting the presence of a PR domain in a larger protein sequence.
As mentioned above, the plant PR-1 protein family is involved in systemic
acquired resistance, which has similarities to the innate immune system in
animals (Kitajima and Sato,
1999; Klessig et al.,
2000
; Maleck et al.,
2000
; Zhang et al.,
1999
). The innate immune system is the first line of defence and
is similar to the plant immune system; a primary challenge is the
discrimination of pathogens from self with the use of a restricted number of
receptors. We find high levels of GAPR-1 expression in immunocompetent cells
and organs. Consistent with a possible role in the innate immune system, we
find high levels of expression in leukocytes, monocytes, lung and spleen.
Another interesting finding is the high expression level of GAPR-1 in
embryonic tissue. This is also consistent with its homology to plant PR-1
proteins, which are highly expressed during cell division and differentiation
events in the development of plant roots and flowering
(Fraser, 1981
;
Kitajima and Sato, 1999
;
Memelink et al., 1990
).
Another human family member of PR-1 proteins, glioma pathogenesis-related
protein or GLIPR, is highly expressed in glial tumors and glioma-derived cell
lines. On the basis of these findings and induced expression upon phorbol
ester treatment in macrophages, Murphy et al. speculated about a function for
PR family members in the human immune response
(Murphy et al., 1995).
Unique features of GAPR-1 within the superfamily of PR-proteins
On the basis of sequence homology, GAPR-1 belongs to the superfamily of
plant pathogenesis-related proteins. Biochemical analysis of this protein,
however, revealed properties that are unique to GAPR-1 and not found in other
family members. The most prominent difference is that, in contrast to GAPR-1,
all other family members studied so far are encoded by DNA that contains a
signal sequence for translocation across the endoplasmic reticulum during
protein synthesis, resulting in their secretion, storage in secretory
granules, or accumulation in vacuoles (Bol
et al., 1990; Kjeldsen et al.,
1996
; Kratzschmar et al.,
1996
; Linthorst,
1991
; Lu et al.,
1993
; Magdaleno et al.,
1997
; Schreiber et al.,
1997
; Schuren et al.,
1993
). The other human family member, named GLIPR or RTVP-1
(Murphy et al., 1995
;
Rich et al., 1996
), is
preferentially expressed in glial tumours and also contains a probable signal
sequence (Rich et al.,
1996
).
GAPR-1 is the first family member that does not have a signal sequence, and
as a result has an intracellular cytoplasmic localization. In addition, GAPR-1
is the first family member shown to bind to membranes. The involvement of
myristoylation in membrane binding of GAPR-1 has been discussed above.
Acylation of other members of the protein superfamily has not been reported.
Finally, the association of GAPR-1 with microdomains is an unexpected finding,
which provides new possibilities to study a function of GAPR-1, in particular
with respect to interactions with other proteins localized to these
microdomains. An important role of microdomains is their function in signal
transduction by clustering of specific proteins
(Brown and London, 1998a;
Simons and Ikonen, 1997
). The
immune response is also regulated by the association of proteins with
microdomains (reviewed in Brown and London,
2000
; Simons and Toomre,
2000
). Studies on the association of GAPR-1 with microdomains and
the possible link with the immune response might provide valuable information
on the function of this protein and probably of other members of the
superfamily as well.
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Acknowledgments |
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