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Originally published In Press as doi:10.1074/jbc.M209137200 on October 30, 2002

J. Biol. Chem., Vol. 278, Issue 3, 1957-1965, January 17, 2003
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Fragmentation of the Golgi Apparatus

A ROLE FOR beta III SPECTRIN AND SYNTHESIS OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE*

Anirban SiddhantaDagger , Andreea RadulescuDagger §, Michael C. Stankewich, Jon S. Morrow, and Dennis ShieldsDagger ||**

From the Department of Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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 beta III spectrin modulate the structural integrity of the Golgi apparatus.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Ibeta (11), and phospholipase A2 (12, 13) all cause vesiculation of the Golgi apparatus.

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 Ibeta 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.

A possible link between inositol phospholipids and the maintenance of Golgi structure comes from observations that a specific form of the cytoskeletal protein beta  spectrin, designated beta 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) alpha - and beta -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 beta 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). beta 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 beta 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 beta 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 beta III spectrin undergoes phosphorylation and that intracellular redistribution of phosphorylated beta III spectrin coincides with fragmentation of the organelle.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta III spectrin (Pab-R-beta III7-11) was generated to a glutathione S-transferase-beta III spectrin fusion protein representing spectrin repeat units 7-11 (codons 1019-1464). Hyperimmune serum was affinity-purified against the beta III 7-11 recombinant peptide after the peptide was freed of glutathione S-transferase by digestion with thrombin (24).

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 -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.

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 [gamma -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.

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-beta III spectrin antibodies.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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 [gamma -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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  		Fig. 2.   Inhibition of PtdIns(4,5)P2 and PtdIns(4)P synthesis in isolated Golgi membranes. Golgi membranes isolated from rat liver were incubated with [gamma -32P]ATP and rat brain cytosol (2 mg/ml) for 0, 0.5, 1.0, 1.5, 2.0, 5, 10, 15, and 20 min at 37 °C with or without 1% 1-BtOH. For PA rescue, Golgi membranes were preincubated with 300 µM of C8-PA for 30 min at 22 °C, after which the membranes were incubated as above. Following incubation, total phospholipids were extracted, and the radiolabeled lipids were resolved by TLC followed by autoradiography ("Materials and Methods"). The intensities of the spots corresponding to PtdIns(4,5)P2 (A) and PtdIns(4)P (B) were quantified by densitometry. The values are the averages of duplicate samples from three separate experiments. A and B, black-square, control incubation; black-triangle, incubation with 1-BtOH; , membranes pretreated with C8-PA and incubated in the presence of 1% 1-BtOH. C, Golgi membranes were incubated with [gamma -32P]ATP and rat brain cytosol for 10 min at 37 °C (as above), after which the radiolabeled membranes were separated by centrifugation through a 0.3 M sucrose cushion. The membrane pellet was resuspended in buffer ("Materials and Methods") and incubated in the absence of ATP with or without rat brain cytosol (2 mg/ml) for 15 min at 37 °C, after which the lipids were extracted and analyzed by TLC and quantitated as above. PIP, PtdIns(4)P), down diagonal hatching; PIP2, PtdIns(4,5)P2, up diagonal hatching.

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 beta III Spectrin from Golgi Membranes-- Previous work demonstrated that PtdIns(4,5)P2 levels mediate the association of beta 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 beta III spectrin in control and alcohol-treated GH3 cells (Fig. 3). In agreement with previous studies (24), beta 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); beta III spectrin staining became more disperse and was mostly evident at the cell periphery (Fig. 3D). Significantly, there was little beta 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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  		Fig. 3.   Localization of beta III spectrin in control and alcohol treated cells. GH3 cells were incubated with medium alone (A-C) or with 1.0% 1-BtOH (D-F) for 40 min at 37 °C. Following incubation, the cells were fixed with -20 °C methanol/acetone and prepared for indirect double immunofluorescence microscopy ("Materials and Methods") using an affinity-purified rabbit antibody to beta III spectrin (A and D) and monoclonal antibody to the medial-Golgi marker mannosidase II (53FC3) (B and E). Mannosidase II (Man II) was visualized using a fluorescein isothiocyanate-conjugated goat anti-mouse IgG and beta III spectrin localized using a Cy3 sheep anti-rabbit IgG. Each sample is from the same field of cells. To demonstrate overlap of beta III spectrin and mannosidase II, CY3 (red) and fluorescein isothiocyanate (green) images were merged (C and F). Yellow indicates regions of colocalization. The arrows indicate colocalization of beta III spectrin and mannosidase II. The arrowheads indicate fragmented Golgi with little beta III spectrin colocalization. All of the micrographs are deconvolved images from single optical sections. Scale bar, 10 µm.

If beta 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 beta 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 beta 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 beta III spectrin was quite dispersed (Fig. 4, D-F). At 10 min after alcohol washout, beta 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 beta III spectrin was observed (Fig. 4, H and I). Significantly, between 20 and 40 min after washout, beta 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-beta III spectrin staining was still evident in the perinuclear region (Fig. 4, P-R). Together these data suggest that beta III spectrin plays a role in the reformation or remodeling of the Golgi apparatus following its fragmentation.


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  		Fig. 4.   Colocalization of beta III spectrin with the Golgi apparatus during Golgi reformation. NRK cells were incubated with medium alone (A-C) or with 1% 1-BtOH for 30 min at 37 °C (D-F), after which the alcohol was removed and replaced with fresh medium for 10 min (G-I), 20 min (J-L), 40 min (M-O), or 60 min (P-R). The cells were prepared for immunofluorescence microscopy with rabbit antibody to beta III spectrin (A, D, G, J, M, and P) and monoclonal antibody 53FC3 to the medial Golgi marker mannosidase II (B, E, H, K, N, and Q). beta III spectrin and Man II were visualized as outlined in the legend to Fig. 3. To demonstrate colocalization, the images were merged (C, F, I, L, O, and R); the yellow regions indicate complete overlap. The arrows indicate beta III spectrin enrichment in the region of the reforming Golgi apparatus and overlap between beta III spectrin and Man II, respectively. The arrowheads indicate fragmented Golgi with minimal beta III spectrin colocalization. All of the micrographs are projected Z series using a cooled CCD camera. Bar, 10 µm.

The diffuse staining of beta 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 beta III spectrin by Western blotting. Consistent with previous observations (24) in control cells, beta 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 beta 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 beta 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 beta 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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  		Fig. 5.   Subcellular distribution of beta III spectrin in GH3 cells. A, cells were either untreated (Control) or treated for 40 min with 1% 1-BtOH and homogenized, and the homogenate was fractionated on an equilibrium floatation gradient (46). An aliquot of each gradient fraction was analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti-beta III spectrin antibodies. The blots were stripped and reprobed with anti-GM130 antiserum. In control cells, GM130 was present in fractions 2 and 3 (arrows), whereas beta III spectrin (~220 kDa; asterisk) was enriched in fractions 2-5. A slightly slower migrating form of beta III spectrin-immunoreactive polypeptide (diamond) was visible in fractions 3-6. In membranes isolated from 1-BtOH-treated cells, the intensity of the ~220-kDa beta III spectrin band was either abolished completely (fraction 2) or greatly diminished (asterisk, fractions 3-11). The intensity of the slower migrating beta III spectrin (diamond) was increased, and this species was now prominent in fractions 9-11, which correspond to the cytosol. The arrowheads indicate the migration of GM130. B, the intensities of the two forms of beta III spectrin (slow and faster migrating bands) were determined using a computing densitometer and their ratios calculated for each gradient fraction. C, the levels of the two forms of beta III spectrin (phosphorylated and nonphosphorylated, respectively) in each gradient fraction were determined by densitometry and expressed as: sum of phosphorylated spectrin from each fraction/total nonphosphorylated + phosphorylated spectrin) × 100.

Phosphorylation of beta III Spectrin-- It was possible that the higher molecular weight form of beta 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 beta III spectrin (Fig. 6A). In control membranes only the lower 220-kDa form of beta 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 beta III spectrin increased in response to 1-BtOH (lane 3).


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  		Fig. 6.   In vitro phosphorylation and dephosphorylation of beta III spectrin. A, Golgi membranes isolated from GH3 cells were incubated with rat brain cytosol (2 mg/ml) either alone (lane 1) or with 1 mM ATP (lane 2) or with ATP and 1% 1-BtOH (lane 3) for 10 min at 37 °C. Following incubation, each sample was analyzed as above. B, aliquots of gradient fractions 9-11 isolated from 1-BtOH-treated cells were incubated with either buffer alone (lane 1) or with the following protein phosphatases: PP1 (lane 2); PP2A (lane 3); PP1 and PP2A (lane 4); protein-tyrosine phosphatase 1B (lane 5); or Yersinia tyrosine phosphatase (lane 6) as outlined under "Materials and Methods." Following incubation each sample was analyzed as described for A. Only treatment with PP1 increased the mobility of the slower migrating form of beta III spectrin (diamond).

Our observation of a more slowly migrating form of beta 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 beta 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 beta 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 beta III spectrin. Strikingly, treatment with PP1 dramatically altered the gel migration of the higher molecular weight beta III spectrin polypeptide, which now comigrated with the faster migrating species (lanes 2 and 4). Furthermore, in alcohol-treated cells the level of phosphorylated beta III spectrin increased ~4-fold compared with control cells, from about 17% to 70% of the total beta III spectrin in the cells (Fig. 5C). Together, these data strongly suggest that the beta III spectrin polypeptide is phosphorylated in response to diminished PtdIns(4,5)P2 synthesis.

If BtOH-induced phosphorylation of beta 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 beta 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, beta 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 beta III spectrin. Indeed, significantly, more of the beta 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 beta III spectrin leads to its partial dissociation from Golgi membranes, thereby destabilizing the structure of the organelle.


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  		Fig. 7.   beta III spectrin dissociates from Golgi membranes in okadaic acid-treated cells. A, NRK cells were incubated with medium alone (A-C) or with 1 µM okadaic acid for 2 h (D-F). The cells were prepared for immunofluorescence with rabbit antibody to beta III spectrin (A and D) and monoclonal antibody 53FC3 to mannosidase II (B and E). beta III spectrin was visualized with Alexa fluor 568 goat anti-rabbit antibody, and Man II was localized using Alexa fluor 488 goat anti-mouse antibody. To demonstrate colocalization of beta III spectrin and Man II, the images were merged (C and F). The yellow regions indicate overlap. The arrows indicate colocalization of beta III spectrin and mannosidase II. The arrowheads (E and F) indicate fragmented Golgi with minimal beta III spectrin colocalization. All of the micrographs are projected Z series using a cooled CCD camera. Bar, 10 µm. B, control GH3 cells or those treated with okadaic acid (as above) were homogenized, and the homogenate was fractionated on an equilibrium floatation gradient (Fig. 5). Aliquots of each gradient fraction were analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti-beta III spectrin antibodies. The blots were stripped and reprobed with antibodies to GM130, a cis-Golgi marker. Asterisk, mobility of nonphosphorylated beta III spectrin; diamond, phosphorylated beta III spectrin. The arrows indicate the position of GM130 in gradients from control cells, and the arrowheads indicate the positions of GM130 in gradients from okadaic acid-treated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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 beta 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 beta 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 beta III spectrin in this process. Our data show that diminished PtdIns(4,5)P2 synthesis resulted in the generation of a phosphorylated form of beta III spectrin, as determined by its sensitivity to phosphatase PP1 (Fig. 6). Much of the phosphorylated form of beta 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 beta 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 beta -spectrin, but not the alpha -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 beta -spectrin was shown to alter the mechanical stability of erythrocyte membranes, whereas decreased phosphorylation enhanced membrane stability (33).

The correlation between formation of phosphorylated beta 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 beta 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 beta 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 beta III spectrin and disassembly of the Golgi apparatus. Together these observations support our hypothesis (Fig. 8) that in part the phosphorylation state of beta III spectrin modulates Golgi structure. Although the site(s) of beta 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 beta 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 Ialpha 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 Ialpha or a closely related Golgi isoform of the enzyme functions in regulating the binding of beta III spectrin to the surface of the organelle; experiments are currently in progress to test this hypothesis.


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  		Fig. 8.   Model for PtdIns(4,5)P2 regulation of beta III spectrin phosphorylation. When PtdIns(4,5)P2 levels are at steady state most beta III spectrin is nonphosphorylated and membrane-bound resulting in normal Golgi morphology. As PtdIns(4,5)P2 levels decrease, a protein kinase negatively regulated by PtdIns(4,5)P2 is recruited to and/or activated on the Golgi membrane. Phosphorylation of beta III spectrin by the putative kinase causes phosphospectrin to dissociate from the membrane, resulting in fragmentation of the Golgi apparatus.


    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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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