Article |
Address correspondence to Hisao Kondo, Cambridge Institute for Medical Research (Rm. 5.36), University of Cambridge, Wellcome Trust/MRC Bldg., Hills Rd., Cambridge CB2 2XY, UK. Tel.: 44-1223-762632. Fax: 44-1223-762640. E-mail: hk228{at}cam.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Golgi; p47; p97; phosphorylation; mitosis
* Abbreviations used in this paper: GalT, ß1,4-galactosyltransferase; Ser, serine; Thr, threonine.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This disassemblyreassembly process is regulated by phosphorylation (Shorter and Warren, 2002), but there are continuing debates as to which kinases are responsible. Suggested kinases include Cdc2 kinase (Lowe et al., 1998; Kano et al., 2000), MAPK kinase (Acharya et al., 1998; Kano et al., 2000), and Polo-like kinase 1 (Sutterlin et al., 2001). Since the Golgi apparatus has a complicated structure, its disassembly is thought to occur in several steps. These kinases might be involved in these disassembly steps. Golgi disassemblyreassembly requires the blocking of membrane fusion at early mitosis and its unblocking at late mitosis (Warren, 1993). Experiments using an in vitro assay showed that Golgi reassembly requires at least two distinct membrane fusion pathways: the NSF pathway and the p97/p47 pathway (Rabouille et al., 1995c). NSF binds to SNAREs via -SNAP and activates it for membrane fusion (Sollner et al., 1993). NSF also needs the assistance of p115-GM130 tethering (Nakamura et al., 1997). At early mitosis, GM130 is phosphorylated on Serine (Ser)*-25 by Cdc2 kinase. This phosphorylation disrupts the tethering of p115-GM130, resulting in the mitotic inhibition of the NSF pathway (Lowe et al., 1998). Similar to NSF, p97 binds to SNAREs via the cofactor p47 (Kondo et al., 1997) and dissociates the SNARE complex with the assistance of the valosin-containing protein (p97)/p47 complexinteracting protein (VCIP135) during membrane fusion (Uchiyama et al., 2002). It has also been suggested that the interaction between p47 and monoubiquitin might be important (Meyer et al., 2002). In contrast to NSF, p97 does not require p115-GM130 tethering (Rabouille et al., 1995c). This leads to the question: how is the p97 pathway regulated mitotically during the Golgi disassemblyassembly?
In this paper, we first observed the main localization of p47 in the nucleus, which suggests that the major population of p47 does not function in the maintenance of the Golgi apparatus during interphase. Next, we found that mitotic phosphorylation of p47 inhibits its binding to Golgi membranes. This phosphorylation was shown to be required for Golgi disassembly in vitro. Finally, we succeeded in maintaining Golgi stacks in mitotic mammalian cells by the microinjection of p47(S140A), which is unable to be phosphorylated. Using this model cell, we tested whether the fragmentation-dispersion of Golgi during mitosis is essential for its equal partitioning.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Although the major population of p47 was localized to the nuclear region, a small amount of p47 was still observed in the cytoplasm (Fig. 1 A, right). The next question was whether the tiny amount of p47 in cytoplasm functions in Golgi membrane fusion in interphase cells. To address this, we microinjected anti-p47 antibodies into the cytoplasm of interphase cells and investigated Golgi morphology. When prophase cells were injected with the same antibody (8 g/l) and incubated for 1.5 h, Golgi reassembly at the end of mitosis was completely inhibited. Only clusters of vesicles and tubules, not stacks, were observed in daughter cells (Uchiyama et al., 2002). Since the amount of cytoplasmic p47 in interphase cells is much less than that in mitotic cells, this concentration (8 g/l) of the antibody must be sufficient to block the function of cytoplasmic p47. Fig. 1 C shows Golgi staining with anti-GM130 antibodies in the injected cells. Here, the Golgi apparatus was still located in perinuclear regions without dispersion (Fig. 1 C, second and fourth panels). Next, we investigated Golgi ultrastructure by EM. Interphase cells were injected with this antibody at 8 or 16 g/l and incubated for 2 h. As shown in Fig. 1 D (the injected antibody, 16 g/l), the Golgi apparatus still showed normal stacking structures without fragmentation (right). Thus, we could not find any obvious evidence of a Golgi maintenance function mediated by p97/p47. We concluded that the fusion activity mediated by p97/p47 is less critical during interphase than at the end of mitosis.
Once the cell enters mitosis, the nuclear envelope is broken down and p47 comes out from the nucleus, which enables p47 to function in Golgi membrane fusion. However, Golgi disassembly at early mitosis requires the inhibition of membrane fusion. This leads to the question: how is the p97/p47 pathway blocked during early mitosis?
p47 is mitotically phosphorylated
To investigate whether there was mitotic phosphorylation of p47, NRK cells were synchronized and incubated in medium containing [32P]orthophosphate. Mitotic cells were collected by mechanical shake-off and used for the preparation of 32P-labeled mitotic cytosol. Hoechst DNA staining showed that >95% of cells were mitotic (unpublished data). Nonsynchronized cells were used for the preparation of interphase cytosol. p47 was isolated from the cytosol by denatured immunoprecipitation (Fig. 2 A). Western blotting showed that the band of mitotic p47 was shifted upwards (Fig. 2 A, top), and autoradiography revealed that p47 was strongly phosphorylated at mitosis (Fig. 2 A, bottom).
|
The consensus motif for Cdc2 is Ser/Thr-Pro-XArg/Lys, with Pro at the +1 position being absolutely critical, and a basic residue at the +3 position preferred but not essential for kinase recognition (Holmes and Solomon, 1996). p47 has one Thr and three Ser residues with Pro at the +1 position: T57, S114, S140, and S272. His-p47 was mutated at each phosphorylation site and tested for phosphorylation. As presented in Fig. 2 E, all p47 mutants except p47(S140A) were phosphorylated by mitotic cytosol. Similar results were obtained by using purified Cdc2 instead of mitotic cytosol (unpublished data). Together, we concluded that p47 was mitotically phosphorylated on Ser-140 by Cdc2.
p47 forms a complex with p97 at mitosis
After determining the phosphorylation of p47 at mitosis, this led to the question of what effect the phosphorylation would have on the function of p47. p47 forms a tight complex with p97, which is essential for in vitro Golgi reassembly (Kondo et al., 1997). We investigated the effect of phosphorylation on the binding of p47 to p97. To test whether p97 binds phosphorylated p47, p97 and p47 were incubated in the presence of Cdc2 and [-32P]ATP (Fig. 3 A). p47 bound to p97 was coimmunoprecipitated with antibodies to the His tag on p97. Phosphorylated p47 was detected by autoradiography. Phosphorylated p47 was coimmunoprecipitated together with p97 (Fig. 3 A, lane 2), suggesting that phosphorylated p47 bound to p97. p97 is also reported to be phosphorylated at mitosis (Madeo et al., 1998), and therefore, we tested whether phosphorylated p97 bound to p47 (Fig. 3 B). Synchronized NRK cells were labeled with [32P]orthophosphate and used for the preparation of mitotic cytosol. p47 and its binding proteins were coimmunoprecipitated from the 32P-labeled mitotic cytosol using polyclonal anti-p47 antibodies and protein A beads. p47-binding proteins were eluted from the beads by 1 M KCl. p97 was isolated from the eluate by denatured immunoprecipitation using polyclonal anti-p97 antibodies, and phosphorylated p97 was detected by antoradiography. As shown in Fig. 3 B, lane 2, phosphorylated p97 bound to p47. We next compared p47 distributions in interphase and mitotic cell extracts. Extracts were size fractionated by gel filtration, and the localization of p97 and p47 were studied (Fig. 3 C). In both the interphase and mitotic extracts, p97 eluted at
670 kD, and p47 eluted at
670 and
200 kD. (Note that the bands of p47 were double in the mitotic extract.) Since p97-associated p47 is eluted at
670 kD (Kondo et al., 1997), these results indicate that p47 forms a complex with p97 in the mitotic extract as well as in the interphase extract. Thus, all the data from the binding experiments and gel filtration show that p47 phosphorylation has no effect on complex formation with p97.
|
|
We first investigated whether phosphorylated p47 bound to Golgi membranes. p47 was phosphorylated by the incubation with Cdc2 and then its phosphorylation condition was frozen by the addition of staurosporine and microcystin-LR. The phosphorylated p47 was used for binding experiments with salt-washed Golgi membranes. As presented in Fig. 4 C, Cdc2-mediated p47 phosphorylation inhibited its binding to the membranes (Fig. 4 C, middle, lane 3). This inhibition was rescued by olomoucine, an inhibitor of Cdc2 (Fig. 4 C, middle, lane 4). These inhibition and rescue were also observed when a p97/p47 complex was added instead of p47 (unpublished data). Next, the mutated p47, p47(S140A), which cannot be phosphorylated in Ser-140, was used for the binding experiments (Fig. 4 D). p47(S140A) bound to Golgi membranes (Fig. 4 D, lane2), and Cdc2 had no effect on this binding (Fig. 4 D, lane 3). We finally tested another p47 mutant, p47(S140D), which mimics the phosphorylation of Ser-140, for the binding experiments (Fig. 4 E). Much less p47(S140D) bound to Golgi membranes (Fig. 4 E, top, lanes 4 and 5) when compared with p47wt (Fig. 4 E, top, lanes 2 and 3) and p47(S140A) (Fig. 4 E, top, lanes 6 and 7). In summary, our biochemical data showed that phosphorylation of Ser-140 in p47 inhibited its binding to Golgi membranes.
We also further investigated the localization of p47 in mitotic cells (Fig. 5). As shown in Fig. 1 A, when NRK cells were directly fixed using PFA, anti-p47 antibodies displayed very strong nuclear staining and weak cytoplasmic staining. However, when such cells were washed with 0.01% saponin-containing buffer to remove p47 that was not membrane-bound before fixation, perinuclear staining was prominent in addition to nuclear staining (Fig. 5 A, arrows). In interphase cells as shown in Fig. 5 A (the star-marked cells), the staining of p47 showed clear colocalization with the staining of GM130, a Golgi marker, at perinuclear regions. In early prometaphase cells (the top left cell in Fig. 5 A and its magnified images shown in Fig. 5 B), p47 staining did not colocalize with GM130 staining. These results strongly support the biochemical data that mitotically phosphorylated p47 does not bind to Golgi membranes. Fig. 5 C presents the localization of p47 in the cell at early telophase. Once the cell entered telophase, p47 staining again showed colocalization with GM130 (Fig. 5 C, arrowheads). It has been reported that GM130 is phosphorylated by Cdc2 at prophase and dephosphorylated at early telophase (Lowe et al., 2000). Similarly, it is likely that p47 was dephosphorylated at early telophase and rebound to Golgi membranes.
|
If p47 is not phosphorylated at early mitosis, are Golgi membranes fragmented? We used mutated p47, p47(S140A), which cannot be phosphorylated on Ser-140. The early biochemical binding experiment had shown that p47(S140A) could bind to Golgi membranes in the presence of Cdc2 (Fig. 4 D). Purified Golgi membranes and mitotic cytosol were incubated with p97/p47wt or p97/p47(S140A) for the in vitro Golgi disassembly assay. Cisternal fragmentation was defined by the loss of cisternal membranes, with fragmentation induced by mitotic cytosol set to 100%. As shown in Fig. 6 A, the addition of p97/p47(S140A) inhibited cisternal fragmentation almost completely, whereas the addition of p97/p47wt had no effect. This indicates that the phosphorylation of Ser-140 in p47 is required for Golgi disassembly.
|
Phosphorylation of Ser-140 in p47 is required for Golgi fragmentation-dispersion during mitosis in living cells
The in vitro Golgi disassembly assay showed that the addition of p97/p47(S140A) to mitotic cytosol inhibited Golgi fragmentation (Fig. 6 A). We next investigated the effect of p47(S140A) in living cells. Either p97/p47wt or p97/p47(S140A) was microinjected into prophase (or early prometaphase) cells and fixed at several intervals. Fig. 7 A shows Golgi staining with anti-GM130 antibodies after injection. In the cells injected with p97/p47wt, the Golgi membranes dispersed all over the cell at prometaphase and metaphase, although there remained some small blobs, and they were reorganized to form Golgi clusters at telophase (Fig. 7 A, top). The cells injected with p97/p47(S140A) showed entirely different Golgi staining: the dispersion of Golgi membranes was mostly suppressed, and Golgi clusters showing bright staining were observed through all mitotic phases (Fig. 7 A, bottom). The Golgi clusters induced by p97/p47(S140A) were doubly stained with antibodies to GM130 and ß1,4-galactosyltransferase (GalT), markers of cis- and trans-Golgi, respectively (Rabouille et al., 1995a). As shown in Fig. 7 B, some parts of the structures were stained by only one antibody, although others were stained by both. This suggests that these Golgi clusters were polar structures.
|
The double staining images presented in Fig. 7 B suggest that the Golgi clusters induced by p97/p47(S140A) may be organized polar structures. Therefore, we investigated the ultrastructure of injected cells by EM. Fig. 8 A shows representative images of the injected cells. In the cells injected with p97/p47wt, Golgi stacks were almost lost and fragmented at late prometaphase, similar to uninjected cells (Fig. 8, left). In contrast, the cells injected with p97/p47(S140A) had highly organized Golgi stacks at the same mitotic phase: 80.1% of cisternal membranes were found in stacks (Fig. 8, right). The connections between stacks were lost, and the Golgi ribbon was broken into discrete stacks. Note that the nuclear envelope had been lost and chromatin was well condensed in this cell (Fig. 8, right, inset). Fig. 8 B presents the quantitative results. The injection of p97/p47(S140A) significantly increased the percentage of Golgi membranes in cisternal structures threefold and significantly decreased the percentage of Golgi membranes in vesicles by a third compared with the injection of p97/p47wt. The amount of tubules/tubular networks did not change. Neither Golgi stacks nor cisternae are generally observed in mitotic cells, whereas the injection of p97/p47wt also allowed a few cisternae to remain at early mitosis (Fig. 8 B). Some of the injected p47wt might not be phosphorylated, which enabled occasional cisternae to remain.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphorylation of Ser-140 in p47 inhibited its binding to Golgi membranes (Fig. 4). The Ser-140 is in a highly unstructured region (Yuan. X., and S. Matthews, personal communications) which could become structured upon binding to SNAREs. The phosphorylation could alter the nature of the unstructured loop region, affecting the conformational changes. Also, the additional negative charges on phosphorylated Ser-140 could alter the local conformation of the structure, masking the interaction site with SNAREs. Our data show that p47 binds to Golgi membranes at interphase, comes off at prometaphase, and rebinds to the membranes at telophase (Fig. 5). These results indicate that p47 is phosphorylated at early mitosis and dephosphorylated at late mitosis, although we have no data on the responsible phosphatase at present. This is consistent with the results of the in vitro function assays which showed that Golgi disassembly requires the phosphorylation of p47 and that Golgi reassembly requires the dephosphorylation of p47 (Fig. 6). Therefore, we propose that the phosphorylation-dephosphorylation cycle of p47 at mitosis as well as its nuclear localization at interphase play key roles in Golgi disassemblyreassembly during the cell cycle. Fig. 9 shows a schematic model. In an interphase cell, p47 is mainly in the nucleus. Once the cell enters mitosis, its nuclear envelope is broken down, and p47 enters the cytoplasm. At the same time, p47 is phosphorylated by mitotically activated Cdc2 kinase. Since the phosphorylated p47 cannot bind to Golgi membranes, p97/p47-mediated membrane fusion is inhibited, resulting in the fragmentation of Golgi membranes. At late mitosis, p47 is dephosphorylated and binds to Golgi membranes. This allows p97/p47-mediated membrane fusion to reassemble the Golgi apparatus. When nuclear envelopes are formed in daughter cells, p47 moves to the nucleus, and the membrane fusion mediated by p97/p47 is suppressed. Hetzer et al. (2001) has reported that p97/p47 is also required for nuclear envelope formation. This implies that, when the nuclear envelope as well as the Golgi apparatus are reassembled and p97/p47-mediated fusion is not required anymore, membrane fusion will be suppressed by the nuclear envelope that p97/p47-mediated fusion assembled. This feedback control system seems to be very tight. Moreover, Ser-140 in p47 is conserved in Drosophila, Xenopus, mouse, and human but not in yeast. In yeast, the nuclear envelope is not broken down at mitosis, and hence, the phosphorylation-dephosphorylation of the yeast p47 homologue shp1p may not be necessary.
|
Normal prometaphase NRK cells had neither Golgi stacks nor cisternae, and vesiculated Golgi membranes were dispersed all over the cytoplasm. In contrast, the p97/p47(S140A)-injected prometaphase cell had Golgi cisternae and stacks (Fig. 8) and seemed to have very little absorption of Golgi vesicles into ER (Fig. 7 C). The injection of p97/p47(S140A) had no effect on cell cycle progression (e.g., chromatin segregation and cytokinesis were pursued normally; see Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200303048/DC1). Hence, we used these p97/p47(S140A)-injected cells as model cells and tested whether Golgi fragmentation-dispersion is required for equal partitioning in mammalian cells. As a result, we found that the p97/p47(S140A)-injected cells achieved equal Golgi partitioning. Therefore, we propose that Golgi fragmentation-dispersion is not obligatory for its equal partitioning even in mammalian cells. Generally, organelles adopt one of two partitioning strategies: stochastic or ordered. Our result strongly suggests that the partitioning of the Golgi is controlled in a highly ordered way. A separate stack can be a minimum unit for partitioning even in mammalian cells. This is similar to the cases of algae and protozoa (Munro, 2002). Stochastic and ordered ways of partitioning need not be mutually exclusive and our results do not belittle the significance of mitotic Golgi fragmentation-dispersion. This might assist the ordered mechanism to increase the accuracy of partitioning. Sutterlin et al. (2002) also reported that Golgi fragmentation-dispersion is required for entry into mitosis, showing that this process is essential for mammalian cells. Taking our results into account, the fragmentation-dispersion of Golgi may have an important, presently unknown, significance besides partitioning.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the expression of HA-tagged p47 in cultured cells, p47 cDNA was subcloned to pCG-N-BL vector (a gift from Dr. Taketani, Kyoto Institute of Technology, Kyoto, Japan). Point mutations were directly introduced into the p47 cDNA in pQE30 or pCG-N-BL by PCR reactions, using the Quick-change mutagenesis kit (Stratagene). All clones were verified by DNA sequencing.
Polyclonal antibodies to p47 and syntaxin5 were prepared as described (Hui et al., 1997; Kondo et al., 1997). A rabbit anti-p97 polyclonal antibody was raised using His-p97(1198) as an antigen. Monoclonal anti-GalT antibodies and polyclonal anti-GM130 antibodies were gifts from Dr. Suganuma (Miyazaki Medical College, Miyazaki, Japan) and Dr. Nakamura (Kanazawa University, Kanazawa, Japan), respectively. mAb to p97, His-tag, and GM130 were purchased from Progen, QIAGEN, and BD Transduction, respectively.
The following reagents were purchased from Calbiochem; staurosporine, olomoucine, PD98059, SB203580, KT5720, calphostin C, microcystin-LR. Cdc2 kinase (p34cdc2/cyclin B) was from New England Biolabs, Inc.
In vivo metabolic 32P labeling
For enrichment of mitotic NRK cells, aphidicolin (2.5 µg/ml) was added to the medium for 14 h. The cells were washed with fresh medium, released from the S phase block for 2 h, and labeled with [32P]orthophosphate (200 µCi/ml) for another 4 h at 37°C. Mitotic cells were flushed from the dish, washed with PBS, and extracted with buffer (20 mM Hepes, 0.1 M KCl, 5 mM EDTA, 2 mM EGTA, 40 mM ß-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 1 mM DTT, protease inhibitor cocktail, 0.5% Triton X-100, 10% glycerol, pH 7.4). The lysate was cleared by centrifugation and used for the native or denatured immunoprecipitation with anti-p47 antibodies.
In vitro phosphorylation
p47 or its mutant was incubated in buffer A (50 mM Tris, 50 mM KOAc, 10 mM MgOAc, 20 mM ß-glycerophosphate, 0.2 mM DTT, 40 µM ATP, 30 µCi/µl [-32P]ATP, pH 7.4) with mitotic cytosol or purified kinase for 30 min at 30°C followed by denatured immunoprecipitation. The reactions were terminated by adding an equal volume of the buffer (100 mM Tris, 2 mM EDTA, 2% SDS, pH 7.4) and boiled for 4 min. After adding 20 vol of buffer (50 mM Tris, 0.15 M KCl, 0.5% Tween-20, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 40 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT, pH 7.4), p47 or its mutant was immunoprecipitated with antibodies to a His tag and protein G beads followed by SDS-PAGE and autoradiography.
For the binding experiments of p47 to salt-washed Golgi membranes, 1 mM ATP was added to buffer A instead of 40 µM ATP and 30 µCi/µl [-32P]ATP. After incubation, the reactions were supplemented with microcystin-LR (10 µM) and staurosporine (25 µM) followed by the binding experiments using Golgi membranes.
Depletion of Cdc2 kinase was performed using p13-suc1 beads (Upstate Biotechnology) as described (Hirota et al., 2000).
Binding experiments and gel filtration
Golgi membranes were purified from rat liver (Hui et al., 1998). 1 M KCl-washed Golgi membranes and mitotic Golgi fragments were prepared as described previously (Uchiyama et al., 2002).
p47 or its mutant was incubated with salt-washed Golgi membranes in buffer (0.1 M KCl, 20 mM Tris, 1 mM MgCl2, 1 mM ATP, 0.4 g/l BSA, 0.2 M sucrose, pH 7.4) for 1 h on ice, and then the membranes were recovered by centrifugation.
Interphase and mitotic cytosol were prepared from HeLa cells (Rabouille et al., 1995b). The homogenizing buffer was supplemented with microcystin-LR (10 µM) and staurosporine (25 µM) in order to maintain their phosphorylation conditions. Cytosolic proteins were fractionated by a Superose 6 in the presence of microcystin-LR (2 µM) and staurosporine (5 µM). Molecular weight markers were thyroglobulin (1,340 and 670 k), apoferritin (440 k), and ß-amylase (200 k).
In vitro Golgi disassembly and reassembly assays
The in vitro Golgi disassembly and reassembly assays were performed as reported previously (Lowe et al., 1998; Shorter and Warren, 1999).
Immunofluorescence microscopy
NRK cells grown on coverslips were fixed with 3% PFA/PBS for 20 min at room temperature and stained with antibodies after permeabilization, unless stated otherwise. 0.1% Triton X-100 and 0.1% saponin were used for the permeabilization of interphase and mitotic cells, respectively.
For the staining of mitotic cells with antibodies to p47 and GM130, cells were incubated in buffer (80 mM PIPES, 1 mM MgCl2, 5 mM EGTA, 0.01% saponin, pH 7) for 7 min at room temperature before the fixation with 3% PFA (Zerial et al., 1992). Saponin (0.01%) was used instead of Triton X-100 for the following permeabilization and labeling with antibodies.
The total cellular fluorescence of GM130-containing structures was calculated using a series of sections (1 µm in thickness) throughout a whole cell: 911 sections for an interphase cell and 1719 sections for a mitotic cell. For the calculation, well-isolated cells were chosen. Background was subtracted and a threshold introduced to exclude diffuse cytoplasmic staining. All pixels above this threshold value were counted as GM130-containing structures in all sections and summed up. The resulting value was normalized to a uninjected interphase cells on the same coverslip.
Microinjection into living cells
The microinjection was performed as described previously (Uchiyama et al., 2002). Fluorescence of coinjected Cy3-BSA (1 µg/µl) was used as an injection marker to identify injected cells. For the injection into mitotic cells, the synchronized cells were used as described above. Prophase (or early prometaphase) cells were microinjected with p97/p47 samples 4 h after the release from aphidicolin block: a phasecontrast microscope was used for the identification of their phases. The injected cells were incubated for another 0.51.5 h and fixed.
For EM study, cells were grown on a coverslip on which a square area of 1 mm x
1 mm was outlined by a diamond pen (Uchiyama et al., 2002). The cells within the area were injected. In case of the injection into mitotic cells, all injections were done within 510 min. Uninjected cells within the area were completely removed by an injection needle using fluorescence of coinjected Cy3-BSA as a marker. After fixation and embedding into Epon, the cells within the area were ultrathin sectioned. Membrane profiles in the Golgi area were divided into three categories: cisternae, tubules, and vesicles. The relative proportion of each category of membranes was counted using an intersection method.
Online supplemental material
Table S1, showing the cell cycle progression after microinjection, is available at http://www.jcb.org/cgi/content/full/jcb.200303048/DC1.
![]() |
Acknowledgments |
---|
E. Jokitalo is supported by Biocentrum Helsinki. This work was supported by a Wellcome Trust grant to H. Kondo.
Submitted: 7 March 2003
Revised: 9 May 2003
Accepted: 9 May 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acharya, U., A. Mallabiabarrena, J.K. Acharya, and V. Malhotra. 1998. Signaling via mitogen-activated protein kinase kinase (MEK1) is required for Golgi fragmentation during mitosis. Cell. 92:183192.[Medline]
Bevis, B.J., A.T. Hammond, C.A. Reinke, and B.S. Glick. 2002. De novo formation of transitional ER sites and Golgi structures in Pichia pastoris. Nat. Cell Biol. 4:750756.[CrossRef][Medline]
Hetzer, M., H.H. Meyer, T.C. Walther, D. Bilbao-Cortes, G. Warren, and I.W. Mattaj. 2001. Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly. Nat. Cell Biol. 3:10861091.[CrossRef][Medline]
Hicks, G.R., and N.V. Raikhel. 1995. Protein import into the nucleus: an integrated view. Annu. Rev. Cell Dev. Biol. 11:155188.[CrossRef][Medline]
Hirota, T., T. Morisaki, Y. Nishiyama, T. Marumoto, K. Tada, T. Hara, N. Masuko, M. Inagaki, K. Hatakeyama, and H. Saya. 2000. Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor. J. Cell Biol. 149:10731086.
Holmes, J.K., and M.J. Solomon. 1996. A predictive scale for evaluating cyclin-dependent kinase substrates. A comparison of p34cdc2 and p33cdk2. J. Biol. Chem. 271:2524025246.
Hui, N., N. Nakamura, B. Sonnichsen, D.T. Shima, T. Nilsson, and G. Warren. 1997. An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal. Mol. Biol. Cell. 8:17771787.[Abstract]
Hui, N., N. Nakamura, P. Slusarewicz, and G. Warren. 1998. Purification of rat liver golgi stacks. Cell Biology: A Laboratory Handbook. Vol. 2. J. Ceils, editor. Academic Press, Orlando, FL. 4655.
Jokitalo, E., N. Cabrera-Poch, G. Warren, and D.T. Shima. 2001. Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum. J. Cell Biol. 154:317330.
Kano, F., K. Takenaka, A. Yamamoto, K. Nagayama, E. Nishida, and M. Murata. 2000. MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopus extracts. J. Cell Biol. 149:357368.
Kondo, H., C. Rabouille, R. Newman, T.P. Levine, D. Pappin, P. Freemont, and G. Warren. 1997. p47 is a cofactor for p97-mediated membrane fusion. Nature. 388:7578.[CrossRef][Medline]
Lowe, M., C. Rabouille, N. Nakamura, R. Watson, M. Jackman, E. Jamsa, D. Rahman, D.J. Pappin, and G. Warren. 1998. Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis. Cell. 94:783793.[Medline]
Lowe, M., N.K. Gonatas, and G. Warren. 2000. The mitotic phosphorylation cycle of the cis-golgi matrix protein GM130. J. Cell Biol. 149:341356.
Lucocq, J.M., E.G. Berger, and G. Warren. 1989. Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway. J. Cell Biol. 109:463474.[Abstract]
Madeo, F., J. Schlauer, H. Zischka, D. Mecke, and K.U. Frohlich. 1998. Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p. Mol. Biol. Cell. 9:131141.
Mayr, P.S., V.J. Allan, and P.G. Woodman. 1999. Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase. Eur. J. Cell Biol. 78:224232.[Medline]
Mellman, I., and K. Simons. 1992. The Golgi complex: in vitro veritas? Cell. 68:829840.[Medline]
Meyer, H.H., J.G. Shorter, J. Seemann, D. Pappin, and G. Warren. 2000. A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19:21812192.
Meyer, H.H., Y. Wang, and G. Warren. 2002. Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4. EMBO J. 21:56455652.
Muller, J.M., H.H. Meyer, C. Ruhrberg, G.W. Stamp, G. Warren, and D.T. Shima. 1999. The mouse p97 (CDC48) gene. Genomic structure, definition of transcriptional regulatory sequences, gene expression, and characterization of a pseudogene. J. Biol. Chem. 274:1015410162.
Muller, J.M., J. Shorter, R. Newman, K. Deinhardt, Y. Sagiv, Z. Elazar, G. Warren, and D.T. Shima. 2002. Sequential SNARE disassembly and GATE-16GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion. J. Cell Biol. 157:11611173.
Munro, S. 2002. More than one way to replicate the Golgi apparatus. Nat. Cell Biol. 4:E223E224.[CrossRef][Medline]
Nakamura, N., M. Lowe, T.P. Levine, C. Rabouille, and G. Warren. 1997. The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Cell. 89:445455.[Medline]
Pelletier, L., C.A. Stern, M. Pypaert, D. Sheff, H.M. Ngo, N. Roper, C.Y. He, K. Hu, D. Toomre, I. Coppens, et al. 2002. Golgi biogenesis in Toxoplasma gondii. Nature. 418:548552.[CrossRef][Medline]
Peters, J.M., M.J. Walsh, and W.W. Franke. 1990. An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF. EMBO J. 9:17571767.[Abstract]
Rabouille, C., N. Hui, F. Hunte, R. Kieckbusch, E.G. Berger, G. Warren, and T. Nilsson. 1995a. Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides. J. Cell Sci. 108:16171627.
Rabouille, C., T. Misteli, R. Watson, and G. Warren. 1995b. Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system. J. Cell Biol. 129:605618.[Abstract]
Rabouille, C., T.P. Levine, J.M. Peters, and G. Warren. 1995c. An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell. 82:905914.[Medline]
Rabouille, C., H. Kondo, R. Newman, N. Hui, P. Freemont, and G. Warren. 1998. Syntaxin 5 is a common component of the NSF- and p97-mediated reassembly pathways of Golgi cisternae from mitotic Golgi fragments in vitro. Cell. 92:603610.[Medline]
Seemann, J., E.J. Jokitalo, and G. Warren. 2000. The role of the tethering proteins p115 and GM130 in transport through the Golgi apparatus in vivo. Mol. Biol. Cell. 11:635645.
Seemann, J., M. Pypaert, T. Taguchi, J. Malsam, and G. Warren. 2002. Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells. Science. 295:848851.
Shorter, J., and G. Warren. 1999. A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system. J. Cell Biol. 146:5770.
Shorter, J., and G. Warren. 2002. Golgi architecture and inheritance. Annu. Rev. Cell Dev. Biol. 18:379420.[CrossRef][Medline]
Sollner, T., S.W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. Geromanos, P. Tempst, and J.E. Rothman. 1993. SNAP receptors implicated in vesicle targeting and fusion. Nature. 362:318324.[CrossRef][Medline]
Sutterlin, C., C.Y. Lin, Y. Feng, D.K. Ferris, R.L. Erikson, and V. Malhotra. 2001. Polo-like kinase is required for the fragmentation of pericentriolar Golgi stacks during mitosis. Proc. Natl. Acad. Sci. USA. 98:91289132.
Sutterlin, C., P. Hsu, A. Mallabiabarrena, and V. Malhotra. 2002. Fragmentation and dispersal of the pericentriolar Golgi complex is required for entry into mitosis in mammalian cells. Cell. 109:359369.[Medline]
Uchiyama, K., E. Jokitalo, F. Kano, M. Murata, X. Zhang, B. Canas, R. Newman, C. Rabouille, D. Pappin, P. Freemont, and H. Kondo. 2002. VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo. J. Cell Biol. 159:855866.
Warren, G. 1993. Membrane partitioning during cell division. Annu. Rev. Biochem. 62:323348.[CrossRef][Medline]
Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Golgi membranes are absorbed into and reemerge from the ER during mitosis. Cell. 99:589601.[Medline]
Zerial, M., R. Parton, P. Chavrier, and F. Rainer. 1992. Localization of Rab family members in animal cells. Methods Enzymol. 219:398408.[Medline]
Related Article