From the Department of Developmental and Molecular
Biology and
Anatomy and Structural Biology, Albert Einstein
College of Medicine, Bronx, New York 10461 and the ¶ Department of
Pathology, Yale University School of Medicine,
New Haven, Connecticut 06520
Received for publication, September 6, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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Phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2) synthesis has been implicated in
maintaining the function of the Golgi apparatus. Here we demonstrate
that the inhibition of PtdIns(4,5)P2 synthesis in
vitro in response to primary alcohol treatment and the kinetics
of Golgi fragmentation in vivo were very rapid and tightly
coupled. Preloading Golgi membranes with short chain phosphatidic acid
abrogated the alcohol-mediated inhibition of PtdIns(4,5)P2 synthesis in vitro. We also show that fragmentation of the
Golgi apparatus in response to diminished PtdIns(4,5)P2
synthesis correlated with both the phosphorylation of a Golgi form of
In mammalian cells the Golgi apparatus consists of a series of
flattened cisternal stacks located in the perinuclear region of the
cell. Several conditions cause the lace-like ribbon structure to
undergo profound morphological changes. For example, during mitosis,
the Golgi complex fragments reversibly into vesicles and tubules that
are distributed equally to daughter cells during cytokinesis (1).
Recent evidence has demonstrated that the Golgi apparatus undergoes
irreversible fragmentation during apoptosis, in part as a result of
cleavage of the high molecular weight peripheral membrane protein GM160
and GRASP65 by members of the caspase family of proteases (2, 3).
Several pharmacological agents and pathological conditions also cause
fragmentation of the Golgi apparatus (4). These include the fungal
metabolite brefeldin A, whereby the Golgi apparatus collapses
into tubules and vesicles that fuse with the endoplasmic reticulum (5)
and the protein phosphatase inhibitor okadaic acid, which causes the
morphology of the Golgi apparatus to resemble that of mitotic cells
(6). In addition, overexpression of several proteins that regulate vesicle trafficking including mutant forms of Rabs (7),
ADP-ribosylation factor 1 (ARF-1)1 (8), ARF GDP-GTP
exchange factors ARNO-1 or -3 (9, 10), a dominant negative form of
PtdIns 4-kinase I Inositol phospholipids play key roles not only in mediating signal
transduction events but also in regulating intracellular vesicular
transport (reviewed in Refs. 14-17). Several observations suggest that
disassembly of the Golgi apparatus might result from changes in
inositol phospholipid metabolism. Overexpression of mutant forms of
PtdIns 4-kinase I A possible link between inositol phospholipids and the maintenance of
Golgi structure comes from observations that a specific form of the
cytoskeletal protein Antibodies--
Rabbit antibody to TGN38 was a generous gift
from Dr. Sharon Milgram (University of North Carolina, Chapel Hill,
NC); mouse monoclonal antibody to mannosidase II (53FC3) was kindly
provided by Dr. Brian Burke (University of Calgary, Calgary, Canada). A rabbit antibody to Other Reagents--
Protein phosphatase 1 (PP1) and protein
phosphatase 2A (PP2A) were obtained from Upstate Biotechnology.
Protein-tyrosine phosphatase 1B and Yersinia pestis tyrosine
phosphatase were generous gifts from Dr. Z. Y. Zhang (Albert
Einstein College of Medicine).
Cell Culture--
Rat anterior pituitary GH3 cells were grown as
described previously (30). Normal rat kidney (NRK) cells were grown as
before (24).
Immunofluorescence Microscopy--
GH3 cells grown on
poly-L-lysine-coated glass coverslips were either untreated
or pretreated with 1.0% 1-butanol for the indicated times and fixed in
either 3% paraformaldehyde or by treatment with Electron Microscopy--
The samples were treated with or
without 1-BtOH and fixed with 2.5% glutaraldehyde in 0.1 M
cacodylate buffer, postfixed with 1% osmium tetroxide followed by 1%
uranyl acetate. The samples were then dehydrated through a series of
graded ethanol concentrations and embedded in LX112 resin (LADD
Research Industries, Burlington, VT). Ultrathin sections were cut on a
Reichert Ultracut E, stained with uranyl acetate followed by lead
citrate, and viewed on a JEOL 1200EX transmission electron microscope
at 80 kV.
Subcellular Fractionation--
Approximately 107 GH3
cells treated with or without 1-BtOH were homogenized using a stainless
steel ball bearing homogenizer. The homogenate was loaded onto a step
gradient to separate Golgi membranes from the endoplasmic reticulum.
Fractions (1 ml each) were collected from the top of the gradient, and
the aliquots were precipitated with an equal volume of ice-cold 20%
(w/v) trichloroacetic acid, dissolved in SDS gel buffer, resolved by
SDS-PAGE, transferred to polyvinylidene difluoride membranes, and
probed with appropriate antibodies.
Measurement of PtdIns(4)P and PtdIns(4,5)P2 in
Vitro--
Golgi membranes isolated from GH3 cells or rat liver were
incubated without or with rat brain cytosol (final concentration, 2.2 mg of protein/ml) in 20 mM Hepes, pH 7.3, 125 mM KCl, 2.5 mM MgCl2, 2 mM ATP, and [ Protein Phosphatase Treatment--
Equal aliquots of sucrose
gradient fractions (above) from alcohol-treated cells were incubated
without or with PP1 (0.4 unit/reaction), PP2A (0.4 unit/reaction),
protein-tyrosine phosphatase 1B (10 µg/reaction) and Y. pestis phosphatase (6.8 µg/reaction) for 30 min at 30 °C.
Following incubation, the reactions were terminated by boiling the
samples in SDS gel loading buffer. The samples were then analyzed by
SDS-PAGE followed by immunoblotting with anti- Recent data from one of our laboratories demonstrated that upon
treatment with low concentrations of 1-BtOH, the Golgi apparatus in
mammalian cells undergoes reversible fragmentation in vivo and in vitro (18-20). Fragmentation and reassembly of the
Golgi apparatus correlated with changes in the synthesis of the
inositol phospholipid, PtdIns(4,5)P2 (18, 20). However, in
these previous studies the kinetics of Golgi fragmentation and
PtdIns(4,5)P2 synthesis were not examined, and we reasoned
that if these two events were linked, then their kinetics should be
tightly coupled.
To determine whether the Golgi apparatus underwent sequential or
immediate fragmentation in response to decreased
PtdIns(4,5)P2 synthesis, we examined its morphology in
response to alcohol treatment for different times (Fig.
1). Surprisingly, as early as after 3 min
of exposure to 1-BtOH, the Golgi apparatus manifested significant morphological changes resulting in the appearance of dilated cisternae and numerous 150-250-nm vesicles (Fig. 1A). By 9 min of
alcohol treatment, the Golgi apparatus fragmented into very large
swollen cisternae (diameter, ~250-500 nm) and numerous smaller
invaginated ~100-150-nm vesicles in greater than 80% of the cells
(Fig. 1B, arrows). Although some dilated Golgi
saccules were evident at 15 min of alcohol treatment, in all of the
cells the Golgi apparatus was fragmented (Fig. 1C) and had
completely disappeared by 30 min to be replaced by 50-100-nm vesicles
(Fig. 1D). As noted previously (18), 1-BtOH had little
effect on the morphology of other organelles (endoplasmic reticulum,
mitochondria, and nuclei), although the plasma membrane did exhibit
some blebbing (Fig. 1A). No changes in Golgi structure were
seen in cells treated with t-BtOH, which does not affect
PtdIns(4,5)P2 synthesis (18).
III spectrin, a PtdIns(4,5)P2-interacting protein, and
changes in its intracellular redistribution. The data are consistent
with a model suggesting that the decreased PtdIns(4,5)P2
synthesis and the phosphorylation state of
III spectrin
modulate the structural integrity of the Golgi apparatus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(11), and phospholipase A2 (12,
13) all cause vesiculation of the Golgi apparatus.
disrupts the Golgi apparatus architecture (11). In
addition, by exploiting the transphosphatidylation activity of
phospholipase D1 (PLD1), it has been shown that incubation with 1-BtOH
results in complete fragmentation of the Golgi apparatus in
vivo and in vitro (18-20). In the presence of 1-BtOH,
phosphatidylbutanol rather than phosphatidic acid is synthesized;
phosphatidic acid stimulates Type I PtdIns(4)P 5-kinases, the final
enzymes in the phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2) biosynthetic pathway (21). The absence of
phosphatidic acid production led to decreased PtdIns(4,5)P2
synthesis and correlated with fragmentation of the Golgi complex (18).
However, the mechanism whereby the Golgi apparatus underwent
fragmentation in the absence of PtdIns(4,5)P2 biosynthesis
was unclear from our previous studies.
spectrin, designated
III, associates with
distinct regions of the Golgi, as well as with other vesicular
organelles (22-25) and interacts directly with PtdIns(4,5)P2 (26, 27). In mammalian cells spectrin
functions in controlling plasma membrane shape, organization, and
stability and has been best characterized in erythrocytes (reviewed in
Refs. 28 and 29). Plasma membrane spectrin is a heterotetramer
consisting of two high molecular weight (~220,000-240,000)
- and
-chains that form long flexible proteins that interact with
actin as well as other proteins including ankyrin and dynactin. Each
spectrin subunit possesses multiple repeats of ~106 amino acids that
assemble into triple helix bundles (29). The Golgi apparatus
III
spectrin is a ~270-kDa polypeptide with conserved actin, protein 4.1, and ankyrin-binding domains and has been postulated to function in maintaining Golgi structure (22, 24).
III spectrin has N- and
C-terminal membrane association domains, designated MAD-1 and -2, respectively; the C-terminal MAD-2 region includes a PH domain that
binds to PtdIns(4,5)P2 containing liposomes in
vitro (29). Significantly, ARF-1 has been shown to enhance the
synthesis of PtdIns(4,5)P2 in Golgi membranes, and the
presence of this lipid is a prerequisite for
III spectrin binding to
membranes in vitro (26, 27). Furthermore, brefeldin A, which
inhibits specific ARF-guanine nucleotide exchange factor
activities, induces rapid release of spectrin from the Golgi apparatus
to the cytoplasm during disassembly of the organelle (22, 26). These
observations suggest that interactions between
PtdIns(4,5)P2 and
III spectrin are required to maintain
the structure of the Golgi apparatus. Here we have tested this
hypothesis and demonstrate that in the absence of ongoing
PtdIns(4,5)P2 synthesis, Golgi
III spectrin undergoes
phosphorylation and that intracellular redistribution of
phosphorylated
III spectrin coincides with fragmentation of the organelle.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
III spectrin (Pab-R-
III7-11) was generated to
a glutathione S-transferase-
III spectrin fusion protein
representing spectrin repeat units 7-11 (codons 1019-1464).
Hyperimmune serum was affinity-purified against the
III 7-11
recombinant peptide after the peptide was freed of glutathione
S-transferase by digestion with thrombin (24).
20 °C
methanol/acetone. The samples were incubated for 1 h at room
temperature with primary antibodies diluted in solution I (0.5% bovine
serum albumin, 0.2% saponin, 1% fetal calf serum in
phosphate-buffered saline) prior to use (18). The samples were then
treated with appropriate secondary antibodies also diluted in solution
I. After washing, the coverslips were mounted onto slides and examined
using an Olympus (Melville, NY) IX 70 microscope with 60× N.A. 1.4 planapo optics using a Photometrics (Tucson, AZ) Sensys cooled CCD
camera. Z series images were obtained through the depth of cells using
a step size range of 0.1-0.4 µm and projected using the maximum
pixel method. Deconvolution was performed with Vaytek (Fairfield, IA)
PowerHazeBuster running on a Macintosh G3, and maximum pixel
projections were rendered with I.P. Lab Spectrum (Scanalytics, Fairfax,
VA). The images were processed using Adobe Photoshop software at
identical settings. The controls were imaged to exclude background
fluorescence or bleed-through between Cy3 and fluorescein
isothiocyanate channels.
-32P]ATP at a final specific
activity of 70 µCi/µmol. The samples were incubated for the
indicated times at 37 °C, and the reactions were terminated by
addition of 1 N HCl followed by CHCl3:MeOH (1:1). The chloroform phase was separated by centrifugation,
transferred to a fresh tube, and washed with methanol:HCl (1 N) (1:1). The organic phase was vacuum-dried and
resuspended in chloroform:methanol:HCl (12 N) (200:100:1),
and spotted onto TLC plates impregnated with oxalic acid. The plates
were developed with
CHCl3:MeOH:H2O:NH4OH (65:47:11:1.6), and the radiolabeled phospholipids were identified by
autoradiography and their comigration with nonradioactive standards.
III spectrin antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequential fragmentation of the Golgi
apparatus. GH3 cells were treated with 1% 1-BtOH for 3, 9, 15, and 30 min (A, B, C, and D,
respectively). The cells were then fixed and prepared for transmission
electron microscopy. The arrows indicate invaginated
vesicles characteristic of the early stages of Golgi fragmentation
(B). Note that mitochondria and nuclei were unaffected by
treatment with alcohol. Bar, 500 nm.
PtdIns(4,5)P2 Synthesis and Golgi
Fragmentation--
We reasoned that if the rapid fragmentation of
Golgi apparatus resulted from decreased PtdIns(4,5)P2
synthesis, then at early times following alcohol treatment there should
be a diminution in the synthesis of this lipid but not other Golgi
inositol phospholipids. To test this idea, isolated Golgi membranes
were incubated with 1-BtOH for different times in the presence of
[-32P]ATP, the lipids were extracted, and the levels
of PtdIns(4)P and PtdIns(4,5)P2 were determined (Fig.
2). In control Golgi membranes PtdIns(4,5)P2 synthesis was very rapid and reached a
plateau by ~2-3 min of incubation. The initial rate of
PtdIns(4,5)P2 synthesis was identical in Golgi membranes
treated with 1-BtOH. However, by 2 min of 1-BtOH treatment,
PtdIns(4,5)P2 synthesis was only ~50% of control levels,
and thereafter its synthesis declined rapidly such that by 10 min of
incubation there was a 10-fold difference between control and alcohol
treated membranes (Fig. 2A). We presume that the initial
synthesis of PtdIns(4,5)P2 in the presence of 1-BtOH was
stimulated by a small pool of endogenous PA present in Golgi membranes;
however, because this was rapidly replaced by PtdBtOH,
PtdIns(4,5)P2 synthesis was inhibited. It was possible that
the inhibition of PtdIns(4,5)P2 synthesis in the presence
of 1-BtOH resulted from nonspecific effects of the alcohol or
denaturation of PLD and/or other enzymes rather than as a consequence
of decreased PA synthesis. To test this idea, isolated Golgi membranes
were pretreated with short chain PA prior to incubation with 1-BtOH.
Our rationale was that if PtdIns(4,5)P2 synthesis were
inhibited as a consequence of PtdBtOH formation and hence the absence
of PA, then preloading the membranes with PA should rescue
PtdIns(4,5)P2 synthesis in the presence of alcohol. Consistent with this idea, in the presence of 1-BtOH,
PtdIns(4,5)P2 synthesis in Golgi membranes preloaded with
PA was about 7-fold higher than in membranes not pretreated with PA
(Fig. 2). In addition, PtdIns(4,5)P2 synthesis was ~75%
of control incubations, i.e. those not treated with 1-BtOH
(Fig. 2). These results strongly suggest that the effects of 1-BtOH
treatment were specific and resulted from the absence of PA rather than
a consequence of enzyme denaturation.
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In contrast to PtdIns(4,5)P2, there was little change in the kinetics of PtdIns(4)P in the absence or presence of 1-BtOH. In both control and alcohol-treated membranes, there was a sharp decrease in PtdIns(4)P synthesis as it was rapidly (~60 s) converted to PtdIns(4,5)P2 (Fig. 2B). Whereas in control Golgi membranes the level of PtdIns(4)P reached a plateau at 90 s, in samples incubated with 1-BtOH the level of PtdIns(4)P was slightly higher consistent with a decrease in its conversion to PtdIns(4,5)P2. Most significantly, the decrease in PtdIns(4,5)P2 synthesis corresponded to the timing of Golgi fragmentation (Fig. 1).
It is possible that the incorporation of 32P into PtdIns(4,5)P2 (Fig. 2A) resulted from its turnover in Golgi membranes. To investigate this possibility and determine whether Golgi membranes possess PtdIns(4)P and/or PtdIns(4,5)P2 phosphatase activities, membranes containing 32P-labeled PtdIns(4)P and PtdIns(4,5)P2 (synthesized as above) were incubated in the absence or presence of cytosol, and the levels of both lipids were determined (Fig. 2C). In the absence of cytosol, no turnover of PtdIns(4)P was observed, and even in its presence hydrolysis was minimal. In contrast, by 15 min of incubation ~60% of radiolabeled PtdIns(4,5)P2 turned over; furthermore, this was independent of cytosol (Fig. 2C). Based on this result, we conclude that the plateau in the level of PtdIns(4,5)P2 synthesis in vitro (Fig. 2A) resulted from the rate of synthesis matching that of its degradation rather than the PtdIns(4)P substrate being limiting (Fig. 2B). Significantly, our data suggest that rat liver Golgi membranes possess a putative PtdIns(4,5)P2 5-phosphatase activity.
Redistribution of III Spectrin from Golgi
Membranes--
Previous work demonstrated that
PtdIns(4,5)P2 levels mediate the association of
III
spectrin both with Golgi membranes (26) and liposomes (27). Because
spectrin functions in maintaining membrane shape and structure (28,
29), we hypothesized that the fragmentation of the Golgi apparatus
observed in response to decreased PtdIns(4,5)P2 synthesis
might result at least in part from spectrin dissociation from the
organelle. We therefore examined the distribution of
III spectrin in
control and alcohol-treated GH3 cells (Fig.
3). In agreement with previous studies
(24),
III spectrin had a vesicular and perinuclear distribution and partially colocalized with the medial Golgi enzyme mannosidase II (Fig.
3, A-C). Upon brief treatment with 1-BtOH, the Golgi apparatus fragmented (Fig. 3E);
III spectrin staining
became more disperse and was mostly evident at the cell periphery (Fig. 3D). Significantly, there was little
III spectrin
staining of the vesicles generated as a result of Golgi fragmentation
and much less colocalization with mannosidase II than in control cells (Fig. 3, D-E).
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If III spectrin functioned in maintaining the Golgi apparatus
structure, then it would be expected to be recruited onto membranes during alcohol washout when the organelle reassembles (18, 20). To test
this idea and exclude the possibility that our observations were unique
to endocrine cells, we followed the time course of
III spectrin
localization to the reforming Golgi apparatus in NRK cells treated with
1-BtOH (Fig. 4). As in GH3 cells, in
control untreated NRK cells
III spectrin exhibited a diffuse, loose
perinuclear staining pattern that partially colocalized with
mannosidase II (Fig. 4, A-C). Following alcohol treatment
(30 min), in most cells the Golgi apparatus became fragmented, and the
distribution of
III spectrin was quite dispersed (Fig. 4,
D-F). At 10 min after alcohol washout,
III
spectrin still exhibited a diffuse distribution (Fig. 4,
G-I); in contrast, mannosidase II staining already began to
coalesce, and in some cells partial overlap with
III spectrin was
observed (Fig. 4, H and I). Significantly,
between 20 and 40 min after washout,
III spectrin immunoreactivity
was evident in the perinuclear Golgi region of the cells, and there was
enhanced colocalization with mannosidase II staining (Fig. 4,
J-O). Between 40 and 60 min after alcohol removal, when
normal Golgi apparatus morphology had been partially restored,
significant immunoreactive-
III spectrin staining was still evident
in the perinuclear region (Fig. 4, P-R). Together these
data suggest that
III spectrin plays a role in the reformation or
remodeling of the Golgi apparatus following its fragmentation.
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The diffuse staining of III spectrin suggested that it had
redistributed from the Golgi membrane into the cytoplasm, and we used
cell fractionation to analyze its distribution. A homogenate from
control and 1-BtOH-treated cells was applied to a sucrose density
floatation gradient (Fig. 5), and each
fraction was assayed for Golgi marker proteins and immunoreactive
III spectrin by Western blotting. Consistent with previous
observations (24) in control cells,
III spectrin was present in the
Golgi fractions (Fig. 5A, fractions 2-4) as
determined by its cofractionation with the cis-Golgi marker
GM130; it was also evident in fractions 5 and 6, which correspond to
endosomal compartments (data not shown). In addition to the major
immunoreactive
III spectrin band (Mr = ~220,000; Fig. 5A, asterisk), a second minor
polypeptide that migrated more slowly on SDS gels was also evident
particularly in fractions 4-6 (Fig. 5A,
diamond); this was absent from the cytosol (fractions
9-11). A similar pattern of two closely spaced immunoreactive
III
spectrin bands was also seen in 35S-labeled Madin-Darby
canine kidney and NRK cells (data not shown). In membranes isolated
from 1-BtOH-treated cells, the distribution of both
III spectrin
forms was significantly different from that of control cells (Fig. 5,
A and B). In most fractions, the lower form (220 kDa) was either absent, or its level was diminished (e.g.
fractions 2-4, corresponding to Golgi membrane; asterisk), and the slower migrating band was now the dominant
spectrin-immunoreactive polypeptide, with significant levels of this
protein (~42%) being present in fractions 9-11 corresponding to the
cytosol (the gradient load zone) (Fig. 5, A and
B, fractions 3-6 and 8-11,
diamond).
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Phosphorylation of III Spectrin--
It was
possible that the higher molecular weight form of
III spectrin and
its altered distribution on sucrose gradients following Golgi
fragmentation occurred because it was derived from another organelle
that cofractionated with the Golgi after alcohol treatment. To exclude
this possibility we analyzed isolated Golgi membranes for the presence
of both forms of
III spectrin (Fig.
6A). In control membranes only
the lower 220-kDa form of
III spectrin was evident (lane
1). Surprisingly, in these isolated Golgi membranes the higher
molecular weight species was only observed following incubation with a
cytosolic extract and ATP (Fig. 6A, lane 2). It
is noteworthy that the level of the slowly migrating
III spectrin increased in response to 1-BtOH (lane 3).
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Our observation of a more slowly migrating form of III spectrin
whose level was increased in vitro by the presence of ATP and 1-BtOH suggested that it corresponded to a phosphorylated form of
the polypeptide. To test this idea, samples of fractions 9-11 from the
sucrose gradient (taken from cells treated with 1-BtOH) were incubated
with different protein phosphatases specific for phosphoserine,
phosphothreonine, or phosphotyrosine residues (Fig. 6B). In
the untreated samples (Fig. 6B, lane 1), two
III spectrin polypeptides were evident in which the slower migrating band was the predominant form. Incubation with the
phosphotyrosine-specific phosphatases protein-tyrosine phosphatase 1B
or Yersenia p. phosphatase had no effect on
III spectrin
gel mobility (lanes 5 and 6). Similarly protein
phosphatase 2A had no effect on the distribution or mobility of the two
forms of
III spectrin. Strikingly, treatment with PP1 dramatically
altered the gel migration of the higher molecular weight
III
spectrin polypeptide, which now comigrated with the faster migrating
species (lanes 2 and 4). Furthermore, in
alcohol-treated cells the level of phosphorylated
III spectrin
increased ~4-fold compared with control cells, from about 17% to
70% of the total
III spectrin in the cells (Fig. 5C).
Together, these data strongly suggest that the
III spectrin
polypeptide is phosphorylated in response to diminished
PtdIns(4,5)P2 synthesis.
If BtOH-induced phosphorylation of III spectrin caused its partial
dissociation from Golgi membranes and the concomitant fragmentation of
the organelle, then treatment of cells with protein phosphatase
inhibitors should produce a similar result, namely accumulation of
phosphorylated
III spectrin and disassembly of the Golgi apparatus.
Indeed, much earlier reports have demonstrated fragmentation of the
Golgi apparatus in cells treated with okadaic acid, an inhibitor of
protein phosphatases (6). NRK or GH3 cells were incubated with okadaic
acid (Fig. 7), and in agreement with
earlier reports (6), the Golgi apparatus underwent fragmentation, and
its lace-like appearance was disrupted (Fig. 7A,
panels B and E). Furthermore,
III spectrin
exhibited a much more diffuse localization that contrasted with its
staining in control untreated cells (panels A and
D). Subcellular fractionation (Fig. 7B)
demonstrated that the majority of the material isolated from okadaic
acid-treated cells corresponded to the slower migrating phosphorylated
form of
III spectrin. Indeed, significantly, more of the
III
spectrin was present in the cytosol (fractions 8-11) than in
1-BtOH-treated cells, and virtually all of this immunoreactive spectrin
was of the higher molecular weight phosphorylated form. Together these results are consistent with our model that phosphorylation of
III
spectrin leads to its partial dissociation from Golgi membranes, thereby destabilizing the structure of the organelle.
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DISCUSSION |
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Studies from several laboratories, including our own (11, 16, 18, 20) have implicated inositol phospholipids in maintaining the structure and function of the Golgi apparatus in mammalian cells. Treatment of cells with 1-BtOH for 40 min led to complete fragmentation of the Golgi apparatus (18). We demonstrated that in the presence of 1-BtOH, PLD hydrolysis of PC yielded PtdBtOH rather than PA; the latter stimulates the Type I PtdIns(4)P, 5-kinase activities, the final step in the PtdIns(4,5)P2 biosynthetic pathway (21). Consequently, in the presence of alcohol, the absence of PA resulted in rapid inhibition of PtdIns(4,5)P2 synthesis (Fig. 2 and Refs. 18 and 20). However, the mechanism whereby the Golgi apparatus fragments in the absence of PtdIns(4,5)P2 was not apparent from our previous studies.
Fragmentation of the Golgi Apparatus Occurs Rapidly following Decreased PtdIns(4,5)P2 Synthesis-- Butanol-induced fragmentation of the Golgi apparatus was significantly more rapid than previously noted; at 5 min of alcohol treatment, dilated cisternae and large invaginated vesicles were evident in many cells (Fig. 1). By 15 min of alcohol treatment, the Golgi apparatus had fragmented completely in greater than 90% of cells examined. In the presence of 1-BtOH, PtdIns(4,5)P2 synthesis in isolated Golgi membranes was inhibited rapidly (~5 min) and by 10 min of incubation was 10-fold lower than control membranes (Fig. 2). It is noteworthy that the kinetics of PtdIns(4,5)P2 inhibition in vitro exactly mirrored those of Golgi fragmentation in vivo, suggesting a role for this lipid in maintaining Golgi architecture (see below). Recent evidence has demonstrated that the majority of cellular PtdIns(4,5)P2 localizes to the plasma membrane, whereas the Golgi apparatus, endosomes, and endoplasmic reticulum have a small but discernable pool of this lipid (41). Although Golgi membranes support PtdIns(4,5)P2 synthesis in vitro (Fig. 2 and Refs. 11, 18, 20, and 43) and undergo fragmentation in the presence of alcohol (18, 20), at present it is unclear in whole cells which of the PtdIns(4,5)P2 pools leads to fragmentation of the Golgi in response to 1-BtOH treatment. In this context, a decrease in either or both PtdIns(4,5)P2 pools could cause dissociation of cytoskeleton proteins (e.g. spectrin; see below) leading to Golgi fragmentation (29). Furthermore, our data suggest that Golgi membranes possess potent PtdIns(4,5)P2 5-phosphatase activity (Fig. 2C) that could itself play a role in regulating Golgi structure; currently, we are investigating this possibility. Alternatively, the presence of PA itself may be a necessary requirement to maintain Golgi structure. Consistent with this idea, our very recent work has shown that both PLD1 and PLD2 localize to the Golgi complex and are also present on the plasma membrane (19, 42). PLD1 is distributed throughout the stacks, whereas Golgi associated PLD2 resides on cisternal rims exclusively (42), suggesting that these enzymes and their product PA play a role in Golgi membrane dynamics (44) and structure.
It might be argued that the inhibition of PtdIns(4,5)P2 synthesis in vitro in response to 1-BtOH or treatment of cells with the alcohol resulted from inactivation or denaturation of PLDs (45) and/or PtdIns(4)P 5-kinases, or that PtdBtOH itself inhibited these activities independently of PA. Several lines of evidence argue against this idea. First, preloading isolated Golgi membranes with PA abrogated the effects of 1-BtOH (Fig. 2), suggesting that the absence of PA rather than enzyme inactivation per se caused inhibition of PtdIns(4,5)P2 synthesis. Second, the activities of other enzymes, e.g. PtdIns 4-kinases, were relatively unaffected by alcohol treatment (Fig. 2B and Ref. 18). Indeed, PtdIns(4)P levels were slightly higher than in controls, because in the absence of PtdIns(4)P 5-kinase activity, this substrate was not converted to PtdIns(4,5)P2 (Fig. 2B). Third, our previous work demonstrated that t-BtOH or secondary alcohols had no effect on Golgi structure in vivo (18). Further, the effects of 1-BtOH were selective for the Golgi apparatus and plasma membrane where changes in cell shape occur (Fig. 4 and Ref. 18), consistent with the localization of PLD2 (31, 42). Finally, the transport of vesicular stomatitis virus G protein, which is inhibited quantitatively in response to 1-BtOH-mediated Golgi fragmentation (18), was rapidly reversible following alcohol wash-out, as was Golgi morphology and PtdIns(4,5)P2 synthesis (18, 20). Taken together our results argue that under the conditions employed for these experiments, the effect of 1-BtOH did not result from nonspecific enzyme inactivation. Consequently, the present data are consistent with our earlier results that had implicated decreased PtdIns(4,5)P2 synthesis in mediating fragmentation of the Golgi apparatus (18).
Phosphorylation of III Spectrin--
The plasma membrane of all
mammalian cells, particularly the erythrocyte, possesses a spectrin
cytoskeleton that functions in maintaining the structural integrity and
domain organization of the plasma membrane (28, 29). The association of
spectrin with membranes is a multivalent process involving several
protein-protein interactions and at least two membrane association
domains (23, 29) as well as a C-terminally disposed PH domain, which
binds PtdIns(4,5)P2 (29). Previous evidence has
demonstrated that ARF-1 stimulates PtdIns(4,5)P2 synthesis
and the concomitant binding of
III spectrin to Golgi membranes
in vitro (26). In the absence of PtdIns(4,5)P2
or in the presence of a competing PH domain peptide, spectrin binding
to Golgi membranes is inhibited (26). Similarly, spectrin mediates the
linkage between acidic phospholipid vesicles and dynactin in squid
axons, an activity also inhibited by
III spectrin PH domain peptides
(27).
The above observations and our demonstration of Golgi
fragmentation in the absence of ongoing PtdIns(4,5)P2
synthesis prompted us to investigate a possible role for III
spectrin in this process. Our data show that diminished
PtdIns(4,5)P2 synthesis resulted in the generation of a
phosphorylated form of
III spectrin, as determined by its
sensitivity to phosphatase PP1 (Fig. 6). Much of the phosphorylated
form of
III spectrin was present in the cytoplasm rather than on
Golgi membranes (Figs. 5 and 7); this was particularly evident in
response to okadaic acid treatment (Fig. 7). We speculate that the
redistribution of phosphorylated
III spectrin contributed to the
fragmentation of the Golgi apparatus. Our present results are similar
to earlier reports demonstrating that in Chinese hamster ovary and HeLa
cells the plasma membrane
-spectrin, but not the
-subunit,
undergoes increased phosphorylation during mitosis, and this correlated
with its redistribution from the cell surface to the cytosol (32).
Interestingly, phosphorylation of plasma membrane
-spectrin was
shown to alter the mechanical stability of erythrocyte membranes,
whereas decreased phosphorylation enhanced membrane stability (33).
The correlation between formation of phosphorylated III spectrin and
fragmentation of the Golgi apparatus is consistent with earlier
suggestions of a structural role for spectrin in the Golgi (22).
Significantly, spectrin dissociated from the Golgi apparatus in
response to brefeldin A treatment and also during mitosis when the
organelle undergoes extensive fragmentation (22). The phosphorylation of
III spectrin as a mechanism for its detachment from Golgi membranes is also reminiscent of the interaction of the Golgi tethering
protein p115 with Golgi membranes and the matrix protein GM130 (34).
During interphase, phosphorylated p115 is present in the cytosol,
whereas the nonphosphorylated form is associated with Golgi membranes
(35). Phosphorylation of GM130 at its N terminus during mitosis
inhibits its binding to p115 resulting in the disassembly of the Golgi
apparatus (36). Similarly, we suggest that the interaction of
III
spectrin with other Golgi cytoskeletal proteins, in particular Golgi
isoforms of ankyrin (23, 37), and the membrane may be weakened by its
phosphorylation; in turn, this could lead to the partial dissociation
of a putative scaffolding structure from Golgi membranes.
Although okadaic acid can inhibit numerous phosphatases, which could
affect Golgi structure via multiple pathways, it is noteworthy that
both butanol and okadaic acid, two agents with quite different modes of
action, led to increased phosphorylation of III spectrin and
disassembly of the Golgi apparatus. Together these observations support
our hypothesis (Fig. 8) that in part the
phosphorylation state of
III spectrin modulates Golgi structure.
Although the site(s) of
III spectrin phosphorylation remain to be
determined, digestion with several protein phosphatases suggests that
tyrosine phosphorylation was not significant. Hydrolysis by only PP1
implied that specific Ser and/or Thr residues were phosphorylated;
their identity is currently being determined. We propose that
III
spectrin phosphorylation is regulated by a putative kinase and
phosphatase whose activities are inversely related to the level of
PtdIns(4,5)P2 synthesis in the Golgi membrane (Fig. 8).
Indeed, much earlier work (38, 39) had identified a species of casein
kinase I activity in erythrocyte membranes whose activity is regulated by the level of PtdIns(4,5)P2 and that could utilize
spectrin among other polypeptides, as a substrate. However, in these
earlier reports the function of the casein kinase I activity was
unclear. Subsequently, these investigators demonstrated that in neurons casein kinase I
was localized to vesicular structures including the
endoplasmic reticulum and Golgi apparatus and that isolated synaptic
vesicles were highly enriched in the enzyme; significantly, this enzyme
activity is regulated by PtdIns(4,5)P2 (40). We speculate
that a casein kinase I
or a closely related Golgi isoform of the
enzyme functions in regulating the binding of
III spectrin to the
surface of the organelle; experiments are currently in progress to test
this hypothesis.
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ACKNOWLEDGEMENTS |
---|
We thank Michael Cammer, Frank Macaluso, and Leslie Gunther for expert technical help with immunofluorescence and electron microscopy. We thank Dr. Z. Y. Zhang (Albert Einstein College of Medicine) for generous gifts of protein-tyrosine phosphatase 1B and Y. pestis tyrosine phosphatase. We thank Drs. Sharon Milgram and Brian Burke for generous gifts of antibodies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK21860 (to D. S.) and DK38979 and DK43812 (to J. S. M.). Core support was provided by National Institutes of Health Cancer Center Grant P30CA13330 (to D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Training Grant T32 GM07491.
** To whom correspondence should be addressed: Dept. of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2653; Fax: 718-430-8567; E-mail: shields@aecom.yu.edu.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M209137200
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ABBREVIATIONS |
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The abbreviations used are: ARF, ADP-ribosylation factor; PA, phosphatidic acid; BtOH, butanol; PtdBtOH, phosphatidylbutanol; PtdIns, phosphatidylinositol; PtdIns(4)P, phosphatidylinositol-4-phosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PLD, phospholipase D; PH, pleckstrin homology; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; NRK, normal rat kidney; Man II, mannosidase II.
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