* Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla Apdo. 1095, 41080-Sevilla, Spain; and Institut
Curie, Section Recherche, UMR144 du CNRS, 75248 Paris, Cedex 05, France
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Abstract |
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We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.
Key words: autoantigens; Golgi apparatus; microtubule-associated proteins; microtubules; centrosome ![]() |
Introduction |
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INTERPHASE microtubules are believed to play important roles in establishment of cell polarity and organization and distribution of organelles in higher eukaryotic
cells. It has become clear that all classes of membrane-bound, cytoplasmic organelles maintain an intimate and
dynamic association with the microtubule cytoskeleton (Cole and Lippincott-Schwartz, 1995). The endoplasmic
reticulum extends tubular membrane structures towards
cell periphery by using microtubule network as a scaffold
(Dabora and Sheetz, 1988
). The Golgi complex colocalizes
with the minus ends of microtubules which are associated
with the centrosome (Kreis, 1990
). Late endosomes and
lysosomes, generally located in the perinuclear area, also display tubular shapes (Matteoni and Kreis, 1987
). Recently, involvement of microtubules in the spatial organization of peroxisomes and in the fast directional movement of a small subpopulation of these organelles has
been reported (Wiemer et al., 1997
). In each case, the
morphology and distribution of the organelles are disrupted by microtubule-depolymerizing drugs.
Microtubules also support the movement of the membranous organelles and their transport intermediates.
These intracellular movements that occur in opposite directions along microtubules are thought to be driven by
two microtubule motors, kinesin (Vale et al., 1985a) which
moves towards the plus ends of microtubules (Vale et al.,
1985b
), and cytoplasmic dynein which moves towards the
minus ends (Paschal and Vallee, 1987
). Tubular lysosome
extension and anterograde lysosome membrane transport
require kinesin activity (Hollenbeck and Swanson, 1990
;
Nakata and Hirokawa, 1995
), and there exist evidences
consistent with a role for kinesin in the plus end-directed
extension of ER tubules to the cell periphery (Feiguin et
al., 1994
). Retrograde transport from the Golgi apparatus to the ER is a microtubule-dependent plus end-directed
movement also. Consistent with this, kinesin has been
shown to be present on membranes that constitutively cycle between the ER and the Golgi and microinjection of
an anti-kinesin antibody inhibited Golgi to ER but not ER
to Golgi membrane transport (Lippincott-Schwartz et al.,
1995
). In contrast, several studies suggest that movement of membranous organelles towards the centrosome is
driven by cytoplasmic dynein. The movement of the Golgi
elements towards the minus ends of microtubules has been
shown to require cytoplasmic dynein in semi-intact cells
(Corthésy-Theulaz et al., 1992
). Injection of anti-DHC2
antibodies, a cytoplasmic dynein heavy chain predominantly localized on the ER-Golgi intermediate compartment (Vaisberg, E.A., P.M. Grissom, S.R. Gill, T.A.
Schroer, and J.R. McIntosh. 1996. Mol. Biol. Cell. 7:403a),
induced the dispersion of the Golgi complex (Vaisberg et
al., 1996
). Overexpression of the dynamitin subunit of the
dynactin complex led to dispersion of the Golgi complex,
endosomes, and lysosomes (Burkhardt et al., 1997
) and blocked the ER to Golgi transport (Presley et al., 1997
).
More recently, cDHC
/
blastocysts have been shown to
display a vesiculated Golgi complex, distributed throughout the cytoplasm, although individual Golgi fragments
still attached to microtubules (Harada et al., 1998
). A similar effect was observed on endosomes and lysosomes distribution. These results indicate that molecules other than
cDHC might mediate attachment of the Golgi complex
and endosomes and lysosomes to microtubules.
Although the role of motors in these processes is now
well established, recent data argue that motors alone are
insufficient to mediate interactions of organelles with microtubules. Indeed, in studies where the static binding of
organelles to microtubules have been examined, binding
have been attributed to nonmotor proteins (Rickard and
Kreis, 1996). Comparison of the results obtained in these
studies revealed a number of differences suggesting that there are specific nonmotor microtubule-associated proteins for different organelles, that have been named
cytoplasmic linker proteins (CLIPs)1. The only CLIP extensively characterized to date is CLIP-170, a microtubule-binding protein associating transiently with endosomes during early to late endosome transport (Pierre et al.,
1992
). Recently, it has been found that ch-TOG, the human homologue of XMAP-215, a previously described
microtubule-associated protein of Xenopus eggs, is associated to ER membranes in interphase human cells
(Charasse et al., 1998
). In addition, p63, an integral membrane protein of the reticular subdomain of the ER, has
been shown to bind microtubules in vivo and in vitro and it
could contribute to the positioning of the ER along microtubules (Klopfenstein et al., 1998
). A cytosolic factor of
150 kD responsible for phagosome-microtubule binding
has also been described (Blocker et al., 1996
).
We have previously described a peripheral Golgi autoantigen of 210 kD whose localization is restricted to the
cis-Golgi network (CGN). Interestingly, this protein exhibits an unusual behavior when cells are treated with nocodazole (NZ) or Brefeldin-A. NZ induces a specific and
early segregation of many p210-associated vesicles or tubules from the Golgi apparatus. Upon Brefeldin-A treatment, p210 does not redistribute in the ER but exhibits a vesicular pattern characteristic of proteins residing in the
CGN (Rios et al., 1994; Alcalde et al., 1994
; Nakamura et
al., 1995
). In this study we report isolation of the full-length cDNA encoding p210. We show that this protein associates with the Golgi apparatus via its NH2 terminus and
directly interacts with microtubules through its COOH-terminal domain. For these reasons, we have designated it
GMAP-210 (Golgi microtubule-associated protein 210).
Functional analyses of intact and mutant forms indicate
that GMAP-210 is a minus end microtubule-binding protein that plays a role in maintaining the structural integrity
of the Golgi apparatus.
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Materials and Methods |
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Cell Culture and Antibodies
HeLa and COS-7 cells were grown under standard conditions. IgG fraction (10 mg/ml final concentration) from RM autoimmune serum (AS)
and affinity-purified anti-GMAP-210 antibodies were obtained as described in Rios et al. (1994). CTR433, a mouse monoclonal antibody, is a
marker of medial-Golgi (Jasmin et al., 1989
). Polyclonal antibody anti-
-tubulin has been previously characterized (Tassin et al., 1998
). Anti-detyrosinated tubulin (
T12 and SG) and anti-p115 antibodies were supplied by Drs. Kreis, Bulinski, and Sztul, respectively.
Molecular Cloning and Sequencing of GMAP-210
106 recombinants of a ZAPII human HeLa cell random-primed cDNA
expression library (P. Chambon, IGBMC, Universitè Louis Pasteur, Strasboug, France) were screened with the autoimmune serum diluted 1:1,500
followed by anti-human IgG alkaline phosphatase conjugated (Promega
Corp.), and positive plaques were detected by incubation with BCIP and
NBT. Positive clones were plaque purified through several rounds of rescreening. Affinity-purified antibodies against each of the three major autoantigens recognized by the serum (Rios et al., 1994
) were obtained and
used to group the clones obtained in immunologically related families.
pBluescript phagemid containing the cloned cDNA inserts were excised
from
ZAPII by coinfection with R408 helper phage as described in the
manufacturer's instructions (Stratagene). The 3' end of GMAP-210 was
cloned by RT-PCR. HeLa mRNA was prepared with a Quick prep
mRNA purification kit (Pharmacia Biotech). First-strand cDNA synthesis
was performed with an oligo-(dT) primer by using a First-strand cDNA
synthesis kit (Pharmacia Biotech). The completed first-strand reaction
was amplified directly by PCR with specific primers. Clone RT-10, containing the 3' end of GMAP-210 cDNA, was obtained with the primers P1
and P2 as indicated in Fig. 1 A.
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A series of overlapping restriction fragments from the clones obtained
by immunoscreening were subcloned into pBluescript SK or pTZ19R.
Double-stranded cDNA in pBluescript or pTZ19R was sequenced on
both strands with an automatic sequencer (Pharmacia) by the dideoxy termination method (Sanger et al., 1977). Sequence data were compiled and
analyzed using the University of Wisconsin Genetics Computer Group
package version 8.1 for Unix computers and the EGCG extensions to the
Wisconsin package version 8.1.0. Comparisons to known sequences were
performed using tFASTA and BLAST. Primary and secondary structure
analysis was conducted with Motifs, PeptideStructure, PepStats, and PepCoil.
The full-length open reading frame of GMAP-210 was assembled in the
eukaryotic expression vector pECE (Ellis et al., 1986) using clones R2,
R3, RT-7 (obtained by RT-PCR using primers P3 and P4), R6, and RT-10
(see Fig. 1 A). To obtain an HA epitope-tagged GMAP-210 protein, primers P5 and P6 were used to create an EcoRI site in the appropriate frame
for cloning into pECE-HA. GMAP-210 cDNA fragments coding for
amino acids 1-375 (
C375) and 1,778-1,979 (
N1778) were cloned in fusion with the green fluorescent protein (GFP) into the eukaryotic expression vectors pEGFPN1 and pEGFPC1, respectively. Full-length or partial
GMAP-210 cDNAs were also cloned into the prokaryotic expression plasmids pGEX. The synthesis of the glutathione-S-transferase (GST) fusion
proteins was induced by addition of 1 mM isopropyl-
-D-thiogalactoside
and the fusion proteins were isolated from bacterial lysates by affinity
chromatography with glutathione-agarose beads (Sigma).
Northern Blot Analysis
Northern analysis was performed on poly(A)+ RNA from human tissues (commercial multiple Northern blot; Clontech). DNA fragments corresponding to nucleotides 418-1,130 and 5,364-5,800 were radiolabeled and used as probes.
Production of Polyclonal Antibodies
After cloning DNA fragments coding for amino acids 375-611 or 618-803 into pGEX vectors, GST-fusion polypeptides were expressed and purified as described above. Fusion proteins were dialyzed in PBS and two rabbit polyclonal antibodies were generated (Agrobio). Sera were collected and Ig fraction purified by ammonium sulfate precipitation. These anti- GMAP-210 polyclonal antibodies were named RM127 and RM130.
Electrophoresis, Immunoblotting, and Immunoprecipitation
Gel electrophoresis and immunoblotting (IB) were performed as described (Rios et al., 1994). For immunoprecipitation experiments, HeLa
cells were incubated for 30 min on ice in order to depolymerize microtubules and then treated with 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40
containing protease inhibitors. Soluble fraction was clarified at 15,000 g
for 15 min and preadsorbed with 5 µl of normal human serum or 5 µl of
each preimmune rabbit sera on 100 µl of protein A-Sepharose. GMAP-210 was immunoprecipitated from supernatants with 5 µl AS, 5 µl RM127,
or 5 µl RM130 on 100 µl of protein A-Sepharose. After incubation and
washing, the presence of GMAP-210 in the bead pellets was detected by
IB with RM130 polyclonal antibody.
Preparation of Microtubule-binding Proteins from HeLa Cells
2 × 108 HeLa cells were trypsinized and harvested at 500 g for 10 min at
room temperature. Subcellular fractionation in order to obtain a high
speed supernatant (HSS) was carried out as described by Rickard and
Kreis (1990). The protein concentration of the HSSs obtained ranged between 6-8 mg/ml when measured using the Bradford assay. The HSS was
adjusted at 3-4 mg/ml with PEM buffer (0.1 M K-Pipes, 2 mM EGTA, 1 mM MgSO4, pH 6.8), incubated for 30 min on ice and then incubated for
15 min at 37°C in the presence of 10 µM TaxolTM (Paclitaxel), 1 mM DTT,
and 1 mM GTP. Microtubules were centrifuged at 30,000 g for 30 min at
30°C through a cushion of 10% sucrose in PEM containing 10 µM taxol, 1 mM DTT, and 1 mM GTP. Microtubules were washed and resuspended in
the same buffer (0.1-0.05 times the original HSS volume). Proteins were
eluted from the microtubules by adding 10 mM ATP, 10 mM GTP, 0.5 M
NaCl, or 2 M urea, respectively, and incubation at 37°C for 30 min followed by centrifugation.
In Vitro Microtubule Binding Assays with Endogenous GMAP-210 or with GST-Fusion Polypeptides
Tubulin was purified from bovine brain by two cycles of polymerization followed by chromatography on phosphocellulose. 10-20 µg of pure tubulin/assay were polymerized with 20 µM taxol, 1 mM GTP, and 1 mM DTT for 15 min at 37°C. Microtubules were centrifuged through 10% sucrose, washed, and finally resuspended in PEM containing GTP and taxol.
After cloning different DNA fragments into pGEX vectors, GST-fusion polypeptides were expressed and isolated from bacterial lysates by
affinity chromatography with glutathione-agarose beads. Microtubules
were then added to glutathione-agarose beads linked to similar amounts
of GST alone, GST-fusion polypeptides or GST-GMAP-210 (<0.5 µg).
After incubation for 2 h at room temperature, beads were low speed sedimented (1,000 g) and washed in PEM buffer. The presence of microtubules in the bead pellets was detected with anti--tubulin antibodies. Alternatively, GMAP-210 was immunoprecipitated from HeLa cells NP-40
supernatants using 5 µl AS, 5 µl RM127, or 5 µl RM130 coupled to 100 µl
of protein A-Sepharose as described in a previous section. After washing,
beads were incubated for 2 h at room temperature with taxol-polymerized
microtubules. Then, beads were low speed sedimented (1,000 g) and
washed in PEM buffer. The presence of microtubules in the bead pellets
was detected with anti-
-tubulin antibodies.
Identification of Microtubule-associated Proteins by Selective Extraction of Cultured Cells
HeLa cells were incubated with 10 µM NZ or 10 µM taxol for 4 h at 37°C.
Untreated, NZ- or taxol-treated HeLa cells were washed twice with PBS
at room temperature and once with a microtubule-stabilizing buffer
PM2G (0.1 M K-Pipes, 2 M glycerol, 5 mM MgCl2, and 2 mM EGTA, pH
6.9). Then, washed cells were extracted for 5 min at room temperature
with 0.1-0.3% NP-40 in the same buffer to obtain NP-40 soluble fractions
(fraction S). The detergent-extracted preparations, that remained attached to substrate, were washed with PM2G without detergent and incubated for 30 min at 4°C in the same buffer containing 5 mM CaCl2 to
depolymerize microtubules (fraction Ca). The detergent and calcium-insoluble preparations were also harvested (fraction I) and all fractions
obtained analyzed by SDS-PAGE and IB. All buffers contained protease
inhibitors (1 mM PMSF and 1 µg/ml each of aprotinin, pepstatin, chymostatin, and leupeptin). In the case of cells pretreated with drugs, each of
the washes contained the same concentration of the drug. In parallel,
HeLa cells grown on coverslips were treated and successively extracted
with NP-40 and calcium as described above. Cell ghosts were then processed for immunofluorescence. The presence of microtubules was revealed by using an anti--tubulin antibody.
Binding of GMAP-210 Constructs to Golgi Membranes
Fractions enriched in Golgi membranes were isolated from HeLa cells by
flotation in a sucrose gradient as described (Rios et al., 1994). Before further processing, Golgi-enriched fractions were diluted by addition of several volumes of 10 mM Tris-HCl, pH 7.4 and protease inhibitors were
added. The suspensions were then centrifuged at 200,000 g for 2 h at 4°C.
To obtain GMAP-210-depleted Golgi membranes, membrane pellets
were incubated with 2 M NaCl for 30 min on ice, high speed centrifuged,
and finally resuspended in 10 mM Tris-HCl, pH 7.4, containing 250 mM
sucrose. GST-fusion polypeptides were purified on glutathione-agarose
beads. Preliminary experiments suggested that the presence of GST in the
NH2 terminus of the fusion proteins could decrease the association of the
recombinant proteins to membranes. Therefore, polypeptides were released from the fusion proteins by treatment with 1% thrombin (wt/wt).
Full-length GMAP-210 and
N1597 construct could not be tested because
they were degraded by thrombin treatment. Binding to Golgi membranes was performed using 10 µg of Golgi membranes and <0.2 µg of each recombinant polypeptide. Since binding of GMAP-210 to membranes was
shown to resist 0.5 M NaCl we performed the binding assay in the presence
of this salt concentration. After sedimentation of Golgi membranes, binding
of the recombinant polypeptides was detected by IB using the AS. In the
absence of Golgi membranes, any recombinant polypeptide sedimented.
Transient Transfection and Immunofluorescence Microscopy
Plasmid DNA used for transfection was purified through two preparative
CsCl/ethidium bromide equilibrium gradients, followed by phenol extraction and ethanol precipitation. COS cells were split 24 h before transfection so that they were 60-80% confluent on the day of transfection. 2-5 × 106 cells/assay were resuspended in 200 µl of 15 mM Hepes-buffered serum-containing medium, mixed with 50 µl 210 mM NaCl containing 5-20
µg plasmid DNA and electroporated using a Bio-Rad Gene Pulser. 6 h after electroporation, medium was replaced by fresh medium and cells were
processed for immunofluorescence (IF) staining after 24-48 h. Indirect IF
was carried out as described (Rios et al., 1994). For GFP-fluorescence,
cells were fixed in
30°C methanol for 3 min. The samples were observed
either using a Leica microscope and a Hamamatsu chilled CCD camera or
using a TCS4D confocal laser scanning microscope based on a DM microscope interfaced with an Argon/Krypton laser. Simultaneous double fluorescence acquisitions were performed using the 488-nm and the 568-nm
laser lines to excite FITC and TRITC dyes using a 63× or 100× oil immersion PlanApo objectives. Images were transferred to a Macintosh computer for editing. All documents presented except Fig. 8 (CCD images) are two-dimensional projections of images collected at all relevant z-axes.
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In Vitro Microtubule Cosedimentation Assay
Microtubules were assembled in vitro from phosphocellulose-purified bovine brain tubulin as described above. To generate more polymer ends while keeping the polymer mass constant, microtubules were sheared by repetitively passing them through a 26-gauge hypodermic needle attached to 1-ml insulin syringe.
HeLa cells were incubated with 20 µM taxol for 4 h at 37°C and then
extracted with 1% NP-40 in PEM buffer. NP-40 soluble fraction from
taxol-treated cells (fraction TS) contained a pool of GMAP-210 and
-tubulin but no
-tubulin as revealed by IB (see Fig. 12, top). Identical
amounts of TS fraction were mixed with equal amounts of microtubules
before (
) or after (+) shearing, incubated for 30 min at room temperature and then centrifuged through a 10% sucrose cushion at 60,000 g for
30 min at 4°C. TS fraction and microtubule pellets were analyzed by IB for
the presence of GMAP-210,
-tubulin and
-tubulin.
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Results |
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cDNA Cloning and Sequence Analysis of GMAP-210
cDNA clones for GMAP-210 (Fig. 1 A) were obtained after immunoscreening of a HeLa cDNA library with the
AS (clones R1-R6 and R8) and rescreening with an R8
fragment as a probe (clone R9). Clones were routinely expressed in fusion with GST and analyzed by IB using affinity-purified anti-GMAP-210 antibodies. Sequence analysis revealed that the 3' end was missing but a sequence
database search detected several expressed sequence tags
whose 5' ends were identical to the 3' end of R9. Clone
RT-10, containing the stop codon and the polyadenylation
signal, was obtained by RT-PCR using primers P1 and P2
(from the partial cDNA sequence L40380; Lee et al.,
1995). This clone showed a very weak positive reaction in
blots revealed with affinity-purified anti-GMAP-210 antibodies, explaining why the 3' end of the protein was not
obtained by screening of the cDNA library with the AS.
The combined sequence derived from the overlapping
clones R2, R3, R5, R6, and RT-10 comprises a total of
6452 bp and contains an open reading frame of 5,937 bp.
The predicted protein sequence consists of 1,979 amino
acid residues (Fig. 1 B) and has a calculated molecular
mass of 223,902 and a predicted pI of 5.1, consistent with
the molecular weight and pI of GMAP-210 determined by
one- and two-dimensional gel electrophoresis (Rios et al.,
1994). The PROSITE Dictionary of Proteins Sites and
Patterns revealed multiple consensus motifs for phosphorylation and two leucine zipper patterns between amino
acids 413-448 and 1,440-1,461. No known consensus nucleotide-binding motifs were found consistent with the
lack of a direct effect of ATP or GTP on the binding of
GMAP-210 to microtubules.
Analysis of GMAP-210 amino acid sequence with PepCoil program showed that a significant portion of the protein had a high probability of forming an extended coil
(Fig. 1, B and C). The secondary structure and charge display of GMAP-210 was predicted using the programs PeptideStructure and PepStats. According to this analysis,
GMAP-210 appears as a very long coil encompassing most
of the sequence although interrupted by a 100-amino acid stretch poorly structured and slightly basic. The NH2 terminus is highly acidic and contains a small coiled-coil domain whereas the COOH terminus is a highly basic proline and glycine-rich domain (Fig. 1, B and C). The two
longest coils are interrupted by two short segments that
contain proline residues. Since proline usually disrupts an
-helix, GMAP-210 seems to be organized into five coiled-coil segments that are separated by non-helical linkers (Fig. 1, B and C). The presence of two leucine-zippers
each localized in a long coiled-coil domain, further supports the possibility of an oligomeric form for GMAP-210
in vivo. Finally, the high content in proline and glycine
(19%) in the basic COOH-terminal domain suggests a
globular structure for this portion of the protein.
Potential protein sequence similarities between GMAP-210 and known proteins were analyzed by searching the GenBank and SwissProt data libraries using tFASTA and BLAST. 20% identity of amino acid sequence was found between GMAP-210 sequence predicted to be coiled-coil and several structural and motor proteins of the cytoskeleton that contain coiled-coil domains such as CENP-E and CENP-F, NuMA, CLIP-170, myosin heavy chains, kinesins and dyneins, and the yeast proteins Nuf1, Uso1p, and Cut14. Comparison of GMAP-210 sequence with those of previously reported Golgi autoantigens such as p230 (golgin 245), GM130 (golgin 95), or GCP170 (golgin 160) revealed no significant similarity (18% through coiled-coil regions).
The tissue expression pattern of GMAP-210 mRNA was studied by Northern blot analysis using two clones as probes, corresponding to 5' and 3' ends. Both probes revealed a single transcript of >7 kb that was more abundant in testis, heart, placenta, skeletal muscle and pancreas although it could be also detected in other tissues (not shown). The size of revealed mRNA easily exceeds that required to encode a protein of 223 kD, supporting that the isolated cDNA codes for GMAP-210.
To definitively demonstrate that the cDNA sequence corresponded to that of the p210 autoantigen, we have generated two polyclonal antibodies against two GST-fusion proteins containing distinct regions of GMAP-210 (amino acids 375-611 and 618-803; Fig. 1 B). These polyclonal antibodies, named RM127 and RM130, revealed by IB a band of identical electrophoretic mobility as that recognized by the AS (Fig. 2 A) and decorated the Golgi apparatus by IF (see Figs. 7-11). Most importantly, GMAP-210 was immunoprecipitated from PNS fractions of HeLa cells using the AS, RM130, and RM127 antibodies and immunoprecipitates were further analyzed by IB using RM130 polyclonal antibody. As expected GMAP-210 was detected in all cases (Fig. 2 B), clearly demonstrating that the isolated cDNA encodes GMAP-210.
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GMAP-210 Is a Microtubule-binding Protein
The first indication that GMAP-210 could be a microtubule-binding protein came from its behavior during cell treatment with microtubule-active drugs (Fig. 3 A). In untreated HeLa cells, GMAP-210 distributed in both Triton X-100 soluble and insoluble fractions matching the distribution of tubulin in these fractions. In taxol-treated cells, all tubulin became insoluble whereas it was still possible to detect part of GMAP-210 in the soluble fraction. Finally, prolonged treatment of HeLa cells with NZ induced complete depolymerization of tubulin and concomitantly complete solubilization of GMAP-210. As a control, distribution of actin was analyzed and as expected, remained unchanged under all of these treatments.
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The association of GMAP-210 with microtubules was further analyzed in vitro. We investigated whether GMAP- 210 cosedimented with microtubules purified from HeLa cells using taxol. A post-nuclear supernatant was prepared and centrifuged at high speed in order to obtain a supernatant depleted of most of membrane organelles. This high speed supernatant (HSS), that contained a pool of GMAP-210 (Fig. 3 B), was incubated with taxol to polymerize endogenous tubulin. Microtubules were further purified by sedimentation through a sucrose cushion. GMAP-210 cosedimented with microtubules as revealed by IB using the AS (Fig. 3 B). When HeLa cytosol was incubated instead with NZ to prevent tubulin polymerization, neither tubulin nor GMAP-210 sedimented through the sucrose cushion, indicating that GMAP-210 sedimentation was microtubule-dependent (not shown). To investigate the microtubule-binding characteristics of GMAP-210, taxol-polymerized microtubules were incubated with 0.5 M NaCl, 10 mM ATP, or 10 mM GTP, centrifuged through sucrose, and then the supernatants and pellets analyzed for the presence of GMAP-210 by IB (Fig. 3 C). Under all these conditions GMAP-210 was detected in the microtubule-containing pellet. Similar results were obtained when these agents were added to cytosol before incubation with taxol in order to prevent the association (not shown). Only when 2 M urea was added to taxol-polymerized microtubules, could GMAP-210 be detected in the supernatant but, in these conditions, approximately half of tubulin was unpolymerized (Fig. 3 C). From these experiments, we conclude that GMAP-210 binds tightly to microtubules in vitro, in a nucleotide-insensitive manner.
GMAP-210 Binds Directly to Microtubules via Its COOH-terminal Domain
To determine whether GMAP-210 binding to microtubules is direct and to decide which domain(s) is responsible for this interaction, a panel of GST-fusion proteins
containing different portions of GMAP-210 or the complete protein was prepared (Fig. 4 A, top) and immobilized on glutathione-agarose beads. The immobilized proteins were allowed to interact with microtubules assembled from bovine brain phosphocellulose-tubulin with
taxol. The presence of microtubules bound to wild-type or
mutant proteins was detected by IB using an anti--tubulin antibody (Fig. 4 A, bottom). These experiments demonstrated a direct binding of GMAP-210 to microtubules and further established that the microtubule binding
site(s) corresponded to the region containing amino acids
1,712-1,979, the basic COOH-terminal domain.
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A similar experiment was then carried out but endogenous GMAP-210 was used instead of recombinant GST- GMAP-210 fusion protein. GMAP-210 was immunoprecipitated from HeLa cell lysates using AS, RM127, or RM130 antibodies precoupled to Sepharose beads. Beads were then incubated with microtubules assembled from bovine phosphocellulose-tubulin with taxol. Fig. 4 B shows that endogenous GMAP-210 efficiently interacted with microtubules. In the absence of added microtubules, no tubulin was detected (RM130-Mts) indicating that GMAP-210 did not bind soluble tubulin present in the cell lysates.
GMAP-210 Associates Preferentially with a Subpopulation of Stable Microtubules
To further characterize the association of GMAP-210 with
microtubules in situ, we analyzed the microtubular cytoskeleton by selective extraction of cells (Solomon, 1986;
Fig. 5). To do so, untreated (Control), 10 µM NZ-, or 10 µM
taxol-treated cells were permeabilized with a non-ionic detergent in order to extract cytoplasmic proteins (fraction
S). Then, microtubules were depolymerized and extracted
with a cold-calcium-containing buffer (fraction Ca). All
fractions obtained together with the detergent- and calcium-insoluble fractions (fraction I) were processed by IB
to analyze the distribution of tubulin and GMAP-210. Fig.
5 A shows that, unexpectedly, tubulin was present in detergent- and calcium-insoluble fractions corresponding to
control and taxol-treated cells. This result suggested that a
subpopulation of microtubules could resist the depolymerization induced by cold-calcium. To confirm this assumption, untreated-, NZ-, and taxol-treated cells were extracted with NP-40 and cold-calcium. Cell ghosts were
then processed for IF and labeled with an anti-
-tubulin monoclonal antibody (Fig. 5 B). According to biochemical
data, some microtubules growing from the centrosome
could be observed in untreated cells and no microtubules
were present in NZ-treated cells whereas many microtubules resisted extraction in taxol-treated cells. GMAP-210
was absent from all Ca-soluble fractions but present in
insoluble fractions corresponding to control and taxol-treated cells (Fig. 5 A). This result indicates that GMAP-210 does not bind to all assembled microtubules in situ but
interacts preferentially with a subset of cold-calcium-resistant microtubules. GMAP-210 was also detected in the
soluble fractions under all conditions suggesting that binding of GMAP-210 to microtubules in vivo must be regulated since all GMAP-210 did not bind to microtubules
even when all tubulin became polymerized with taxol (see
also Fig. 3 A).
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To characterize this subpopulation of stable microtubules, we quantitatively analyzed the distribution of detyrosinated -tubulin (glu-tubulin) with respect to total tubulin (
-tubulin) in the three fractions obtained from
control cells, using the same subfractionation procedure
(S, Ca, and I; Fig. 5 C). In untreated cells, tubulin was
found to distribute as follows: 35% was soluble, 52% was
polymerized in microtubules that were sensible to cold-calcium (corresponding to 80% of total microtubules), and
13% in microtubules resistant to these conditions (corresponding to 20% of microtubules). This subpopulation of
more stable microtubules contained 29% of the total detyrosinated
-tubulin whereas 80% of more labile microtubules contained 67% of glu-tubulin. These results indicate
that resistant microtubules contain 1.8 times more glu-tubulin than the rest. Thus, GMAP-210 appears preferentially associated in situ to a subpopulation of stable microtubules that are enriched in detyrosinated
-tubulin.
GMAP-210 Associates with Golgi Membranes via Its Acidic NH2 Terminus
The binding site for Golgi membranes on GMAP-210 was
also mapped by deletion analysis. A series of NH2-terminal and COOH-terminal truncation mutants were
constructed and expressed as GST-fusion proteins (Fig.
6, top). They were immobilized on glutathione-agarose beads and further treated with thrombin to release recombinant proteins from GST. Golgi membranes were purified by flotation in a sucrose gradient, washed with 2 M
NaCl to remove endogenous GMAP-210 and centrifuged
at high speed. 10 µg of washed Golgi membranes were incubated with <0.2 µg of different fragments of GMAP-210 in the presence of 0.5 M NaCl. After centrifugation, both supernatants and pellets were analyzed by IB using
the AS (Fig. 6). The constructs C611 and
C375 appeared enriched in Golgi membranes containing pellets,
whereas fragments lacking NH2-terminal domain mostly
remained in supernatants. Binding of GMAP-210 NH2 terminus to Golgi membranes was further confirmed in vivo
by transfection experiments (see below).
|
Functional Analysis of GMAP-210 In Vivo
To approach the function of GMAP-210 in the organization of the Golgi apparatus, we transiently expressed intact or mutant proteins in COS cells. Full-length cDNA coding for GMAP-210 with or without an NH2-terminal HA-epitope was introduced in cells by electroporation. Cells were incubated, fixed, double labeled and finally observed in a confocal laser microscope. An effect on the structure of the Golgi apparatus and microtubule network could be observed depending on the expression level of the transfected protein. At low expression level, the full-length GMAP-210 localized to a compact juxtanuclear reticulum characteristic of the Golgi apparatus as revealed by an anti-HA antibody (not shown). The labeling pattern overlapped exactly with that exhibited by the AS that recognized both exogenous and endogenous proteins, indicating that the expressed protein is targeted to the same place as the endogenous GMAP-210, namely the CGN. No significant changes in the structure of the Golgi complex or the microtubule network could be appreciated (not shown).
By contrast, overexpression of GMAP-210 dramatically
perturbed the organization of the Golgi apparatus. Under
these conditions, transfected cells could be easily distinguished from the surrounding nontransfected cells by IF,
due to the high amount of exogenous GMAP-210. Several
features could be observed. First, a dramatic enlargement
of the Golgi apparatus that could remain as a single pericentrosomal structure (Fig. 7 a) or as multiple fragments centrosomally localized but also distributed throughout
the cytoplasm (Fig. 7 d). Double labeling for GMAP-210
and the medial Golgi marker CTR433 (Jasmin et al., 1989;
Fig. 7, a-c) or the cis-Golgi marker p115 (Barroso et al.,
1995
; Fig. 7, d-f) revealed that not only the CGN but also
the other compartments of the Golgi apparatus were distorted by overexpression of GMAP-210. Remarkably, the
spatial distribution of GMAP-210 and the Golgi markers
was largely coincident, although they demonstrated a
graded inverse distribution: GMAP-210 labeling increased
towards the periphery whereas the intensity of CTR433
and p115 decreased. A careful analysis of optical cuts suggested an hypertrophy of the CGN extending towards the
cell periphery whereas the remnant of the Golgi stacks remained close to the nucleus (Fig. 7, c and f). Second, the
array of microtubules in the centrosomal area appeared
modified in overexpressing cells. The microtubule aster
became undefined and many microtubules appeared to
emanate from the enlarged Golgi area rather than from the centrosome (Fig. 7, g-i). In overexpressing cells in
which the Golgi complex was fragmented, perturbation
was more pronounced and microtubules appeared randomly distributed.
We further showed that the Golgi perturbation provoked by GMAP-210 overexpression depended upon the
microtubule-binding capacity of the protein: when a truncated form of GMAP-210 lacking the COOH-terminal microtubule-binding domain (C1778) was overexpressed, it
clearly accumulated at the Golgi apparatus although an
homogeneous cytoplasmic staining was also observed (Fig.
8, a-c). Comparison between a
C1778 overexpressing
cell (Fig. 8, d, f, and h) and a nontransfected cell (Fig. 8, e,
g, and i) revealed no significant differences in the Golgi
apparatus or the microtubule network structure. It must
be noted that overexpression of this mutant had no detectable effect on the distribution of endogenous GMAP-210.
We also studied the effect of microtubule-active drugs on the phenotype displayed by GMAP-210 overexpressing cells. When transfected cells were incubated with NZ, the Golgi apparatus became fragmented and scattered throughout the cytoplasm in all overexpressing cells and complete colocalization with CTR433 and p115 was observed (not shown). Transfected cells were also incubated with taxol to promote the formation of microtubule bundles. In the presence of taxol, GMAP-210-enriched membranes preferentially redistributed at the periphery of the cells, whereas the rest of the Golgi complex remained juxtanuclear (Fig. 9, a-c). GMAP-210-enriched membranes were found to be always located at one end of microtubule bundles (Fig. 9, d-f). Higher magnifications at the cellular periphery allowed to distinguish individual microtubules and GMAP-210-enriched membranes could be seen associated with them (Fig. 9, g-i). Thus, taxol-induced microtubules were able to tear the GMAP-210-enriched elements away from the rest of the Golgi apparatus.
To further characterize the functional domains of
GMAP-210, both NH2-terminal and COOH-terminal domains were cloned in fusion with the green fluorescent
protein and transiently expressed in COS cells. C375-GFP mutant appeared mostly cytosolic but also localized
in punctate structures clustered in the Golgi region (Fig.
10 a). Labeling with an antibody that specifically recognized endogenous GMAP-210 revealed partial colocalization (Fig. 10, a-c). This result indicated that the truncated
chimera was able to bind to Golgi membranes. At higher
expression level, endogenous GMAP-210 was hardly visible consistent with the truncated mutant displacing the endogenous protein from membranes (Fig. 10, d-f). Under
these conditions, the medial Golgi marker CTR433 also
showed an altered distribution (Fig. 10, g-i). Instead of the
normal compact Golgi seen in the untransfected cell in this
field, CTR433 appeared in some punctate structures in the
Golgi area and the labeling intensity had considerably decreased. Therefore, Golgi compartments other than the
CGN appeared morphologically perturbed in the absence of membrane-bound GMAP-210. These data together
with those presented in Fig. 6 demonstrate that the NH2
terminus of GMAP-210 is involved in targeting the protein
to Golgi membranes and strongly suggest that GMAP-210
is required for processes involved in maintaining the integrity of the Golgi apparatus.
Similar experiments were carried out with the microtubule-binding domain for which GFP was placed in the
NH2 terminus of the mutant protein. Contrary to our expectations, the GFP-N1778 mutant protein did not
decorate microtubules. At low expression level, one or
two spots that colocalized with the centrosomal marker CTR453 (Bailly et al., 1989
) could be observed (Fig. 11, a-c). With increasing expression levels of the mutant, increasing
accumulation of GFP at the centrosome occurred together
with the dispersion of the pericentriolar material that became almost undetectable with the monoclonal antibody
CTR453 (Fig. 11, a1-3, b1-3, and c1-3). Centrioles were
present in the center of the GFP-labeled centrosomes, as
revealed by anti-detyrosinated tubulin antibodies (not
shown). After longer periods of expression, GFP-
N1778 accumulated massively at the centrosome (Fig. 11, d-f) although heterogeneous sized bright foci of GFP scattered
throughout the cytoplasm were also observed. The Golgi
apparatus was displaced to the periphery of the massive
centrosomal aggregate (Fig. 11, e and f) that appeared surrounded by a dense network of detyrosinated microtubules (Fig. 11, g-i).
GMAP-210 Binds to Microtubule Ends
The finding that the COOH-terminal microtubule-binding
domain of GMAP-210 localizes in vivo to centrosome
strongly suggested that GMAP-210 might interact only
with the minus ends of microtubules. One prediction of
this hypothesis is that for constant polymer mass, GMAP-210 binding to microtubules in vitro will increase when
more ends are present. To test this, we sheared a constant mass of microtubules (polymerized from phosphocellulose-bovine brain tubulin using taxol) by passing them
through a hypodermic needle attached to 1-ml syringe. In
parallel, we prepared a NP-40 soluble fraction from HeLa
cells that had been treated with taxol for 4 h in order to
completely polymerize the endogenous tubulin. This tubulin-free fraction (TS) contained a pool of GMAP-210 and -tubulin, as revealed by IB (Fig. 12, top). Equal amounts
of fraction TS were mixed with equal amounts of microtubules before (
) or after (+) shearing. After incubation,
microtubules were centrifuged through a sucrose cushion
and microtubule-pellets analyzed by IB. A typical experiment is shown in Fig. 12 (top) and the mean of values of
three independent experiments is represented in Fig. 12
(bottom). Shearing increased the number of microtubules
twice as determined by quantification of bound
-tubulin
before and after shearing. By using the same procedure Li
and Joshi (1995)
obtained an increase in the microtubules
number of 1.5 times, fitting well with our results. The
amount of GMAP-210 bound to microtubules also increased 1.8 times indicating that GMAP-210 interacts only
with the ends of microtubules.
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Discussion |
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Significant progress has been made towards understanding the molecular mechanisms of microtubule-dependent motors and their roles in membrane traffic. By contrast, the possible role of other microtubule-binding proteins lacking motor activity in the organization of the central membrane system has remained elusive. We report here the cloning and functional characterization of a previously described CGN-associated protein, p210.
GMAP-210 Is a Large Coiled-Coil Protein that Binds to Microtubule Ends via Its COOH Terminus and to Membranes through Its NH2-terminal Domain
Several pieces of evidence argue for GMAP-210 being
a microtubule-binding protein. First, the presence of GMAP-
210 in Triton-extracted cell ghosts is always dependent
on the existence of polymerized microtubules. Second,
GMAP-210 cosediments with taxol-polymerized microtubules from HeLa cells and remains associated in situ with stable microtubules. Finally, both endogenous GMAP-210
and purified bacterially expressed GMAP-210 interact
with purified bovine brain microtubules. Interestingly,
GMAP-210 binding to microtubules increases with the
number of free ends in a given mass of microtubule polymers. This was demonstrated by shearing microtubules
into short fragments, hence keeping the polymer mass
constant. Interaction of GMAP-210 with polymerized tubulin appears to be a tight nucleotide-insensitive binding
as it is not released from the microtubule pellet by salt conditions that are known to dissociate most MAPs, or by
ATP or GTP addition. These binding characteristics correlate well with those reported for interaction between the
Golgi complex and microtubules (Karecla and Kreis,
1992) and differ from those exhibited by CLIP-170, motors, or other MAPs.
The complete sequence of GMAP-210 has been obtained (accession number Y12490). Recently, an almost
identical sequence, submitted to data banks after submission of our sequence, has been reported under the accession number AF007217 (Chang et al., 1997). This protein
was reported to be a thyroid hormone receptor coactivator, negatively regulated by the retinoblastoma protein
and named Trip230. We have no explanation for such a
functional discrepancy but we are confident that the sequence we have cloned corresponds to the p210 autoantigen associated to the Golgi apparatus described previously
(Rios et al., 1994
) by all the experimental data presented
in this paper. In addition, a partial cDNA sequence of
a novel gene named CEV-14 (these data are available
from GenBank/EMBL/DDBJ under accession number
AF011368) that corresponds to amino acids 1,188-1,753 of
GMAP-210 has been reported. This partial sequence has
been identified as the NH2-terminal half of a fusion gene
in which the COOH-terminal half is a fragment of the
platelet-derived growth factor receptor containing transmembrane and tyrosine kinase domains. CEV14-PDGFR
fusion gene is generated by a chromosomal translocation
that is associated with acute myelogenous leukemia (Abe
et al., 1997
). Our finding that this fragment corresponds to
the second large coiled-coil domain of GMAP-210 supports the idea proposed by the authors that the mechanism
of transformation is the ligand-independent dimerization
of PDGFR leading to ectopic constitutive tyrosine kinase activation.
Structural features predicted by the molecular characterization of GMAP-210 indicate a long -helical domain
with high potential to form a coiled-coil structure. Such a
long coiled-coil domain has been found in other autoantigens associated with the cytoplasmic face of the Golgi
membrane including giantin or GCP372 (Seelig et al.,
1994
; Sohda et al., 1994
), golgin-245 or p230 (Fritzler et
al., 1995
; Erlich et al., 1996
), golgin-95 or GM130 (Fritzler et al., 1993
; Nakamura et al., 1995
), and golgin 160 or
GCP170 (Misumi et al., 1997
).
GMAP-210 is distinct from previously described microtubule-interacting proteins. GMAP-210 is predicted to
possess a three domain structure consisting of the long
-helical central domain and the nonhelical end domains,
similar to other proteins that interact with the cytoskeleton. The organization of charged regions of GMAP-210 is
also reminiscent of that of several other MAPs. The NH2-terminal end is acidic whereas the COOH-terminal domain has a high pI like MAP2 (Lewis et al., 1988
), MAP4
(West et al., 1991
), tau (Lee et al., 1988
), or NuMA
(Compton et al., 1992
). However, it is inverse to CLIP-170
that contains a basic NH2-terminal domain, a central
coiled-coil domain, and an acidic COOH-terminal domain.
The highly basic COOH-terminal region of GMAP-210
was shown to mediate direct interaction with microtubules. Neither the acidic NH2-end, nor the long
-helical domain associate with microtubules in vitro. Thus,
GMAP-210 behaves like most MAPs characterized so far,
in which basic regions play a significant part in conferring ability to bind microtubules. The microtubule-binding domain of GMAP-210 shows no overall homology to any of
previously characterized sites. It does not contain internal
repeats like MAP2, tau, or CLIP-170, suggesting that only
one binding site is present in each GMAP-210 molecule.
The binding domain of GMAP-210 to the cytoplasmic
side of CGN membranes has been localized in the NH2
terminus of GMAP-210. This region consists of two acidic
nonhelical regions interrupted by a small coiled-coil domain and followed by a large coil. The acidic COOH-terminal domain of CLIP-170 has also been postulated to interact with other proteins on the surface of endocytic
organelles. A molecular design consisting of an elongated
molecule containing a conserved microtubule-binding region at one end and a variable domain at the other end involved in specifying interactions with organelles has been
proposed for nonmotor proteins implicated in linking cytoplasmic organelles to microtubules such as CLIP-170 (Pierre et al., 1992). Although no significant sequence homology with CLIP-170 out of the coiled-coil region has
been found, the molecular design of GMAP-210 fits well
with this model.
GMAP-210 Is Required for Processes Maintaining the Structural Integrity of the Golgi Apparatus
The biochemical data provide strong evidence that GMAP- 210 is a Golgi-associated protein that directly interacts with microtubule ends. Consistent with this, overexpression of GMAP-210 induced perturbations in both the Golgi apparatus and the microtubule network, and these effects increased with the expression level of the protein. Exogenous GMAP-210 was correctly addressed to the Golgi apparatus that appeared dramatically enlarged and sometimes fragmented in structures scattered throughout the cytoplasm. This hypertrophy of the Golgi apparatus occurred together with a modification of the microtubule network that mainly affected the Golgi-centrosome area. In fact, transfected cells could be easily identified by looking at the microtubule network, due to the absence of a distinct microtubule aster at the centrosome. Instead, a dense network of short microtubules could be appreciated colocalizing with the enlarged Golgi apparatus, and cytoplasmic microtubules seemed to emanate from the periphery of this perturbed region. Deletion of the COOH-terminal domain completely abolished both effects, strongly suggesting that the basic COOH terminus is responsible for binding of GMAP-210 to microtubules in vivo as well as in vitro. These data also indicate that the perturbation of the Golgi apparatus produced by GMAP-210 overexpression is primarily due to an excess of binding to microtubules.
It was intriguing that truncated GMAP-210 lacking microtubule-binding domain (C1778) accumulated mostly
in the cytosol whereas intact GMAP-210 associated almost
completely with Golgi membranes. The
C1778 mutant
partially associated with Golgi membranes without apparently displacing endogenous GMAP-210, suggesting that
endogenous GMAP-210 is present at subsaturating levels
in cells and that interaction of GMAP-210 with membranes is saturable. This view is also consistent with the results obtained in vitro in which purified GMAP-210-containing membranes were able to bind a limited amount of
C611 or
C375 mutants. But how to understand that
GMAP-210 can steadily accumulate on CGN membranes
in cells overexpressing the full-length protein if the interaction of GMAP-210 with CGN membranes is saturable?
An amplification mechanism could reasonably explain this
paradoxical observation: the progressive accumulation of
GMAP-210 to the CGN would induce the stabilization of
microtubules in this area that would allow the progressive
enlargement of CGN elements. Such an increase in membrane surface would in turn allow that more GMAP-210 accumulates.
Numerous observations indicate that the size of the
Golgi apparatus is maintained by a fine tuning of the
membrane flux in and out of the Golgi complex. Overexpression of GMAP-210 produces a significant increase in
the Golgi apparatus size. On the contrary, when endogenous GMAP-210 is displaced from Golgi membranes by overexpression of a truncated mutant containing only the
membrane binding domain, the size of the Golgi complex
decreased (C375; see Fig. 10). Thus, GMAP-210 seems
to play a role in determining the size of the Golgi apparatus, probably by regulating the number of microtubule-binding sites on the surface of the CGN. A tighter association of the CGN with microtubules could prevent its
maturation into Golgi cisternae (Bannykh and Balch,
1997
) or impede anterograde vesicular transport from the
CGN to the medial Golgi (Orci et al., 1997
), producing the
enlargement of the CGN by accumulation of newly synthesized or recycling proteins. This could explain the partial colocalization of cis- and medial-Golgi markers with GMAP-210 in the enlarged CGN. At higher expression
levels, translocation of pre-Golgi elements from peripheral ER sites to the Golgi area could be also affected and
fragmentation of the enlarged Golgi apparatus would occur. Consistent with this, microtubules appeared randomly
nucleated in cells in which the Golgi apparatus was fragmented.
The relationship between CGN and microtubules thus
appears as a finely regulated interaction in order to ensure
not only the localization but also the steady state structure
and size of the Golgi apparatus. Many recent studies have
emphasized the role of microtubule motors not only in
membrane traffic but also in determining the localization
and morphology of the Golgi apparatus. It has been proposed that in the steady state the Golgi complex is in dynamic equilibrium between the anterograde force mediated by kinesin family motors and the retrograde force
mediated by cytoplasmic dyneins rather than simply being associated with microtubules near their minus end
(Harada et al., 1998). Our results indicate that association
of CGN membranes with the minus end of microtubules is
also necessary for the proper localization, morphology and size of the Golgi apparatus. They are consistent with a
model in which dynein is responsible for moving Golgi elements towards the centrosome whereas GMAP-210 is regulating the static binding of the Golgi membranes to microtubules.
GMAP-210 Links the CGN to the Minus End of Microtubules
We present here several lines of evidence showing that
GMAP-210 is a minus end microtubule-binding protein.
First, GMAP-210 binding to microtubules in vitro is
related to the number rather than the mass of microtubule polymers. Second, the COOH-terminal domain
of GMAP-210, responsible for the microtubule-binding
property of the protein in vitro, localizes in vivo to the centrosome, where the minus ends of microtubules accumulate. Moreover, it is able to disorganize the pericentriolar
material suggesting that it could displace other centrosomal proteins from the minus ends of microtubules. In the
presence of NZ, GMAP-210 does not remain associated
with the centrosome (Rios et al., 1994). Taxol treatment of
GMAP-210 overexpressing cells induced specific redistribution of GMAP-210 enriched elements to the cell periphery where they associated with one end of microtubule
bundles. Finally, in GMAP-210 overexpressing cells, a
dense network of short microtubules appears in the Golgi
area from which cytoplasmic microtubules seem to emanate, consistent with the notion that binding or capping of the minus ends of microtubules by proteins result in their stabilization.
All data presented here support a model in which
GMAP-210 serves to maintain the structural integrity of
the Golgi apparatus by interacting with the minus end of
microtubules. The NH2-terminal domain binds to CGN
membranes whereas the COOH-terminal domain interacts with the minus end of microtubules at the centrosome. Remarkably, a low expression level of the COOH-terminal domain leads to the centrosomal localization of GFP in
a manner identical to the centrosomal decoration obtained
with an antibody directed against -tubulin (Moudjou et
al., 1996
), or against a
-tubulin binding protein (Tassin et
al., 1998
), i.e., the centriole pair and a domain surrounding
it. From the study of
-tubulin distribution in epithelial
cells, Mogensen et al. (1997)
have proposed that peripheral docking elements distinct from nucleating elements could be closely associated with centrosomes. In their
model, microtubule minus ends would be capped and stabilized before being released from nucleating elements,
which would remain in the juxtacentriolar vicinity to nucleate new microtubules. Control mechanisms would ensure that a microtubule is anchored to the centrosome periphery or released. Several reports have indeed suggested
that centrosomally nucleated microtubules can escape to
other locations, particularly in neurons (Ahmad and Baas,
1995
) but also in other cell types (Vorobjev and Chentsov,
1983
; Belmont et al., 1990
; Keating et al., 1997
).
Based on this idea, one could propose that free microtubule ends could be captured by GMAP-210 via its COOH-terminal domain. This might also account for the existence
of a subpopulation of short, stable microtubules that colocalizes with the Golgi apparatus (Schulze and Kirschner,
1987). This subpopulation is rich in detyrosinated microtubules (Skoufias et al., 1990
) and we have shown that
GMAP-210 is specifically enriched in a microtubule fraction with similar characteristics. In this view, CGN membranes, rather than the centrosome itself, would be the
anchoring sites for microtubules participating in Golgi dynamics and stability.
A direct homologue of GMAP-210 has not been found in the yeast genome database. This is consistent with the fact that in lower eukaryotic cells no spatial segregation between the ER and the Golgi apparatus exists. In such cells, the Golgi complex is often found next to the ER sites and pre-Golgi elements form the first cisternae of the Golgi stack. However, in many eukaryotic cells the Golgi apparatus is centered around the centrosome and actively maintained there. Our data uncover a critical role for the CGN in this process and identify GMAP-210 as an important factor in mediating both the localization and the integrity of this Golgi compartment. During mitosis, the microtubule dynamics changes and the centrosomal localization of the Golgi apparatus is lost. One can speculate that GMAP-210 could be a target for the mitotic regulation of the CGN. Since GMAP-210 remains bound to Golgi membranes during mitosis (unpublished results), the obvious candidate for this mitotic regulation is the microtubule-binding domain. It is interesting to note in this regard that the COOH-terminal domain has two putative cyclin-dependent kinase phosphorylation sites. Experiments are now in progress to test this hypothesis.
![]() |
Footnotes |
---|
Received for publication 2 November 1998 and in revised form 25 February 1999.
Address correspondence to Rosa M. Rios, Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Apdo. 1095. 41080-Sevilla, Spain. Tel.: 34 954557120. Fax: 34 954557830. E-mail: rmrios
@cica.es
This paper is dedicated to the memory of Thomas Kreis.
We thank B. Goud, C. Antony, and J. Salamero for helpful discussions
and J. Salamero for assistance with laser confocal microscopy. We are indebted to Drs. Sztul, Bulinski, and Kreis for kindly providing us with antibodies and Dr. P. Chambon for the generous gift of random and oligo-
(dT) primed ZAP-II libraries. We are grateful to Dr. M. Tortolero for
her support during the progress of this work.
This work was supported by grants from the Junta de Andalucía, Spain and by CNRS (grant Biologie Cellulaire: du Normal au Pathologique, no. 96070) and Institut Curie, France. F. Ramos-Morales was supported by a return grant of the Training and Mobility of Researchers program from the European Union. C. Infante was supported by a Fundación Camara fellowship from the University of Seville.
![]() |
Abbreviations used in this paper |
---|
AS, autoimmune serum; CGN, cis-Golgi network; CLIP, cytoplasmic linker protein; GFP, green fluorescent protein; GMAP, Golgi microtubule-associated protein; GST, glutathione-S-transferase; HSS, high speed supernatant; IB, immunoblotting; IF, immunofluorescence; NZ, nocodazole.
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