Correspondence to: James Shorter, Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK., shorter{at}icrf.icnet.uk (E-mail), 0171-269-3403 (phone), 0171-269-3417 (fax)
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Abstract |
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During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.
Key Words: Golgi apparatus, mitosis, p115, tethering, stack
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Introduction |
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THE Golgi apparatus exists as a series of flattened cisternal membranes that are tightly aligned in parallel to one another to form a stack. Transport vesicles are closely associated, often by fibrous attachments (
To elucidate determinants that are important for the maintenance and establishment of this morphology, we have exploited the fact that during mammalian M phase, this elaborate structure is dramatically transformed into Golgi mini-stacks during prophase, and then a disseminated array of tubulovesicular clusters during metaphase (
From the cell-free system, mitotic disassembly appears to proceed via two independent, concurrent pathways. The coat protein I (COPI)1-dependent pathway proceeds as COPI vesicles continue to bud, but are unable to tether and therefore fuse with their target membrane due to a Cdc2 kinase-mediated event (
A possible molecular explanation for the accumulation of COPI vesicles at mitosis lies in the fact that the binding of p115, a vesicle tethering protein, to Golgi membranes is significantly inhibited at mitosis (
It is postulated that p115 acts to cross-link the acceptor membrane to the incoming COPI vesicle by simultaneously binding its two receptors on Golgi membranes: GM130 on the acceptor membrane and giantin on the COPI vesicle (S at the prevailing donor membrane concentration, whereas GM130 is largely excluded (
At mitosis, the extreme basic NH2 terminus of GM130, comprising the p115 binding site (
This requirement for cyclin B-Cdc2 kinase activity for mitotic Golgi membrane fragmentation is in contrast to results obtained in a semipermeabilized cell system that requires MEK1 activity and not Cdc2 activity (
As cells exit M phase, mitotic phosphorylations are reversed by protein phosphatases facilitating the reversion of mitotic Golgi fragments (MGF) to their original morphology. The first phase of reassembly entails the regeneration of single cisternae (-SNAP,
-SNAP, and p115), and p97 (and its cofactor p47), which seem to contribute nonadditively to cisternal regrowth (
-SNAP for its binding (
As single cisternae begin to form, they align and dock to form stacks. So far, the only factor shown to be required for this event is an NEM-sensitive membrane-bound component (
It is possible to view cisternal stacking and COPI vesicle tethering as functionally equivalent processes. Both require agents that bring and hold membranes in close proximity. The intimacy of these two processes may be reflected by the intimacy of the interaction between GM130 and GRASP65 (
Here we have examined whether p115 plays a direct role in stacking cisternae at the end of mitosis that is distinct from its role in membrane fusion. The results suggest that p115, in conjunction with giantin and GM130, is essential for cisternal stacking and NSF-mediated cisternal regrowth. p115 acts most potently at an early stage in the stacking reaction, upstream of GRASP65, and may facilitate the initial tethering of Golgi cisternae that is a prerequisite for stacking.
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Materials and Methods |
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Materials
All reagents were of analytical grade or higher and purchased from Sigma Chemical Co., Boehringer Mannheim, or BDH Chemicals Ltd., unless otherwise stated. The following antibodies were used in this study: rabbit polyclonal antibodies NN15 against GM130 from N. Nakamura (Department of Molecular Biology, Kyushu University, Fukuoka, Japan); polyclonal antibodies against giantin from M. Renz (Department of Pharmacology, Basel University, Switzerland); mAbs 4H1 and 8A6 against p115 from G. Waters (Department of Molecular Biology, Princeton University, Princeton, NJ); mAbs against p97 from J.M. Peters (IMP, Vienna, Austria); mAb 2E5 against NSF from M. Tagaya (School of Life Science, Hachioji, Japan); rabbit polyclonal antibodies against 1,2-mannosidase I (Mann I) and GRASP65 from F. Barr (University of Glasgow, UK); polyclonal 1946 against
-SNAP from G. Stenbeck (UCL, London, UK); and rabbit polyclonal C-19 against rab6 (Santa Cruz).
Purification of Rat Liver Golgi Membranes
Rat liver Golgi membranes (RLG) were purified as in
Mitotic and Interphase Cytosols
Mitotic and interphase cytosols were prepared from spinner HeLa cells (sHeLa), as in
A 40% ammonium sulphate cut of rat liver cytosol was prepared as in
Depletion of p115 from Rat Liver Cytosol
p115 was depleted from cytosol using either the mAb 4H1 or a biotinylated peptide comprising the NH2-terminal 73 amino acids of GM130 (N73pep), which binds p115 (
1 ml biotinylated N73pep (10 mg/ml in distilled water) was coupled to 2 ml Neutravidin beads (Pierce Chemical Co.). After coupling, beads were blocked with 10 mg/ml soybean trypsin inhibitor. Beads were then packed into a 0.7 x 10-cm Econo-column (Bio-Rad), and the column was equilibrated with 20 ml KHM. 2 ml rat liver cytosol was loaded onto the column and allowed to interact with the resin for 15 min. The column was then eluted with KHM.
In both cases, the mock depletions were made with the same blocked beads without antibody or peptide coupled. 20 µg cytosolic proteins were separated on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose (Hybond C, Nycomed Amersham). Blots were probed with 8A6 to determine the extent of p115 depletion and processed as in
Protein Purification
p115 was purified as in -SNAP and His-tagged
-SNAP were prepared as in
Reassembly Assay
The disassembly reaction was performed as in
To assess the relative polypeptide composition of MGF isolated with or without the MEB/0.5-M sucrose cushion, the 2 µl 2-M sucrose cushion was omitted, and the resulting pellet solubilized in SDS-PAGE sample buffer, boiled for 3 min, and separated on 520% gradient SDS-polyacrylamide gels. The proteins in the gel were transferred to nitrocellulose (Hybond C, Nycomed Amersham) using a semi-dry blotter. Blots were processed as in
For reassembly, the MGF were gently resuspended (final concentrations 0.751 mg/ml) in rat liver cytosol (0.210 mg/ml final concentrations) in KHM buffer (with 2 mM ATP and 1 mM GTP) supplemented with a 10x ATP regeneration system (200 mM creatine phosphate, 10 mM ATP, 2 mg/ml creatine kinase, 0.2 mg/ml cytochalasin B). The final reaction volume was 20 µl. p115-depleted cytosol and p115-depleted cytosol with p115 added back were also used. p115 was estimated to be present at 34 ng/µg cytosol by Western analysis and was added back to this level. Cytosol was replaced by the purified components NSF, -SNAP,
-SNAP, and/or p97, p47, as in
For EM, reactions were fixed, processed, and sectioned as in
For Western blotting, completed reactions were made up to 120 µl with ice-cold KHM and membranes were recovered by centrifugation at 15,000 rpm (13.1 Kgav) for 30 min at 4°C in the horizontal rotor of the Eppendorf centrifuge. The resulting pellet was solubilized in SDS-PAGE sample buffer and processed as for MGF.
In some experiments, the MGF were pretreated with 1 µl anti-GM130 NN15 and/or 1 µl antigiantin for 15 min on ice before resuspension in the purified component reassembly system. The reaction was then allowed to proceed for 60 min at 37°C.
In some experiments, the MGF were pretreated for 15 min on ice with N73pep (080 µM) or soluble GRASP65 (075 ng/µl) and the complete purified reassembly reaction mix (i.e., NSF, -SNAP,
-SNAP, p115, p97, and p47). The reaction was then allowed to proceed at 37°C for 60 min. The effect of N73pep and soluble GRASP65 treatment was also assessed on starting RLG. RLG at 0.75 mg/ml were treated with N73pep (80 µM) or soluble GRASP65 (75 ng/µl) in KHM (with 2 mM ATP and 1 mM GTP) buffer and an ATP regeneration system in a final volume of 20 µl, and incubated for 15 min on ice or 60 min at 37°C. They were then fixed and processed for EM.
To assess the temporal sensitivity of reassembly to N73pep and soluble GRASP65, the complete purified reassembly reaction was allowed to proceed for increasing time at 37°C. At various times, the reaction was transferred to ice and fixed and processed for EM or treated with KHM, 80 µM N73pep, or 75 ng/µl soluble GRASP65 for 15 min on ice. They were then reincubated at 37°C for a total time of 60 min.
Stereology
Stereological definitions were as in
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Results |
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MGF Isolated through a 0.5-M Sucrose Cushion Require Cytosolic Components for Cisternal Regrowth and Stacking
To test whether p115 played a role in cisternal stacking during reassembly, we needed to alter the reassembly assay to make it dependent on added soluble factors. Previously, MGF reassembled into stacked cisternae in buffer alone to the same extent as when cytosol was added, suggesting that everything required for correct reassembly and cisternal stacking was present on the MGF (
Highly purified RLG were incubated with mitotic sHeLa cytosol for 20 min at 37°C and membranes were reisolated by centrifugation in the presence or absence of a 0.5-M sucrose cushion. These membranes were termed MGF and were morphologically similar, whether the 0.5-M cushion was present or not. The percentage total membrane present as Golgi cisternae fell from 77% in RLG to 31% in either set of MGF (Table 1). The most dramatic loss was from stacked Golgi cisternae, which fell from 53% in RLG to <1% in the MGF (Table 1). The 47% loss of membrane from cisternal membranes was accounted for by a concomitant 3035% increase in tubules and 1015% increase in vesicles (data not shown). The mean cross-sectional length of cisternae diminished dramatically by ~70% during the mitotic incubation. The mean cisternal length fell from 1.1 µm in RLG to 0.330.35 µm in the two sets of MGF (Table 1). These MGF do not differ significantly in morphology from those used in previous studies of reassembly (
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The MGF isolated without the 0.5-M sucrose cushion were fusion competent when incubated in KHM buffer alone for 60 min at 37°C (Table 1), as previously reported (
However, both sets of MGF were fusion competent when incubated in rat liver cytosol (10 mg/ml) for 60 min at 37°C (Table 1). The percentage total membrane as cisternae rising from 31 to ~60% for both sets of MGF, and the percentage total membrane present as stacked regions of cisternae from <1 to 2025% (Table 1). The mean cross-sectional length increased to 1.3 µm in cisternae reassembled from MGF isolated with the 0.5-M sucrose cushion and 1.2 µm in cisternae reassembled from MGF isolated without the 0.5-M sucrose cushion (Table 1).
Analysis of the polypeptide composition of the two sets of fragments revealed that the MGF isolated through the 0.5-M cushion were significantly less contaminated with cytosolic factors (data not shown). In fact, the MGF isolated through the 0.5-M cushion contained 65% less protein (data not shown). Western analysis (Figure 1) revealed MGF isolated with or without the 0.5-M sucrose cushion contained similar amounts of Mann I, GM130, and GRASP65 (Figure 1). Therefore, the 0.5-M cushion was not affecting the amount of membranes that were recovered. However, when the MGF are compared with starting RLG, virtually all the Mann I was recovered, but only 4050% of the GM130 and GRASP65 appeared to be recovered. This may be due to a decrease in the reactivity of the antibodies against mitotically phosphorylated GM130 and GRASP65. MGF isolated with or without the 0.5-M sucrose cushion had similar levels of rab6 and -SNAP.
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The presence of the 0.5-M sucrose cushion reduced the MGF p115 levels four- to fivefold, and NSF and p97 levels 2025-fold. The insertion of a 0.5-M sucrose layer separates the mitotic cytosol (of which 1% of total input is shown in the far right lane), which rests on top of this layer, from the MGF, which sediment through this layer on to the underlying 2-M sucrose cushion. This reduces the risk of collecting contaminating cytosolic proteins on collection of the MGF. As the membranes enter the 0.5-M sucrose layer, there may also be some differential removal of the p115, p97, and NSF that are still loosely bound to the membranes. The reduced levels of p115, NSF, and p97 may explain why these MGF are fusion incompetent in buffer alone. This is also consistent with previous observations that NEM treatment or 0.25-M KCl extraction of MGF isolated without a 0.5-M sucrose cushion renders them fusion-incompetent in buffer alone (
p115 Is Essential for the Post-mitotic Stacking of Reassenbling Golgi Cisternae
To assess p115 function in the reassembly process, rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol with purified p115 added back were titrated into the reassembly assay. p115 was depleted >95% from rat liver cytosol using either the mAb 4H1 or N73pep (Figure 2 A). p115 was purified to near homogeneity from rat liver cytosol to add back to this depleted cytosol. MGF were resuspended in cytosol of increasing concentrations and incubated for 60 min at 37°C.
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In rat liver cytosol, cisternal regrowth was near maximal at 1 mg/ml (Figure 2, BD, and 3 A) and the same was true for the mock depleted cytosols (data not shown). At cytosol concentrations below 1 mg/ml, the p115-depleted cytosol supported threefold less cisternal regrowth (Figure 2 E and 3 A). This inhibition was reversed by adding purified p115 back to the depleted cytosol (Figure 2 H and 3 A). Therefore, this loss of activity was due to p115 activity and not the activity of another factor that may have been codepleted from the cytosol by an interaction with p115. However, at cytosol concentrations of 1 mg/ml and above, p115-depleted cytosol supported full cisternal regrowth (Figure 2F and Figure G, and Figure 3 A). Therefore, p115 is not essential for this process, or a p115-independent pathway of cisternal regrowth is operating. We favor the latter explanation because two nonadditive pathways of cisternal regrowth controlled by NSF and p97 have been described previously (
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The stacking process in rat liver cytosol displayed distinct properties to cisternal regrowth in that the number of stacks were still increasing at the highest cytosol concentration tested (10 mg/ml; Figure 2 D and 3 B). This asymmetry may be due to an imbalance in factors required for cisternal regrowth and stacking. This mirrors the disassembly process in that low concentrations of mitotic cytosol are sufficient to inhibit transport (
The most striking effect on reassembly in p115-depleted cytosol at all concentrations tested was the virtual complete absence of stacked Golgi structures at the end of the incubation (Figure 2, EG, and 3 B). This effect could be reversed by adding purified p115 back to the depleted cytosol (Figure 2, HJ, and 3 B), again suggesting that p115 itself was the active component, and not that another factor had been codepleted. Cisternal regrowth and stacking are thus separable processes. The single cisternae formed in the absence of p115 had a more wrinkled, corrugated appearance (asterisks in Figure 2 G), suggesting an involvement of p115 in a membrane smoothing event during the reassembly process. This effect was again reversed by supplementing the depleted cytosol with purified p115. These effects of p115 depletion on reassembly were identical if sHeLa interphase cytosol was used instead of rat liver cytosol (data not shown).
Kinetic analysis revealed the reassembly reaction was complete for both cisternal regrowth and stacking after 60 min in rat liver cytosol (10 mg/ml; Figure 4 I). The first intermediates that formed quickly during the first 15 min of the incubation were single cisternae (Figure 4A and Figure B), frequently with tubular networks at their rims (asterisks in Figure 4 B). By 15 min, these intermediates had begun to dock and align to form the beginnings of stacked Golgi structure (arrowheads in Figure 4 B). The lag in the formation of stacked structures (Figure 4 I) therefore may be considered due to the need to form single cisternae first. By 45 min, this process was well advanced and Golgi stacks with two or more cisternae per stack were prevalent and these discrete stacks were becoming linked via tubular networks (Figure 4 C). By 60120 min, these linkages had been made, the tubular networks were less apparent, and long cisternal stacks were the end product, which often adopted an approximate closed concentric circular morphology (compare Figure 2 D and 4 D).
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In p115-depleted cytosol, single cisternae formed at the start of the reaction, although with a reduced initial rate (Figure 4I and Figure E). Once again these cisternae were blunt-ended, indicating the p97 pathway of reassembly may be dominant (asterisks in Figure 4F and Figure G). By 15 min, these single cisternae were still well separated (Figure 4 F), and even after 4560 min, single, blunt-ended cisternae were the major reaction product (Figure 4G and Figure I). However, after 120 min, even though no more cisternal regrowth occurred, these single cisternae did begin to align and form stacks (Figure 4H and Figure I). Even then, the level of stacking only reached ~50% of that of rat liver cytosol (Figure 4 I), and the intercisternal distance between adjacent cisternae of the stack seemed more variable (compare Figure 4C and Figure H). We conclude that the absence of p115 severely retards both the initial rate and overall extent of the stacking of Golgi cisternae during the reassembly reaction.
Was the time at which p115 added back to the depleted cytosol crucial to reverse the effect on stacking? To assess this, reassembly was conducted in p115-depleted cytosol and p115 was added back to the reaction at 15, 30, or 60 min and the reaction was allowed to proceed for 120 min. If added within the first 30 min, the p115 was able to restore stacking activity to the cytosol. However, if added at 60 min, the p115 only slightly stimulated stacking (Figure 4 J). This suggests that p115 must be present as cisternae are reassembling for it to fulfil its stacking function. Once cisternae have formed completely, it seems p115 is no longer able to stimulate stacking.
The p97 Pathway Generates Only Single Cisternae in the Absence of p115
To more finely discern the role played by p115 in the p97 and NSF pathways of reassembly, and to corroborate the above findings, we moved to the purified system. p115 was titrated into the p97, NSF, and NSF/p97 catalyzed reassembly reactions. The amount of cisternal regrowth using these systems was moderately better than that achieved in rat liver cytosol. The NSF, p97, and NSF/p97 combined reactions generated ~70% total membrane present as cisternae from 31% in MGF after a 60 min incubation at 37°C (Table 1).
Titration of p115 into the p97 reaction revealed that this fusion pathway was insensitive to added p115 (Figure 5 A). However, only single cisternae with a mean cisternal cross-sectional length of 1.4 µm formed in the absence of p115 (Table 1). These single cisternae had a wrinkled appearance reminiscent of those formed in p115-depleted cytosol (compare Figure 2 G and 5 A). Upon addition of p115, these cisternae had a smoother appearance and formed stacks (Figure 5 A). However, the stacks formed rarely had more than two cisternae per stack.
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Titration of p115 into the NSF reaction revealed that p115 was required for both cisternal regrowth and stacking (Figure 5 B). Both processes were saturating, but still rising at the maximum p115 concentration tested (30 ng/µl). The reassembling cisternae formed stacks that usually had three or more cisternae per stack (Table 1). The cisternae formed had a mean cross-sectional length of 0.79 µm (Table 1), which is considerably shorter than those formed by the p97 pathway. A Mann-Whitney test revealed the NSF and p97 cisternal length distributions to be significantly different in location, with P = 0.0023.
When p115 was titrated into the combined NSF/p97 reaction, cisternal regrowth was insensitive to added p115 (Figure 5 C). However, stacking still required p115. In the absence of added p115, only wrinkled single cisternae were formed. On addition of p115, the cisternae formed were smooth and formed stacks with two or three cisternae per stack, similar to the number formed in rat liver cytosol (Table 1). The mean cisternal cross-sectional length was 1.3 µm and this value was similar to that achieved in rat liver cytosol (Table 1). A Mann-Whitney test revealed that these two distributions were not significantly different in location. This provides more correlational evidence that the p97 and NSF pathways operate to reform cisternae in rat liver cytosol.
The p115 Stacking Event Requires GM130 and Giantin
Previously, it has been shown that p115 binds to GM130 and giantin on Golgi membranes, and that these interactions are important for COPI vesicle tethering in vitro (
When the MGF were resuspended for the p97 or NSF/p97 combined pathway, cisternal regrowth was unaffected by antigiantin and/or anti-GM130 (Figure 6 A). In contrast to this, the stacking process was severely inhibited in these two pathways (Figure 6 B) and the preimmune sera had no effect on this process (data not shown). Thus, p115 stacking function requires p115 interactions with GM130 and giantin. Furthermore, this indicates that p115 may be able to tether cisterna to cisterna, as well as COPI vesicle to cisterna.
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When MGF were pretreated with antigiantin or anti-GM130 and resuspended for the NSF pathway, both cisternal regrowth and, as a consequence, stacking were inhibited (Figure 6A and Figure B), and the preimmune sera had no effect (data not shown). That cisternal regrowth is inhibited strongly suggests that the interaction between GM130, p115, and giantin is essential for NSF-mediated Golgi membrane fusion. This may be due to an inhibition of COPI vesicle tethering to Golgi membranes (
p115 Is Required Before GRASP65 at an Early Stage in Stack Formation
An NEM-sensitive membrane-bound component of MGF is essential for the stacking reaction during reassembly. This factor was identified as GRASP65, a highly conserved, N-myristoylated protein that exists as a tight complex with GM130 on the membrane (
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The soluble GRASP65 did not act to remove GM130 from the membranes (Figure 7 A), which is consistent with the fact that the complex between GM130 and GRASP65 is stable and can only be reconstituted if both proteins are cotranslated (
N73pep was also titrated into this assay, and in agreement with the effect of anti-GM130, potently inhibited stacking, but not cisternal regrowth (Figure 7 B). N73pep clearly inhibited the rebinding of p115 to reassembling Golgi membranes (Figure 7 B). The S25D N73pep mutant, which binds p115 with a much lower affinity, had no effect on either process (data not shown).
To assess the temporal sensitivity of the stacking reaction to these two inhibitors, MGF were incubated in the NSF/p97-purified reaction for increasing time at 37°C. At various time points (time of addition, t; Figure 8, AD) the reaction was transferred to ice and either fixed with 2% glutaraldehyde and processed for EM, or treated with buffer (KHM, the GRASP65 and N73pep solvent), N73pep, or soluble GRASP65, and then reincubated at 37°C for a total time of 60 min.
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The time course for the reassembly of stacked regions of cisternae in the NSF/p97 reaction displayed similar characteristics to the rat liver cytosol catalyzed reaction (compare Figure 8 A and 4 I). p115 rebound rapidly to the reassembling Golgi membranes (Figure 8 A).
Addition of KHM buffer had no effect on the stacking reaction or on the amount of p115 bound to the Golgi membranes at the end of the incubation (Figure 8 B). This suggests that the buffer, and transferring the reaction to ice, was not detrimental to the process.
The stacking process was sensitive to N73pep for the first 15 min of the reaction (Figure 8 C). When added at 15 min, the time point when cisternae begin to dock and align (Figure 4 B), the N73pep actually unstacked those stacks that had formed, suggesting that p115 was mediating this event (compare Figure 8A and Figure C). At time points later than 15 min, the reassembled stacks became resistant to added N73pep and normal stacking was able to proceed. Stacked RLG are also unaffected by N73pep treatment (Table 2), suggesting that this is a shared property of reassembled Golgi stacks and starting stacked RLG. At all time points tested, N73pep was able to significantly remove bound p115 from the membranes, such that, at the end of the incubation, only 15% of the p115 was bound, as compared with control reactions (Figure 8 C). Thus, it was not that N73pep could no longer remove p115 from the membranes at later time points. The requirement for p115 appears to be a transient event required for the initial docking and alignment of newly formed single cisternae.
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The stacking process was sensitive to soluble GRASP65 for the first 30 min (Figure 8 D). Soluble GRASP65 acted to unstack Golgi cisternae that had formed before this point (compare Figure 8A and Figure D). However, beyond 30 min, the reassembled stacks became resistant to soluble GRASP65, which is also a property of the starting stacked RLG (Table 2). Soluble GRASP65 treatment of starting RLG did not disrupt their stacked structure. Neither the percentage total membrane as stacked regions of cisternae nor the number of cisternae per stack were affected (Table 2). At no time point did soluble GRASP65 affect p115 binding: the amount bound at the end of the incubation remained constant (Figure 8 D). That the reassembled stacks remain sensitive to soluble GRASP65 longer than they do to N73pep suggests that GRASP65 may act downstream of p115 in the stacking pathway, raising the possibility that the stacking reaction proceeds by an initial p115-dependent tethering step that is followed by a GRASP65-dependent stacking step.
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Discussion |
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We have employed a modified cell-free assay to more closely assess p115 function in cisternal regrowth and stacking. In this assay, the MGF are isolated through a 0.5-M sucrose cushion that renders them incompetent for cisternal regrowth in the absence of added soluble factors, due to the virtual absence of the membrane fusion ATPases, NSF and p97. Previously, treatment of the MGF with the cysteine alkylating reagent NEM inactivated any residual NSF or p97, thus ensuring dependence on added soluble factors (
Several lines of evidence strongly suggest a requirement for p115 in the stacking of reassembling Golgi cisternae. Firstly, p115-depleted cytosol supports full cisternal regrowth at cytosol concentrations above 1 mg/ml, but not cisternal stacking, suggesting that these are separable processes. Cisternal stacking is restored by addition of purified p115 to the depleted cytosol. Reassembly conducted in p115-depleted cytosol at maximum concentration for periods of well over 1 h did support some stacking, but the initial rate and overall extent of stacking were severely retarded. In the reassembly assay conducted with purified fusion components, NSF-dependent reassembly required p115 for stacking and cisternal regrowth. While in the p97-dependent reassembly, p115 was required for stacking, but not cisternal regrowth. Similarly, when the NSF/p97 pathways were combined, p115 was only required for cisternal stacking, as was the case in the reassembly conducted in p115-depleted cytosol. Thus, we conclude that p115 is able to tether cisterna to cisterna, as well as COPI vesicle to cisterna, and in so doing, plays a role in cisternal stacking.
That p115 functions in both the NSF (for membrane fusion and stacking) and p97 (stacking only) pathways suggests that this may be another point where these pathways intersect and are modulated. Syntaxin 5 is also a common component of the two pathways, and may explain why they contribute nonadditively to cisternal regrowth (
The difference in cisternal length produced by the NSF and p97 pathways was not detected when MGF were pretreated with NEM (
Therefore, our working hypothesis is that the NSF pathway reconstitutes the Golgi rims while the p97 pathway reconstitutes the cisternal cores, as has been suggested before (
Another feature of cisternae reassembled in the absence of p115 is their frequent wrinkled, corrugated morphology. This suggests p115 is required for a membrane-smoothing event during the reassembly process. Analogy may be drawn to the post-mitotic reassembly of the nuclear envelope. In a cell-free system that utilizes Xenopus egg extracts and scanning EM to visualize nuclear envelope assembly (
p115-mediated stacking requires both receptors for p115 on Golgi membranes, giantin and GM130. Pretreatment of MGF with antibodies against GM130 and/or giantin precluded cisternal stacking, as well as NSF-mediated cisternal regrowth. Previously, the GM130p115giantin complex had been implicated in tethering COPI vesicles that had been isolated in the presence of GTPS to Golgi membranes (
The fact that GM130 largely appears to be excluded from COPI vesicles, and the relative effects of preblocking COPI vesicles or Golgi membranes with antigiantin or anti-GM130 antibodies on subsequent COPI vesicle tethering, suggested that the tether was made up of giantin on the COPI vesicle linked to GM130 on the target membrane via p115 (
Many of the factors required for the reassembly assay have a predominantly cis-Golgi membrane localization (e.g., GM130 [
Whether the cisternal maturation model or the vesicular transport model is true, both models have a requirement for the transfer of COPI vesicles between successive layers of the stack, even though the directionality/content of these vesicles may vary between models (
Previously, it has been shown that GRASP65, which anchors the COOH terminus of GM130 to the Golgi membrane, is involved in the stacking process (
The 15 min time point of reassembly, where N73pep has its most potent effects, is the stage when single cisternae begin to dock and align to form stacks (
Precisely how GRASP65 acts to stack cisternae and precisely how soluble GRASP65 interferes with this reaction remains obscure. One possibility is that the oligomeric state of GRASP65 may be important for anchoring cisternae together. GRASP65 appears to be either a dimer or a trimer (
Analogy may be drawn to the proposed mechanism of vesicular transport, where p115 acts at an early stage in tethering the COPI vesicle to its acceptor membrane, and then hands it over to the SNAREs to complete the fusion step. Similarly, in cisternal stacking, p115 may act at an early stage in tethering cisternal membranes together, and then hand over to another set of molecules that complete the stacking reaction. GRASP65 is an excellent candidate for one of these downstream factors. The stacking reaction has also been shown to have a microcystin-sensitive component (
Comparison of COPI vesicle production by the Golgi apparatus under interphase and mitotic conditions reveals an apparent capacity to generate twice as many COPI vesicles at mitosis with the same content (
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Footnotes |
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Dr. Warren's current address is Department of Cell Biology, SHM, C441, Yale University School of Medicine, 33 Cedar St., PO Box 208002, New Haven, CT 06520-8002. Tel.: (203) 785-5058. Fax: (203) 785-4301. E-mail: graham.warren@yale.edu
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Acknowledgements |
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We would like to thank Francis Barr, Barbara Dirac-Svejstrup, and Martin Lowe for many constructive comments and support; Hemmo Meyer for p97, Joyce Müller for NSF, Hisao Kondo for p47, Francis Barr for soluble GRASP65, G. Waters, N. Nakamura, M. Tagaya, G. Stenbeck, F. Barr, M. Renz, and J.M. Peters for antibodies, and Rose Watson and Eija Jämsä for help with EM.
James Shorter was supported by a predoctoral research fellowship from the Imperial Cancer Research Fund.
Submitted: March 29, 1999; Revised: May 21, 1999; Accepted: June 4, 1999.
1.used in this paper: COPI, coat protein I; GRASP65, Golgi reassembly stacking protein 65; IQ, illimaquinone; Mann I, 1,2-mannosidase I; MGF, mitotic Golgi fragments; N73pep, NH2-terminal 73 amino acids of GM130 peptide; NEM, N-ethylmaleimide; NSF, NEM-sensitive fusion protein; RLG, rat liver Golgi membranes; sHeLa, spinner HeLa cells; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; t, target; v, vesicle
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References |
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