Calphostin C induces selective disassembly of the Golgi complex by a protein kinase C-independent mechanism

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European Journal of Cell Biology 76, 93-101 (1998, June) . © Gustav Fischer Verlag· Jena

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Calphostin C induces selective disassembly of the Goigi complex by a protein kinase C-independent mechanism Manuel Alonso a , Manuel Muiiiz a , Christine Hall b , Angel Velasco 3 , losefina Hidalgo1)a a b

Department of Cell Biology, Faculty of Biology, University of Seville, Seville/Spain Department of Neurochemistry, University of London, London/UK

Received September 10, 1997 Accepted January 9, 1998

Calphostin C - Golgi complex - Golgi disassembly protein kinase C

Introduction

Intact cells incubated with calphostin C, an inhibitor of the regulatory domain of protein kinase C, showed fragmentation and dispersal of the Golgi complex by a light-dependent mechanism. At the ultrastructural level Golgi stacks were replaced by clusters of vesicles and short tubules that resembled the Golgi remnants present in control mitotic cells. Vesicle-mediated transport processes along both the exocytic and endocytic routes were also inhibited by calphostin C treatment. Golgi disassembly, however, was not due to protein kinase C inhibition since several inhibitors of the catalytic domain did not cause a similar effect. In contrast, pretreatment with phorbol 12-myristate 13-acetate partly protected the Golgi complex from disassembly by calphostin C. The in vitro effect was shown to be reversible, required both cytosol and ATP and it was inhibited by pretreatment of the Golgi membranes with trypsin but not with high salt. These results suggest the interaction of calphostin C with a structural Golgi protein containing a phorbol esterbinding domain and necessary for the stability of this organelle during interphase.

The unique reticular structure of the Golgi complex of interphase cells is the result of a dynamic balance between several vesicle-mediated transport processes [10 , 28]. A basic Golgi organization of interconnected , polarized cisternal stacks provides the structural scaffold necessary to properly process and sort cargo molecules while allowing the accommodation of the different transport routes converging in this organelle [10, 23 , 27]. Golgi organization , however, is not permanent. In order to provide an accurate partitioning of Golgi membranes during cell division Golgi disassembly occurs at the onset of mitosis [37, 38]. Recently, this event was successfully reconstituted in vitro which has enabled the identification of both factors required for Golgi vesiculation and structural intermediates in the process [17, 24, 25]. In spite of this , virtually nothing is known about the molecular mechanism underlying Golgi breakdown during mitosis as well as the integral Golgi components that maintain the stacked organization during interphase [26]. A useful approach in the characterization of these factors involves the use of agents that selectively promote Golgi disassembly without affecting other cell structures [14 , 21, 33], Although little is known about the molecular targets for some of these treatments they are of interest given the limited information available about the fundamentals of Golgi organization. Calphostin C is a fungal metabolite that when activated by light selectively inhibits protein kinase C (PKC) [4 , 15]. Since this kinase activity regulates both constitutive and regulated exocytosis, several studies have recently reported the use of calphostin C as a specific tool to show the influence of PKC activity upon particular vesicle-mediated protein transport steps [5, 7, 9, 29 , 30]. However, the effects of this inhibitor on the secretory pathway are far from being understood since the relevant protein affected by calphostin C treatment during transport inhibition does not seem to be a PKC isoenzyme [9]. Here we describe Golgi disassembly caused by incubation of either intact cells or purified Golgi stacks with light-activated calphostin C. The results indicate that this effect is due to the

Abbreviations: ER Endoplasmic reticulum . - HRP Horseradish peroxidase. - PKC Protein kinase C. - PMA Phorbol 12-myristate 13acetate. - VSV-G Vesicular stomatitis virus G glycoprotein.

I) Prof. Dr. Josefina Hidalgo , Department of Cell Biology, Faculty of Biology, University of Seville , Avd. Reina Mercedes sin , E-41012 Seville/Spain.

94 M. Alonso, M. Muniz, C. Hall et 01. action of calphostin C on a structural Golgi protein that has no PKC activity but probably contains a diacylglycerol/phorbol ester-binding domain.

Materials and methods Materials Recombinant rat n(al)-chimaerin was prepared by cloning a Fok 1 restriction fragment containing the entire coding region [18] in a maltose-binding protein fusion vector [16]. Expression in E. coli, affinity purification on amylose-resin , and cleavage of al-chimaerin from maltose-binding protein was as previously described [16]. PKC inhibitors were purchased from Calbiochem (San Diego , CNUSA) . Phorbol 12-myristate 13-acetate (PMA), ferritin, nocodazole, and horseradish peroxidase (HRP) were from Sigma (St. Louis, MO/USA). Endoglycosidase H was from Boehringer Mannheim (Germany) and fluorescein-labeled goat anti-rabbit IgG secondary antibody from TAGO (Burlingame, CNUSA) . a-Mannosidase II antibody was a gift of Dr. M. G. Farquhar (University of California, San Diego, CN USA).

Cell culture and treatments

NRK cells were maintained in DMEM containing 5 % fetal calf serum, 2mM L-glutamine, 50 units/ml penicillin , and 50Ilg/ml streptomycin. Incubation with agents that modulate PKC activity was performed at 37 D C in serum-free medium. To activate calphostin C, samples were irradiated with a cool-white fluorescent lamp located at 20cm.

Preparation of Goigi membranes and cytosol A subcellular fraction enriched in Golgi stacks was prepared from rat liver according to Slusarewicz et al. [31]. Golgi membranes (1-2 mgt m/) were stored at -70 DC in l00mM potassium phosphate, pH 6.7, containing 250 mM sucrose and 5 mM MgCh. A 80--90-fold enrichment over homogenate was found by immunoblot detection of Golgi amannosidase II. Bovine brain cytosol was prepared as described [12] and dialyzed against 25 mM Tris-HCl, pH 8.0, 50 mM KCI , and 1 mM dithiothreitol. It was centrifuged for I h at 100000g to remove aggregates; aliquots were maintained at -70 DC. Before being used cytosolic proteins were applied to a Econo-PaclODG Bio-Rad column eluted with the buffer used in the Golgi fragmentation assay.

Studies on VSV-G processing

Cells were infected at 32 DC with 10--20 plaque-forming units/cell of the ts045 thermosensitive mutant of vesicular stomatitis virus (VSV) [6]. They were metabolically labeled at the restrictive temperature (39.5 DC) with 100 IlCi/ml Tran35 S-label for 5 min and chased for 30 min at either the restrictive or the permissive temperature (32°C). Processing ofVSV-G glycoprotein to an endogJycosidase H-resistant form was analyzed as described [3].

Studies on endocytosis

Incubation with either cat ionized ferritin or HRP was in serum-free medium. Cells were thoroughly rinsed with cold medium first and then with phosphate-buffered saline (PBS). Determination of HRP activity was performed as described [32] using cells that were previously lysed in 0.1 % SDS. Cells exposed to cationized ferritin were incubated with calphostin C and then processed for electron microscopy.

Goigi fragmentation assay

The standard assay was that described by Misteli and Warren [24]. It contained : 5III of a 40 x concentrated ATP-regenerating system (l00mM ATP, 1 M creatine phosphate, 10mg/ml creatine phosphokinase), 20 III 2 M sucrose in H 20, 40 III Golgi membranes (100 Ilg/ml final concentration), 120 III cytosol (7.5-10 mg/ml final concentration), and buffer (50mM Tris-HCl , pH 7.3, 50mM KCl, lOmM MgCIz, 20 mM ~-glycerophosphate, 15 mM EGTA, 2 mM ATP, 1 mM DTT) to make 200 III final volume. Incubation was performed for 30 min at 37"C. Samples were then fixed and processed for electron microscopy.

Electron microscopy and immunocytochemistry Samples to be processed for conventional electron microscopy were fixed at room temperature in 2.5 % glutaraldehyde in 100 mM cacodylate buffer, pH 7.4, containing 0.1 mM of both CaCl 2 and MgCIz. They were postfixed with 1 % OsOJI % K4Fe(CN)6 and embedded in Epon. For immunofluorescence , cells were fixed at room temperature for 2h in 3 % paraformaldehyde in PBS under white light. Radiation was necessary in order to quench endogenous fluorescence emitted by calphostin C. Fixed cells were permeabilized with PBS containing 0.5 % bovine serum albumin and 0.05 % saponin. Incubation with antibodies was performed in this same buffer for 30 min at 37°C. Cells to be processed for ultracryotomy were fixed in 3 % paraformaldehyde/0.05 % glutaraldehyde in 100 mM phosphate buffer, pH 7.4, and infiltrated with 20 % polyvinylpyrrolidone/2 .3 M sucrose. Immunogold detection of a-mannosidase II was carried out as previously described [36].

Stereology To calculate the surface area of the Golgi clusters in intact cells , photographs printed at a final magnification of 14000 x were scanned with an electronic pen connected to a microprocessor programmed (Videoplan , v.2.S) to estimate surface area. The volume of Golgi clusters was determined from two sets of serial thin sections each containing the entire structure. The mean surface area was multiplied by 70 nm (the average thickness of the sections) and the number of individual sections. The stereological analysis of Golgi fractions was performed from micrographs taken at low magnification and printed at a final magnification of 35000 x. The perimeter of both cisternal (considered to have a length more than twice its diameter) and vesicular profiles were estimated by the Videoplan program. The percentage of membranes in each category was determined by counting the number of intersections of each membrane structure with the lines of a 4mm square grid [24].

Results Goigi disassembly induced by calphoslin C The Golgi complex underwent rapid disassembly after incubation of intact NRK cells with light-activated calphostin C. As seen by the immunofluorescence detection of a-mannosidase II the Golgi complex appeared as a single reticular structure confined to the perinuclear region in both control, untreated cells and cells incubated with calphostin C in the dark (Fig. IA) . In contrast, it became extensively fragmented following incubation with 0.5 ~M activated calphostin C for 30 min (Fig.IB). Calphostin C-induced Golgi fragments or remnants were visualized as small fluorescent spots. Initially located around the nucleus (Fig.lB) , they increasingly dispersed throughout the peripheral cytoplasm at later time points (Fig. Ie). The effect was not only general but irreversible. Most of the cells originally exposed to calphostin C still showed Golgi disassembly after incubation in drug-free medium for several hours (Fig. Ie). At the ultrastructural level, the Golgi stacks were replaced by clusters of vesicles and short tubules (Fig. 2). Clusters had an average surface area of 6.9 ± 0.7 ~m2 (n = 20). Based on reconstitution by serial-thin sections we estimated an average volume of 3.01 ± 0.25 ~m3. In agreement with their detection by immunofluorescence most of them (> 80 % at the 30 min time point) were located in the perinuclear region (Fig. 2A) . Within single clusters two vesicular profiles were distinguished (Fig.2B). Small vesicles of 58 ± 10 nm (n = 72) diameter accounted for 74 % of the vesicular profiles in a cluster. Large vesicles had an average diameter of 135 ± 34 nm (n = 55). Both vesicular profiles appeared uncoated (Fig.2B). Overall, the fraction of total cluster membranes in vesicles was found to be

Goigi disassembly induced by calphostin C

95

Fig. 1. Detection of a-mannosidase II by immunofluorescence in calphostin C-treated cells. NRK cells were incubated at 37°C for 30 min with 0.5 f.lM calphostin C either in the dark (A) or under white light (B-C). Cells were either directly fixed and processed for immunofluo-

rescence (A-B) or additionally incubated for 2 h in the absence of calphostin C to show the irreversible effect of this drug on Golgi disassembly (C). Golgi remnants are indicated with arrows. Bar, 25 f.lm.

80 ± 1.8 %, the remainder being tubules and single cisternal profiles. The identity of these membranes was confirmed by immunogold labeling with anti-Golgi mannosidase II. Some vesicles but not all were labeled (Fig.2C). Other organelles including the nuclear envelope, endoplasmic reticulum (ER), mitochondria and endosomes were unaffected by calphostin C treatment (Fig.2A). Indeed, endosomes preloaded with cationized ferritin did not participate in the generation of calphostin C-induced Golgi clusters (not shown). Golgi complexes which were previously fragmented into stacks by

nocodazole-induced microtubule depolymerization still experienced disassembly following incubation with calphostin C (not shown). Taken together these results indicated the alteration of a structural factor responsible for the integrity of the Golgi stack.

Fig. 2. Ultrastructural alteration of the Golgi complex by calphostin C. Cells incubated with activated calphostin C were fixed and processed for either conventional electron microscopy (A-B) or immuno-

Transport inhibition Calphostin C has been reported to block export ofVSV-G glycoprotein from the ER [9] and to inhibit transport from the trans-Golgi network to the cell surface [5, 9, 30]. In addition,

gold labeling of cryosections with anti-mannosidase II (C). cs, Centrosome; ne, nuclear envelope; er, endoplasmic reticulum; m, mitochondria. Bar, 0.2 f.lm.

96

M. Alonso, M. Muniz, C. Hall et 01.

we observed in calphostin C-treated cells that the ts045 mutant form of VSV-G retained in the ER at nonpermissive temperature did not acquire endoglycosidase H resistance which would have been indicative of Golgi redistribution into the ER (not shown). Calphostin C also inhibited fluid phase endocytosis of HRP (not shown) . Altogether these data indicate that the morphological alterations of Golgi structure caused by calphostin C treatment are accompanied by a general inhibition of transport activities.

EHects of other PKC modulators on Goigi structure Calphostin C binds to the diacylglycerol/phorbol ester site present in the regulatory domain of PKC [4, 15]. To evaluate the involvement of this enzyme in Golgi disassembly we tested other agents known to inhibit PKC by binding to its catalytic domain. Cells incubated with either 1 ~M bisindolylmaleimide 1 (Fig.3A), 3-5 ~M Ro 31-8220 or 1-5 ~M staurosporine showed the typical organization of stacked cisternae characteristic of the Golgi complex in control cells. These are potent inhibitors with affinity for the ATP-binding site [13 , 34, 35]. They were used at high concentrations reported to completely inhibit PKC activity. We therefore concluded that Golgi disassembly induced by calphostin C is not mediated by this kinase activity. However, the process required protein phosphorylation. Cells depleted of ATP or treated with the general protein

Fig. 3. Effects of bisindolylmaleimide 1 incubation and pretreatment with PMA on Golgi organization. (A) Cells were incubated at 37 DC for 1 h with 1 f.lM bisindolylmaleimide I. (8) After 15 min preincubation with 1 f.lM PMA, 0.5 f.lM calphostin C was added and incubation continued for 1 h. Cells were fixed and processed for electron microscopy. Bar, 0.2f.lm.

kinase inhibitor staurosporine prior to incubation with calphostin C did not experience Golgi disassembly (not shown) . We also examined the effects of treating the cells with PKC activators. Cells incubated with PMA did not show significant Golgi alterations (not shown) . However, PMA partly protected Golgi membranes from disassembly. Cells preincubated with PMA prior to exposure to calphostin C showed numerous cisternal profiles, some of which were scattered throughout the perinuclear cytoplasm while others were organized into small Golgi stacks (Fig. 3B). Thus, although PKC phosphorylating activity was not involved in the calphostin C effect these results suggested that a protein with a phorbol ester binding domain could be the molecular target for this agent in the Golgi complex.

Goigi disassembly in vitro We used a cell-free system to analyze the mechanism of action of calphostin C on Golgi structure. The starting material was a Golgi-enriched subcellular fraction prepared from rat liver. These membranes were incubated with bovine brain cytosol and an ATP-generating system in the presence (Fig. 4B) or not (Fig.4A) of activated calphostin C. Golgi disassembly was efficiently reproduced in this assay as judged by the decrease in the length of the cisternal profiles in samples containing activated calphostin C in comparison to those containing nonactivated calphostin C or either no drug at all (Table I) . This was accompanied by an increase in the number of small (50-60 nm) vesicular profiles (Fig. 4B). Whereas 41 ± 4.5 % of total Golgi membranes was present as vesicular profiles in the starting preparation, this value increased to 80 ± 1.8 % after incubation with activated calphostin C. An inverse correlation was found for the percentage of total membrane in cisternae (Fig. 5). Disassembly was dependent on both exogenous cytosol and ATP since many Golgi cisternae remained intact in preparations lacking one of these two components (Fig.4C-D). Furthermore, Golgi breakdown occurred when calphostin C-pretreated Golgi fractions were rinsed and then incubated with cytosol, ATP-generating system and no further addition (not shown). This indicated that once calphostin C becomes intercalated into the Golgi membranes it promotes disassembly by an active process that requires ATP and cytosolic factors. More important, it established that the structural alterations induced by calphostin C were the result of a direct interaction of this agent with the Golgi complex itself rather than the consequence of a block in the ER-to-Golgi transport. Although no ER-to-Golgi transport takes place in this in vitro assay, the basic organization of the Golgi stacks was maintained in the absence of activated calphostin C (Table I, Fig. 5). Interestingly, the concentration of cal ph os tin C necessary to achieve complete Golgi breakdown in vitro was about 5-fold lower than in vivo (Fig. 5). In fact , considerable vesiculation (67 ± 3.08 %) was observed with concentrations as low as 0.01 ~M . The incubation time was also an important factor. Usually, complete disassembly occurred after 30-35 min of incubation at 37°C. During this time period the percentage of Golgi membranes in vesicular profiles almost doubled that in control preparations lacking calphostin C (Fig. 5). At earlier time points, i.e. 10 min, disassembly was negligible irrespective of the concentration of calphostin C used. These observations indicated that binding of calphostin C to Golgi membranes occurred with high affinity. In addition, they showed that the time required for the disassembly process to take

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Goigi disassembly induced by calphostin C

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Fig. 4. In vitro effect of calphostin C on purified Golgi membranes. Th e standard assay consisted of rat live r Golgi membranes incubated at 37 °C for 30 min and under white light with bovine brain cytosol, ATP-generating system, and (J.25f.lM calphostin C (8). The effects of

o mitting either calphostin C (A) , cytosol (C) or ATP (D) are shown. Membranes were fixed, peJleted and processed for electron microscopy. Bar, 0.2 f.lm.

place was independent of the concentration of calphostin C used to induce it.

reverted. As mentioned above in vivo Golgi disassembly induced by calphostin C was shown to be irreversible (Fig. 1). However, this could be an indirect effect derived from PKC inhibition by this agent. Golgi membranes disassembled by the action of calphostin C were rinsed in the presence of either serum or albumin to remove the drug, and then incubated with cytosol and ATP-generating system. Under these conditions they reassembled into cisternae and stacks (Fig. 6,

The calphostin C target is a structural Golgi protein We took advantage of the above in vitro assay to gain insight into the interaction of calphostin C with Golgi membranes . First , we investigated the possibility that the effect could be

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Fig. 6. Reversion of the calphostin C effect. Golgi membranes were subjected to calphostin C-induced disassembly by incubation in the standard in vitro assay (A). Samples were diluted with transport buffer and the membranes collected by centrifugation. They were incubated on ice with transport buffer containing 10 % fetal calf serum for 90 min, pelleted , and then incubated with cytosol and ATP-generating system for 30 min (8). Membranes were finally fixed and processed for electron microscopy. Bar, 0.2 ~m.

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11M Fig. 5. Effects of calphostin C concentration and time of incubation on Golgi disassembly. Golgi membranes were incubated and processed as indicated in Fig. 4. 15-20 photographs were taken for each experi· mental condition in a random fashion and used to determine the relative proportion of both vesicular and cisternal profiles. Data are mean ± SEM.

TableI). Indeed, the percentage of membrane in cisternae showed a 25 % increase during reversal. This provided evidence that binding of calphostin C to the Golgi membranes was not irreversible per se. Additionally, we examined the role played by Golgiassociated proteins in the process. Treatment of Golgi memTab. I.

branes with high salt did not avoid disassembly but it did decrease total fragmentation. Thus , some cisternal profiles remained after incubation of high salt-washed membranes with calphostin C (Fig.7A). In contrast, fragmentation was inhibited when Golgi membranes were pretreated with trypsin . This protease caused the disassembly of the Golgi stacks into single cisternae which were for the most part resistant to calphostin C treatment (Fig. 78). These results supported the interaction of calphostin C with an endogenous Golgi protein. The fact that such a protein could bind not only calphostin C but also PMA (Fig. 3B) indicated that it could have a phorbol ester domain. Proteins with such a domain and different to PKC have been previously described [1, 2]. For instance, nchimaerin is a GTPase-activating protein having the typical cysteine-rich C6H2 motif required for phorbol ester binding [1, 8, 11]. We tested the ability of this protein to competitively inhibit Golgi disassembly induced by calphostin C. Used at 0.5-1!J.M n-chimaerin protected Golgi cisternae from fragmentation induced by calphostin C (Fig. 8). Therefore, these

Change in the cisternal organization of Golgi membranes during in vitro incubation with calphostin C and reversal.

Incubation condition

Number of cisternae/stack

Perimeter of cisternal profiles (J.lm)

No calphostin C Non-activated calphostin C Light-activated calphostin C Reversion from calphostin C treatment

2.30±0.08 2.14±0.09 1.86±0.1 2.10±0.2

2.15±0.07 (n=20)

(n=10) (n=42) (n=50) (n=11)

1.92±0.08 (n=82)* 1.11 ±0.05 (n=70)* 1.86±0.08 (n=25)

Data are expressed as mean ± SEM. n, Number of Golgi stacks or cisternal profiles evaluated. * Value statistically different (p < 0.001) from each other.

Goigi disassembly induced by calphastin C

99

Fig. 7. Effects of high salt wash and trypsin digestion on Golgi disassembly induced by calphostin C. Purified Golgi membranes were incubated on ice for 30 min with either 0.25 M KCI (A) or 0.5 mg/ml trypsin (B). Trypsin digestion was stopped with 10 [.lg/ml soybean tryp-

sin inhibitor and 1 mM phenylmethylsulfonyl fluoride. Membranes were collected by centrifugation and used in the standard in vitro assay. Arrowheads indicate single cisternal profiles remaining after incubation with calphostin C. Bar, 0.2 [.lm.

data strongly supported the interaction of calphostin C with a structural Golgi protein having a phorbol ester-binding domain.

the Golgi mitotic state. However, additional data indicated that PKC was not involved. Inhibitors of the catalytic domain of PKC did not cause a similar Golgi breakdown (Fig.3A). Moreover, protein phosphorylation was required during calphostin C action as shown by pretreatment with the general protein kinase inhibitor staurosporine. In this respect, Golgi disassembly by calphostin C resembles the effect induced by okadaic acid. This phosphatase inhibitor selectively reproduces aspects of Golgi mitotic fragmentation without affecting other organelles [21]. It has been suggested that this is the result of an unbalance between phosphorylation and dephosphorylation of selected proteins which in interphase would remain dephosphorylated by the action of specific phosphatases [21]. It is therefore accepted that like other mitotic events Golgi disassembly is dependent on phosphorylation but the relevant kinase activity and its substrates have not been identified so far. We have been able to analyze in detail the mechanism of action of calphostin C from studies in vitro with purified Golgi fractions. In principle, both light-activated and nonactivated calphostin C can bind to the Golgi membranes where they become intercalated. Binding occurs with high affinity (Fig. S) and is reversible (Fig. 6). Only activated calphostin C, however, is able to promote disassembly (Figs. SA-B). Our data indicate that this would occur by interaction with a structural Golgi protein which in turn would trigger fragmentation. Thus, fragmentation was abolished by pretreatment of the Golgi cisternae with trypsin (Fig. 7B). The disassembly process itself required cytosolic factors (Fig.4C) and ATP (Fig.4D). Our results suggest that the putative calphostin C protein target likely contains a phorbol ester-binding domain similar

Discussion This paper describes the disassembly of the Golgi complex caused by calphostin C, a selective and potent inhibitor of PKC [4, 1S]. The ultrastructural and functional alterations achieved by this agent on the Golgi complex resemble the modifications that it undergoes during mitosis [19, 20]. Thus, under both situations Golgi remnants are originated consisting of clusters of uncoated vesicles and short tubules distributed throughout the cell cytoplasm. Clusters induced by calphostin C, like mitotic Golgi remnants [24], contained two kinds of vesicles: small vesicles of SO-60 nm diameter and large electron-lucent vesicles of 100-1S0nm diameter. Also, protein traffic along both the exocytic and endocytic routes is arrested in both cases [37]. However, unlike the mitotic cells, calphostin C-treated cells did not exhibit vesiculation of the nuclear envelope or the ER. This makes calphostin C a useful reagent to characterize specific factors involved in Golgi disassembly during the cell cycle. It should be noted, however, that prolonged treatments of intact cells with calphostin C are precluded by its irreversible effects on PKC. Thus, even with low (0.OS-D.1 f-lM) doses of this drug after 3-S h incubation cell viability is impaired. Cdc 2 kinase is responsible for the entry of cells into mitosis and reconstitution of Golgi fragmentation in vitro requires phosphorylation [24]. It was therefore surprising that treatment of intact interphase cells with calphostin C could mimic

100 M. Alonso, M. Muniz, C. Hall et al.

Fig. 8. Inhibition of calphostin C-induced Golgi disassembly by nchimaerin. The standard in vitro assay was modified to include (C) or

not (A-B) 0.5 flM recombinant n-chimaerin and incubation was performed either in the dark (A) or under white light (B-C). Bar, 0.2 flm.

to the cysteine-rich C6H2 motif present in PKC, n-chimaerin, and unc-13 proteins. Both PMA and calphostin C could competitively bind to this target although with opposite effects. Binding of PMA stabilized Golgi structure (Fig. 3B) while calphostin C binding promoted disassembly (Fig. 4B). The fact that n-chimaerin abrogated calphostin C action (Fig. 8C) is consistent with this proposal. N-chimaerin like unc-13 has no kinase activity but contains a C6H2 motif to which both phorbol esters and diacylglycerol can bind [1,2,22]. Calphostin C bound with higher affinity to n-chimaerin than it did to its target in the Golgi and apart from this there is no evidence that both proteins can be somehow related. However, the existence of a common phorbol ester-binding domain could potentially be used in the characterization of the calphostin C molecular target in the Golgi and determination of its functional role during both interphase and mitosis.

[3] Balch, W E., M. M. Elliott, D. S. Keller: ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi. J. BioI. Chern. 261, 14681-14689 (1986). [4] Bruns, R. E, ED.Miller, R.L.Merriman, J.J.Howbert, WE Heath, E. Kobayashi, 1. Takahashi, T. Tamaoki, H. Nakano: Inhibition of protein kinase C by calphostin C is light-dependent. Biochem. Biophys. Res. Commun. 176,288-293 (1991). [5] Buccione, R., S. Bannykh, 1. Santone, M. Baldassarre, E Facchiano, Y. Bozzi, G. D. Tullio, A. Mironov, A. Luini, M. A. De Matteis: Regulation of constitutive exocytic transport by membrane receptors. J. BioI. Chern. 271, 3523-3533 (1996). [6] Davidson, H. W, C. H. McGowan, W E. Balch: Evidence for the regulation of exocytic transport by protein phosphorylation. J. Cell BioI. 116, 1343-1355 (1992). [7] De Matteis, M. A., G. Santini, R. A. Kahn, G. D. Tullio, A. Luini: Receptor and protein kinase C-mediated regulation of ARF binding to the Golgi complex. Nature 364, 818-820 (1993). [8] Diekmann, D., S. Brill, M. D. Garrett, N. Totty, J. Hsuan, C. Monfries, C. Hall, L. Lim, A. Hall: Bcr encodes a GTPaseactivating protein for p2lrac. Nature 351, 400-403 (1991). [9] Fabbri, M., S. Bannykh, W E. Balch: Export of protein from the endoplasmic reticulum is regulated by a diacylglycerol/phorbol ester-binding protein. J. BioI. Chern. 269, 26848-26857 (1994). [10] Farquhar, M. G.: Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell BioI. 1,447-488 (1985). [11] Hall, c., C. Monfries, P. Smith, H. H. Lim, R. Kozma, S. Ahmed, V. Vanniasingham, L. Leung, L. Lim: Novel human brain cDNA, encoding a 34000Mr protein n-chimaerin, related to both the regulatory domain of protein kinase C and BCR, the product of the breakpoint cluster region gene. J. Mol. BioI. 211, 1-16 (1990). [12] Hidalgo, J., M. Muniz, A. Velasco: Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum. J. Cell Sci. 108, 1805-1815 (1995). [13] Keller, H. u., V. Niggli: The PKC-inhibitor Ro 31-8220 selectively suppresses PMA- and diacylglycerol-induced fluid phase pinocytosis and actin polymerization in PMNS. Biochem. Biophys. Res. Commun. 194, 1111-1116 (1993).

Acknowledgements. This work was supported by a Spanish DGICYT grant PB92-0674 (to A. Velasco) and FISs grant 93/0824 (to 1. Hidalgo). M. Alonso and M. Muniz contributed equally. M. Alonso was supported by a predoctoral fellowship from Agencia Espanola de Cooperaci6n Internacional and M. Muniz by a predoctoral fellowship from UNICAJAlJunta de Andalucia.

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