Article |
Address correspondence to Graham B. Warren, Dept. of Cell Biology, SHM, C441, 333 Cedar St., New Haven, CT 06520-8002. Tel.: (203) 785-5058. Fax: (203) 785-4301. E-mail: graham.warren{at}yale.edu
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Abstract |
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Key Words: Golgi apparatus; mitosis; p115; SNAREpin; Golgin
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Introduction |
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A well-characterized and highly conserved vesicle tethering protein is p115, which tethers coat protein (COP)*I vesicles to Golgi membranes (Sönnichsen et al., 1998). p115 functions include ER-Golgi transport (Cao et al., 1998), intra-Golgi transport (Waters et al., 1992; Seemann et al., 2000a), and stacking Golgi cisternae (Shorter and Warren, 1999). This myosin-shaped homodimer consists of an NH2-terminal globular head domain, a coiled-coil tail, and a short acidic COOH-terminal domain (Fig. 1 A) (Sapperstein et al., 1995). p115 juxtaposes membranes by simultaneously binding via its acidic COOH-terminal domain two Golgins, GM130 in one membrane and Giantin in the other (Sönnichsen et al., 1998; Shorter and Warren, 1999; Dirac-Svejstrup et al., 2000). GM130 and Giantin are long rod-like fibrous proteins due to an extensive coiled-coil domain structure typical of Golgins (Linstedt and Hauri, 1993; Nakamura et al., 1995). GM130 is restricted to Golgi cisternae, whereas Giantin is also present in COPI vesicles (Nakamura et al., 1995; Sönnichsen et al., 1998; Martinez-Menarguez et al., 2001). Thus, p115 may tether COPI vesicle to cisterna or cisterna to cisterna, depending on the topological restriction of Giantin, and so couple stacked Golgi structure to processive COPI vesicle flow (Linstedt, 1999; Shorter and Warren, 1999; Orci et al., 2000). The Giantin-p115-GM130 tether is mitotically regulated by cyclin B-CDK1, which directly phosphorylates GM130 and precludes p115 binding. This may help explain the accumulation of COPI vesicles that occurs during mitosis as part of the Golgi disassembly and inheritance process (Nakamura et al., 1997; Lowe et al., 1998). In addition, p115 in synergy with Rab1 tethers COPII vesicles to membranes, though the mechanism is obscure (Cao et al., 1998; Allan et al., 2000; Moyer et al., 2001).
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We have examined the significance of the putative p115SNARE homology by using a cell-free system that reconstitutes many aspects of postmitotic Golgi reassembly. Isolated mitotic Golgi fragments (MGFs) will regenerate Golgi cisternae via one of two pathways controlled by the AAA ATPases NSF and p97. The NSF reaction requires Giantin-p115-GM130 tethers and the Golgi v-SNARE GOS-28 (Nagahama et al., 1996) and its cognate t-SNARE syntaxin-5 (Hay et al., 1997; Rabouille et al., 1998). Using this system, we now show that p115 physically couples COPI vesicle tethering (Golgin dependent) to COPI vesicle docking (SNARE dependent) by sequentially linking GM130 to Giantin, followed by GOS-28 to syntaxin-5, to actively catalyze SNAREpin assembly. This direct catalytic role of p115 in SNARE assembly is impelled via its SNARE motif-related region.
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Results |
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The interactions between Golgi SNAREs and p115/CC1 were specific since the TGN/endosomal SNAREs syntaxin-6, syntaxin-11, Vti1a, and Vti1b were not retained (Fig. 1 C). Moreover, if a detergent extract of a rat liver postnuclear supernatant was probed with p115/CC1 beads neither syntaxin-1, SNAP-23, VAMP2 (plasma membrane SNAREs), nor SNAP-29 (multiple compartments) were retained (unpublished data). In contrast, syntaxin-5 and GOS-28 were still retrieved (unpublished data). Therefore, p115/CC1 does not interact indiscriminately with SNAREs but interacts specifically with a subset of Golgi SNAREs, which form SNAREpins that contain syntaxin-5 as their common component (Hay et al., 1997, 1998; Parlati et al., 2000; Zhang and Hong, 2001).
Sly1p (a syntaxin-5 binding Sec1/Munc18 protein [Jahn, 2000]) was also retained on p115/CC1 beads but not on CC2, CC3, CC4, or mock beads and may correspond to the 66-kD protein visible by silver stain (Fig. 1, B and C). Giantin, GM130, and p115 were not retained by CC1-4 beads, suggesting these peptides do not retrieve molecules from the extract simply because they contain coiled-coil domains. The fact that
-SNAP was not retained even though
-SNAP interacts with the SNARE motif of syntaxin-1 (Jahn and Südhof, 1999) further illustrates the specificity of p115/CC1SNARE interactions.
Histail and acidic domain of p115 (TA) and Histail domain of p115 (T; Fig. 1 A) were also used as affinity ligands. In corroboration, both retrieved exactly the same subset of Golgi SNAREs as p115/CC1 (Fig. 1 C), indicating that the globular head domain of p115 is not required for these interactions. p115 and His-TA but not His-T retrieved Giantin and GM130, reinforcing the importance of the acidic domain of p115 for these interactions. Thus, p115 appears to be a multivalent scaffold for both Golgins (Giantin and GM130) and SNAREs.
p115 stimulates the assembly of syntaxin-5 SNARE complexes
Since a common feature of the specific subset of SNAREs retrieved by p115 is that they constitute syntaxin-5 SNAREpins, we tested whether p115 promoted their assembly. To this end, Golgi membranes were salt washed to remove endogenous p115 and incubated with NSF to disassemble preexisting cis-SNARE complexes (Otto et al., 1997). NSF was then inactivated (using NEM), and the membranes were solubilized in Triton X-100 buffer, clarified, and incubated with increasing amounts of p115. GOS-28 or syntaxin-5 was then immunoprecipitated, and the extent of coprecipitation of other Golgi SNAREs was determined by immunoblot (Fig. 2, AD).
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Similarly, increasing p115 concentrations enhanced the coprecipitation of GOS-28, Ykt6p, rSec22p, and membrin with syntaxin-5 from 510% to 1520% of the total SNARE present, and GS15 and Bet1p from 10 to 35% (Fig. 2, B and D). The stimulated coprecipitation of rSec22p and membrin with syntaxin-5 suggests that p115 may also stimulate formation of the well-defined membrinBet1prSec22psyntaxin-5 SNAREpin (Parlati et al., 2000; Xu et al., 2000). The specificity of this enhancement is reinforced by the lack of coprecipitation of syntaxin-6, syntaxin-11, or Vti1b (Fig. 2 B). Vti1a coprecipitated with syntaxin-5 at low levels (Fig. 2 B) consistent with a Vti1pSed5p complex at the cis-Golgi network in yeast that may function in retrograde vesicle transfer between vacuole and the cis-Golgi network (Fischer von Mollard and Stevens, 1999). However, Vti1asyntaxin-5 complex formation was not enhanced by p115 (Fig. 2 B). This may suggest Vti1asyntaxin-6 complexes are more prevalent in rat liver Golgi (Xu et al., 1998).
Identical results were obtained if apyrase was added after quenching the NEM with DTT (unpublished data). Apyrase acts to deplete any remaining ATP in the system. Therefore, p115 was not stimulating SNARE complex assembly by simply inhibiting any residual NSF activity that might remain after NEM treatment.
In both immunoprecipitations, low levels of p115 were present in the retrieved complexes (Fig. 2, A and B), and increasing p115 concentration caused increasing amounts of GM130 and Giantin to be coprecipitated (Fig. 2, A and B). Very similar results were obtained if MGFs were used (unpublished data). This emphasizes that p115 may simultaneously bridge GM130 to Giantin, while coordinating syntaxin-5 SNAREpin assembly.
Addition of CC1 peptide abrogated this enhancement of syntaxin-5GOS-28 SNARE complex formation and blocked the coprecipitation of p115 (Fig. 2, E and F), suggesting that the SNARE motif-related region of p115 mediates the observed stimulation. In contrast, CC2, p115 CT (COOH-terminal 75 aa of p115, which binds to Giantin and GM130) and GM130 NT (NH2-terminal 73 aa of GM130, which binds p115) had no effect (Fig. 2, E and F).
Could p115 also stimulate SNARE complex formation on intact Golgi membranes? This was tested by assaying the formation of SNARE complexes that are preserved in SDS at room temperature. cis-SNARE complexes were disassembled on salt-washed Golgi membranes (as above) and incubated for 30 min at 37°C with or without p115. The extent of GOS-28syntaxin-5 SDS-resistant complex formation was then determined (Otto et al., 1997). Several distinct GOS-28syntaxin-5 SDS-resistant complexes were apparent in the starting material and present at the same level after incubation with buffer (Fig. 2 G). These complexes were preserved at 25°C but disassembled at 95°C (Fig. 2 G). p115 greatly increased the formation of these SDS-resistant complexes, and densitometry revealed this was a fourfold stimulation that was inhibited by addition of CC1 but not CC2 (Fig. 2 G). These effects were also found when Bet1p and Ykt6p but not syntaxin-6 SDS-resistant complexes were analyzed (unpublished data). Intriguingly, p115 caused the formation of a very slow migrating GOS-28syntaxin-5 species (Fig. 2 G, asterisks) that was not immunoreactive to anti-p115 antibodies (unpublished data) and may represent SNAREpin oligomers. Collectively, these data demonstrate that p115 assembles GolgiSNARE complexes that contain syntaxin-5 in either detergent solution or on native membranes, and this assembly is mediated by the SNARE motif-related domain (CC1) of p115.
p115 stimulates assembly of GS15Ykt6pGOS-28syntaxin-5 and membrinBet1prSec22psyntaxin-5 SNAREpins
To determine precisely which SNAREpins were assembled by p115, reactions were performed as in the preceding section except that either GS15 or membrin was immunoprecipitated (Fig. 3). Without incubation before the immunoprecipitation, no SNAREs coprecipitated with GS15 (Fig. 3 A, lane 2). After incubation, only three SNAREs coprecipitated with GS15, namely Ykt6p, GOS-28, and syntaxin-5, strongly suggesting that they comprise a SNAREpin. Formation of this complex was enhanced approximately fivefold by p115 (Fig. 3 A, lane 3 compared with 6). This stimulation was abolished by inclusion of CC1 (Fig. 3 A, lane 8) but not CC2 (Fig. 3 A, lane 10) in the reaction, strongly suggesting that the SNARE-motif like region of p115 drives this assembly. CC1 was not acting to prevent direct SNARESNARE interactions, since SNARE complexes alone (no added p115) assembled at the same background level in the presence or absence of CC1 (Fig. 3 A, lane 3 compared with 7). Therefore, CC1 inhibited only the p115-mediated stimulation of SNAREpin assembly. p115 also coprecipitated with the SNAREs (Fig. 3 A, lanes 46) but was not acting to simply link the SNAREs together, since it could be removed at the end of the immunoprecipitation by either a wash with 10 µM CC1 (lane 12) or 1 M KCl (lane 14; 10 µM CC2 [lane 13] and 150 mM KCl [lane 11] had no effect), without affecting the integrity of the retrieved SNARE complexes. This strongly indicates that p115 assembles SNAREpins but is not required to maintain them, since it can be removed without affecting SNAREpin integrity.
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Essentially identical effects were observed when membrin was immunoprecipitated except that the three SNAREs that coprecipitate with membrin are Bet1p, rSec22p, and syntaxin-5, the components of a well-characterized SNAREpin (Fig. 3 C) (Hay et al., 1997, 1998; Parlati et al., 2000; Xu et al., 2000). Their assembly into SDS-resistant complexes was also enhanced greatly by p115 (Fig. 3 D) in a manner dependent on the SNARE motif-like domain of p115. Thus, p115 assembles at least two distinct SNAREpins comprising GS15Ykt6pGOS-28syntaxin-5 and membrinBet1prSec22psyntaxin-5 via its SNARE motif-related domain.
The SNARE motif-related region of p115 specifically inhibits NSF-driven Golgi reassembly
We next asked if the SNARE motif-related region of p115 plays a role in Golgi membrane fusion. p115 is absolutely required for NSF-catalyzed cisternal regrowth from isolated MGFs, where it stimulates COPI vesicle fusion by linking Giantin on COPI vesicles to GM130 on acceptor tubular remnants. CC1 was added to this reaction and inhibited cisternal regrowth by >90% (Fig. 4 A). The inhibition was dose dependent with an IC50 of 1.9 µM, an 14-fold molar excess over p115 (Fig. 4 A, inset). Even when added at an equimolar concentration to p115, CC1 reduced cisternal regrowth by
30%. This biphasic response to CC1 is indicative of multiple binding sites for CC1 on Golgi membranes and is consistent with the ability of CC1 to bind several Golgi SNAREs and Sly1p (Fig. 1 C). This effect was specific to CC1, since CC2, CC3, and CC4 alone or in combination had little effect (Fig. 4 A). Thus, the CC1 inhibition is not due to nonspecific effects of coiled-coils. The lack of effect of CC4 implies that the weak interactions between CC4 and Golgi SNAREs (Fig. 1 C) are functionally irrelevant, at least in this assay.
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All of the Golgi SNAREs that interacted with p115 were required for NSF-driven Golgi reassembly as demonstrated by antibody inhibition (Fig. 4 B). Antibodies against GOS-28 and syntaxin-5 had the greatest effect, inhibiting cisternal regrowth by 85% (Fig. 4 B). Antibodies against membrin, Bet1p, rSec22p, Ykt6p, and GS15 inhibited by 4075% (Fig. 4 B). Other anti-SNARE antibodies (against syntaxin-6, syntaxin-11, Vti1a, Vti1b, syntaxin-1, SNAP-23, SNAP-29, and VAMP2) had no effect on reassembly. Although antibodies rather than Fab fragments were used in these experiments, they provide corroborative evidence that those SNAREs interacting with p115 are also involved in Golgi reassembly (Fig. 4 B).
The SNARE motif-related domain of p115 does not disrupt Giantin-p115-GM130 tethers
How Giantin-p115-GM130 tether formation was related to the p115-mediated stimulation of syntaxin-5GOS-28 SNAREpin formation was unclear. Was it possible that CC1 was blocking reassembly by disrupting Giantin-p115-GM130 tethers rather than p115SNARE interactions? This seemed improbable, since CC1 beads retained neither GM130, Giantin, nor p115 from Golgi detergent extract (Fig. 1 C). CC1 did not bind to purified p115 directly either (unpublished data). However, to test this further Golgi membranes were solubilized and incubated with or without p115. Giantin was immunoprecipitated, and the extent of coprecipitation of GM130 and p115 was determined. GM130 only coprecipitated with Giantin in the presence of p115 (Fig. 5 A). This linking of Giantin to GM130 was effectively abolished by GM130 NT (Fig. 5 A) but was unaffected by CC1 (Fig. 5 A). This strongly suggests that CC1 does not inhibit reassembly by disrupting Giantin-p115-GM130 tethers.
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p115 assembles GOS-28syntaxin-5 complexes directly via its SNARE motif-related domain
To resolve the role of p115 in SNAREpin formation more acutely, purified SNAREp115 binding assays were established. Recombinant GST- or His-tagged SNAREs were expressed and purified from Escherichia coli. All SNAREs used were purified to 95% homogeneity as judged by Coomassie staining (unpublished data).
We focused on GOS-28 and syntaxin-5, since they were the most active SNAREs in Golgi reassembly (Fig. 4, A and B). The interaction between p115 and the purified cytoplasmic domains of syntaxin-5 or GOS-28 was direct and inhibited by 85% with CC1 but not with CC2 or CC3 (Fig. 6 A). This inhibition implies that p115 binds the two SNAREs via CC1, the SNARE motif-related region. Binding was specific for these SNAREs, since p115 did not bind HisVAMP-2, GSTsyntaxin-1 (Fig. 6 B), or GSTsyntaxin-6 (unpublished data). Furthermore, His-TA and His-T could bind HisGOS-28 and GSTsyntaxin-5 (Fig. 6 B) as could CC1 (unpublished data). Hishead domain of p115 (H; Fig. 6 B), CC2, and CC3 (unpublished data) did not bind purified SNAREs, suggesting they are not required for p115SNARE interactions. In fact, p115 bound to GSTsyntaxin-5, with an apparent Kd of 1.8 µM (Fig. 6 C), and HisGOS-28, with an apparent Kd of 1.5 µM (Fig. 6 D). Addition of CC1 at a fixed concentration of 13 µM greatly reduced the amount of p115 that bound to either SNARE (Fig. 6, C and D). Thus, CC1 may inhibit NSF-driven Golgi reassembly by reducing the probability of specific and productive p115SNARE interactions.
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Next, we added increasing concentrations of HisGOS-28 to a fixed amount of GSTsyntaxin-5 in the presence or absence of CC1. Up to 0.4 moles of HisGOS-28 bound per mole of GSTsyntaxin-5, and CC1 had very little effect on this amount, suggesting that CC1 did not interfere with GOS-28syntaxin-5 binding directly (Fig. 7 C). Addition of p115 increased the amount of HisGOS-28 that bound to GSTsyntaxin-5, especially at low GOS-28 concentrations and up to a maximum of 0.7 moles/mole, suggesting that it acts to increase the efficiency of GOS-28 binding to syntaxin-5 (Fig. 7 D). This effect was abolished by inclusion of CC1 (Fig. 7 D), illustrating that CC1 inhibited the stimulation of GOS-28syntaxin-5 binding by p115 and not GOS-28syntaxin-5 binding itself.
In corroboration, His-H did not stimulate HisGOS-28GSTsyntaxin-5 binding, whereas His-TA and His-T stimulated binding just as well as p115 (Fig. 7 E). Addition of His-TA or His-T to very high levels actually inhibited the binding of HisGOS-28 to GSTsyntaxin-5 (Fig. 7 E). Such inhibition is diagnostic of a tethering event, since very high concentrations of the linking protein will simply suppress formation of any ternary complex at the expense of binary complexes.
p115 catalyzes SNARE assembly and is not required to maintain SNARE complexes
Was p115 simply linking HisGOS-28 to GSTsyntaxin-5 or stimulating a direct interaction between them? To make this distinction, we adopted three independent approaches. First, we sought a condition that releases p115 from HisGOS-28GSTsyntaxin-5. If p115 simply links the two SNAREs, then such release should disrupt the HisGOS-28GSTsyntaxin-5 interaction. If HisGOS-28 and GSTsyntaxin-5 were interacting directly, then p115 release should not disrupt the complex, providing the HisGOS-28GSTsyntaxin-5 interaction was resistant to the condition that released p115. Hence, binding experiments were performed (Fig. 7 A), except that after GSTsyntaxin-5 retrieval complexes were challenged with different washes to try and release p115. p115SNARE complexes were disrupted by 1 M KCl (Fig. 8 A) or 2 M urea (unpublished data). Strikingly, upon p115 release HisGOS-28 remained bound to GSTsyntaxin-5 at the same level as in control washes (Fig. 8 A). Similarly, a 20 µM CC1 wash released p115 without affecting the level of HisGOS-28 retained (Fig. 8 A). This was specific for CC1, since CC2 did not release p115 (Fig. 8 A). Corroboratively, if endogenous GS15 or membrin-containing SNARE complexes were immunoprecipitated from Golgi detergent extracts and challenged with 1 M KCl or 10 µM CC1, p115 was released without affecting SNARE complex integrity (Fig. 3, A and C, compare lanes 11, 12, and 14). Moreover, p115 stimulated formation of multiple GOS-28syntaxin-5 SDS-resistant complexes on Golgi membranes (Fig. 2 G and Fig. 3, B and D) that do not contain p115 (unpublished data). In total, these results imply that p115 stimulates the formation of GOS-28syntaxin-5 complexes but is not required to maintain them.
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Finally, if p115 is a bona fide catalyst for HisGOS-28GSTsyntaxin-5 binding, then it should not be consumed by the reaction. Thus, very low substoichiometric levels of p115 should stimulate the reaction. Hence, HisGOS-28GSTsyntaxin-5 binding was conducted with a p115 concentration three orders of magnitude lower than the SNARE concentration. To detect any effect, the incubation time was increased to 4 h. Even at levels below detection by immunoblot, p115 catalyzed the formation of HisGOS-28GSTsyntaxin-5 complexes (Fig. 8 C). HisGOS-28 was incorporated into the complex in amounts that were at least 20-fold greater than the total amount of p115 present. Kinetic analysis of HisGOS-28GSTsyntaxin-5 binding revealed that this substoichiometric level of p115 greatly enhanced the initial rate of GOS-28syntaxin-5 binding (Fig. 8 D). This suggests that p115 catalyzes SNARE assembly.
These findings were verified with endogenous Golgi SNAREs (Fig. 8 E). Golgi membranes were salt washed to remove endogenous p115 and incubated with NSF to disassemble preexisting cis-SNARE complexes (Otto et al., 1997). NSF was then inactivated (using NEM), and the membranes were solubilized in Triton X-100 buffer, clarified, and incubated with 100 pM p115. GS15 was immunoprecipitated, and the coprecipitation of cognate SNAREs was determined by immunoblot. With the use of recombinant SNAREs as standards, we estimated that rat liver Golgi membranes (RLGs) contain 2 ng/µg Golgi protein of GOS-28 and
4 ng/µg Golgi protein syntaxin-5 (unpublished data). Thus, the endogenous SNAREs are present in the reaction at nM concentrations (
14 nM GOS-28 and 22 nM syntaxin-5) compared with at most pM p115 concentrations. This substoichiometric level of p115 was sufficient to stimulate the formation of the GS15Ykt6pGOS-28syntaxin-5 SNARE complex (Fig. 8 E). Furthermore, kinetic analysis revealed that substoichiometric p115 enhanced the initial rate of GS15Ykt6pGOS-28syntaxin-5 SNAREpin assembly (Fig. 8 F). Thus, p115 catalyzes SNARE assembly.
Sequence of events in NSF-driven Golgi reassembly
To determine the functional sequence of p115Golgin and p115SNARE interactions during reassembly, a range of inhibitors was screened (Fig. 9 B) and the morphology of the reaction products generated determined (Fig. 9, A and C). From the morphology, these inhibitors were grouped into two classes: those that inhibited fusion generating dispersed tubules/vesicles and those that inhibited fusion generating clustered tubules/vesicles.
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The second class of inhibitor induced a population of clustered tubules/vesicles. These inhibitors increased the density of tubules/vesicles more than twofold (Fig. 9 C) and includes chrysin (Fig. 9 B), an inhibitor of a Golgi-associated CKII-like kinase that phosphorylates p115. p115 phosphorylation is required for reassembly and strengthens the Giantin-p115-GM130 tether (Dirac-Svejstrup et al., 2000). This may elicit a transition from tethering to SNAREpin formation. CC1, soluble GOS-28 (Fig. 9 A), and syntaxin-5 also had this effect (Fig. 9 C) as did antiGOS-28 and antisyntaxin-5 antibodies (unpublished data). Since these reagents do not disrupt Giantin-p115-GM130 tethers, it would seem that the fragments are aligned in preparation for fusion by this tether. However, fusion may not occur because productive SNAREpins do not form.
To examine this possibility, the kinetic sensitivity of NSF-driven reassembly to each inhibitor was determined. Thus, various inhibitors were added to NSF-driven reassembly at designated times and allowed to proceed for a total time of 1 h (Fig. 9 D). Alternatively, reactions were terminated by fixation or treated with buffer. This mode of enquiry determines the latest stage of the reaction that is sensitive to each inhibitor. Once the reaction acquires resistance to an inhibitor, this implies the target of the inhibitor has completed its function. For example, an inhibitor which blocks only at early time points affects a target required at early stages of the process. In contrast, an inhibitor that blocks at all time points affects a target required at a terminal phase of the process. Thus, a putative sequence of events can be discerned (Ungermann et al., 1998).
Termination by fixation revealed that reassembly proceeds with approximately linear kinetics for the first 45 min (Fig. 9 D). The reaction was completely insensitive to added buffer (Fig. 9 D). GM130 NT, Gtn1-448 (Giantin NH2-terminal 448 aa), 10-fold dilution, and GDI only inhibited the reaction if added within the incipient 15 min (Fig. 9 D). At later time points, these inhibitors were impotent, implying the reaction had moved to a stage beyond the initial formation of Giantin-p115-GM130 tethers. This is consistent with the kinetic sensitivity of cisternal stacking to GM130 NT (Shorter and Warren, 1999). The reaction remained sensitive to chrysin for slightly longer, as appreciable inhibition occurred at 23 min (Fig. 9 D). CC1 inhibited the reaction up to 30 min (Fig. 9 D), implying that p115-catalyzed GOS-28syntaxin-5 pairing is required up to this stage. In contrast, soluble GOS-28 and syntaxin-5 effectively stopped reassembly whenever they were added (Fig. 9 D), implicating the SNAREs in a terminal phase of membrane fusion. Ultimately, these data suggest that p115 consecutively links Golgins then SNAREs, which leads to SNAREpin assembly and membrane fusion during Golgi reassembly.
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Discussion |
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The assembly of cognate topologically correct SNAREpins (Parlati et al., 2000) is likely a highly orchestrated process entailing the interdependent sequential assembly of SNARE monomers or oligomers to form a four-helical bundle (Xu et al., 2000; Chen et al., 2001; Fasshauer et al., 2002). Using a minimal system, we focused on the interaction between the well-defined cognate SNARE pair GOS-28 and syntaxin-5 (Hay et al., 1997, 1998; Zhang and Hong, 2001), a likely step in the assembly of GOS-28syntaxin-5 SNAREpins. These were the two most active SNAREs in Golgi reassembly, emphasizing the physiological significance of their interaction. p115 stimulated GOS-28syntaxin-5 binding, and this effect was abolished by CC1 peptide. In the absence of p115, CC1 had no effect on the amount of GOS-28syntaxin-5 binding, despite its ability to bind to both SNAREs individually. Thus, CC1 was not affecting Golgi reassembly by blocking SNARE pairing but by inhibiting specific and productive p115SNARE interactions.
p115 was not required to maintain endogenous or purified SNARE complexes, since it could be released at the end of the reaction without affecting SNARE complex integrity. Thus, the endpoint of the reaction is not p115 tethering the SNAREs together. The assembly of GOS-28syntaxin-5 complexes by p115 traverses two kinetic phases. The initial phase is sensitive to CC1 and 1 M KCl, whereas the terminal phase is not. One possibility is that p115 stimulates GOS-28syntaxin-5 binding by first linking the SNAREs together and then allowing them to interact directly. In this way, p115 may enhance GOS-28syntaxin-5 binding by stabilizing an early and otherwise labile reaction intermediate. This putative SNARE tethering role for p115 may help explain why very high concentrations of TA and T actually inhibited GOS-28syntaxin-5 complex formation. Such a response is symptomatic of tethering because a large excess of the linking protein will inhibit ternary complex formation by sequestering the proteins to be linked in binary complexes. In addition, it may help explain why CC1 alone did not stimulate GOS-28syntaxin-5 interactions. This is because CC1 is largely monomeric in contrast to p115, TA, and T that as dimers can link the two SNAREs together. Finally, p115 stimulated the rate of assembly of GOS-28syntaxin-5 complexes at concentrations three orders of magnitude lower than the SNAREs, making it highly improbable that p115 was linking the SNAREs together in a final complex. Since p115 enhances the rate of SNAREpin assembly and is not consumed by the process, it is a catalyst in the purest sense.
It may be that p115 helps to relieve a hysteresis associated with SNARE complex assembly (Barrick and Hughson, 2002; Fasshauer et al., 2002). That is, p115 may relieve a kinetic barrier between the disassembled and assembled states (Baker and Agard, 1994). In the absence of p115, the SNAREs may be kinetically trapped and cannot assemble into SNAREpins on a biologically relevant time scale. Thus, we were unable to derive a dissociation constant for the HisGOS-28GSTsyntaxin-5 interaction in the absence of p115, since the reaction was likely not at equilibrium (Fig. 7 C). This was only revealed by the fact that p115 stimulated the amount of GOS-28syntaxin-5 complex that formed in this time frame (Fig. 7 D) and that p115 was acting catalytically, since it was not required to maintain the final GOS-28syntaxin-5 complex. p115 may help reduce the activation energy required for SNAREpin assembly and therefore enhance assembly rates, perhaps by stabilizing an early and otherwise unstable reaction intermediate. A similar function may be performed by complexin in the assembly of syntaxin-1SNAP-25VAMP SNAREpins (Tokumaru et al., 2001).
Both the SNAREpin assembly activity of p115 and Giantin-p115-GM130 tethers are required for NSF-driven reassembly. Agents that selectively disrupt either p115 activity prevent reassembly. Importantly, CC1 had no effect on Giantin-p115-GM130 tether formation, and GM130 NT had no effect on p115-induced SNARE assembly, thus providing a means to discriminate between these two events. The differential kinetic sensitivity of reassembly to various inhibitors resolved these two consecutive functions of p115. Based on these kinetic data, we propose a sequence of events during NSF-driven Golgi reassembly (Fig. 9 E). First, a COPI vesicle is attached to its acceptor membrane via Giantin-p115-GM130 tethers (GM130 NT, Giantin NH2-terminal 448 aa, 10-fold dilution sensitive). This event is coordinated by a Rab-GTPase (GDI sensitive), possibly Rab1, which interacts with p115 and GM130 (Allan et al., 2000; Moyer et al., 2001; Weide et al., 2001) or Rab2, Rab6, or Rab33b, which interact with GM130 (Short et al., 2001; Valsdottir et al., 2001). Second, p115 phosphorylation is required (chrysin sensitive). Thereafter, p115 may tether GOS-28 to syntaxin-5 (CC1 sensitive) and promote their direct tight interaction during SNAREpin assembly that initiates bilayer mixing (soluble SNARE sensitive).
p115 might promote assembly by incorporating GOS-28 as the unitary v-SNARE into the SNAREpin (Fig. 9 E). Alternatively, this may be in the construction of the three-component t-SNARE on the acceptor membrane, which may be the rate-limiting step in SNAREpin assembly (Fasshauer et al., 2002). Since no membranes are present in many of the binding reactions we performed, the configuration of the complexes may be more akin to cis-SNARE complexes. However, we prefer the former model because syntaxin-5 is enriched on mitotic cisternal/tubular remnants and GOS-28 on COPI vesicles (unpublished data; Orci et al., 2000). Whether other factors are required downstream of SNAREs for bilayer mixing remains unresolved (Peters et al., 2001). The next step is to test the predictions of this model in vivo.
When viewed in this light, SNAREs may be seen as simply short tethers that once assembled into SNAREpins catalyze or signal for membrane fusion. Conversely, one may view the Golgins as extended SNAREs that evolved for the specialized function of long range vesicle capture. We envision p115 to play a pivotal role in membrane docking by gradually bringing the COPI vesicle closer to its target via these successive interactions. First, Giantin-p115-GM130 tethers mediate long range COPI vesicle capture. Upon capture, p115 phosphorylation may fasten the Giantin-p115-GM130 tether (Dirac-Svejstrup et al., 2000). This would enable p115 to engage the cognate SNAREs GOS-28 and syntaxin-5, thus catalyzing SNAREpin assembly and membrane fusion. The fact that p115, GM130, and Giantin coimmunoprecipitate with both GOS-28 and syntaxin-5 (Fig. 2, A and B) suggests they may be components of a large tethering complex.
p115 also contributes to the specificity of vesicle transfer, since it did not promote noncognate SNARE interactions and only interacted with SNAREs required for NSF-driven Golgi reassembly. Furthermore, p115 did not link GOS-28 or syntaxin-5 to themselves. It may be that p115 bound to GOS-28 is restricted to a conformation that is only able to bind syntaxin-5 and not another GOS-28 molecule. Therefore, the p115SNARE tether must be asymmetric in nature. A similar situation may exist for Giantinp115GM130 interactions. Thus, binding of one SNARE to p115 transmits or encodes specificity to any subsequent p115SNARE interaction. In this way, p115 may form part of the syntax that ensures that only cognate topologically correct SNAREpins will assemble and so enhances vesicle transfer specificity.
The characteristics of CC1 that are related to the SNARE motif appear to be maintained in the Drosophila and yeast p115 homologues. Thus, this may be a conserved function of p115. Intriguingly, the globular heads of p115 seem to play no direct role in p115-catalyzed SNAREpin assembly or Giantin-p115-GM130 tether formation (Dirac-Svejstrup et al., 2000), despite containing some of the most conserved parts of the molecule (Sapperstein et al., 1995). Corroboratively, His-TA and His-T retrieve the entire complement of p115-interacting SNAREs from Golgi detergent extract, bind to GOS-28 and syntaxin-5 directly, and can promote GOS-28syntaxin-5 complex formation. Furthermore, His-TA can replace p115 in NSF-driven Golgi reassembly, but reassembly only proceeds with 60% efficiency (Dirac-Svejstrup et al., 2000). Thus, the head domains of p115 may regulate or enhance tail function, perhaps by binding to a Rab-GTPase. In contrast, His-T cannot support NSF-driven Golgi reassembly, reinforcing the importance of the Giantin-p115-GM130 tether in this process (unpublished data).
We can now perceive a sophisticated regulatory network that physically couples the successive phenomena of vesicle tethering, docking, and fusion in the mammalian Golgi apparatus. Central to this network is p115, which executes consecutive linkages, joining first the long tethers, Giantin, and GM130, and then the short tethers, GOS-28 and syntaxin-5, as part of cognate SNAREpin assembly. Thus, p115 ineluctably guides COPI vesicles into contact with their correct final destination for cargo delivery.
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Materials and methods |
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p115 CT, GM130 NT (Dirac-Svejstrup et al., 2000), CC1 (aa 637699 of rat p115), CC2 (aa 728765 of rat p115), CC3 (aa 783827 of rat p115), and CC4 (aa 843930 of rat p115) were synthesized as NH2-terminally biotinylated peptides (Lovering et al., 1993). Superdex-75 gel filtration, laser light scattering (Dirac-Svejstrup et al., 2000), and native gel analyses revealed that CC1, CC2, CC3, and CC4 were mostly monomeric (70%) but with dimeric (
20%) and tetrameric (
10%) subpopulations.
Antibodies
Monoclonal antibodies used in this study were against p115 (G. Waters, Princeton University, Princeton, NJ), Giantin (H.P. Hauri, University of Basel, Basel, Switzerland), GOS-28, -SNAP, syntaxin-6, syntaxin-11, GM130, GS15, Vti1a, Vti1b, Bet1p (Transduction Labs), membrin (Stressgen), syntaxin-1 (G. Schiavo, Cancer Research UK), and hexahistidine (Amersham Pharmacia Biotech). Rabbit polyclonals used in this study were against Sly1p (W. Balch, The Scripps Research Institute, La Jolla, CA), syntaxin-6 (F. Wendler, Cancer Research UK), Rab1, Rab6 (Santa Cruz Biotechnology, Inc.), GOS-28, membrin, rSec22p, Bet1p (J. Rothman, Memorial Sloan Kettering Cancer Center, New York, NY), Ykt6p (W. Hong, Institute of Molecular and Cell Biology, Singapore), SNAP-23, SNAP-29, VAMP2 (Synaptic Systems), GST (Sigma-Aldrich), p115 (M. Lowe, University of Manchester, Manchester, UK), Giantin (L. Pelletier, Cancer Research, UK), and syntaxin-5 (A. Price, Cancer Research UK).
Affinity chromatography from Golgi detergent extracts
Biotinylated p115, CC1-4 were coupled to Neutravidin beads (Pierce Chemical Co.) at 10 µM. 20 µg RLGs (Shorter and Warren, 1999) were extracted for 15 min at 4°C with 200 µl Triton X-100 buffer (20 mM Hepes-KOH, pH 7.3, 200 mM KCl, 5 mM magnesium acetate, 0.1 mM DTT, 0.5% Triton X-100). A Triton X-100 extract of rat liver postnuclear supernatant was also used. Extracts were clarified by centrifugation (14,000 rpm, 10 min, 4°C) and incubated for 1 h at 4°C with 10 µl p115, CC1-4, or mock (no protein) beads, His-TA (0.5 µM), or His-T (0.5 µM). His-TA/T were retrieved at the end of the incubation with Ni-NTA agarose. Recovered beads were washed with Triton X-100 buffer and eluted with SDS-PAGE sample buffer. Eluates were fractionated by SDS-PAGE and silver stained or processed for immunoblot.
Immunoprecipitations
AntiGOS-28, antimembrin, or anti-GS15 monoclonal antibodies were covalently coupled to Affigel-10 (Bio-Rad Laboratories), and antisyntaxin-5 polyclonals were covalently coupled to protein Asepharose with DMP (Pierce Chemical Co.). RLGs/MGFs were resuspended at 0.2 mg/ml in 1 M KCl buffer (25 mM Hepes-KOH, pH 7.3, 1 M KCl, 5 mM magnesium acetate, 0.2 M sucrose, 0.1 mM DTT) for 2 min at 4°C and recovered by centrifugation (10,000 rpm, 30 min, 4°C). Membranes (1 mg/ml) were incubated for 30 min at 37°C in the same buffer (except with 60 mM KCl, 2 mM ATP) with NSF (1.3 µM), -SNAP (0.7 µM),
-SNAP (0.7 µM), and an ATP regeneration system (Rabouille et al., 1998). NEM (2.5 mM) was added for 5 min at 4°C followed by DTT (5 mM) for 5 min at 4°C. In some experiments, apyrase (5 U/ml; Sigma-Aldrich) was then added. Membranes (0.2 mg/ml) were extracted in Triton X-100 buffer for 15 min at 4°C and incubated for 30 min at 4°C with 0100 nM p115 plus or minus 10 µM CC1, CC2, 20 µM p115 CT, or GM130 NT. In kinetic experiments, this incubation time was varied from 30 min to 18 h. 10 µl antiGOS-28-Affigel, antimembrin-Affigel, antiGS15-Affigel, or antisyntaxin-5 protein Asepharose was then applied for 30 min at 4°C. Washed beads were eluted with SDS-PAGE sample buffer minus reducing agents. Eluates were separated from beads, supplemented with 3.3% (vol/vol) ß-mercaptoethanol and processed for immunoblot. Giantin was immunoprecipitated as in Dirac-Svejstrup et al. (2000).
SDS-resistant complexes
1 M KCl-extracted RLGs were treated with NSF and SNAPs as above. After NEM treatment, membranes were incubated for 30 min at 37°C plus or minus p115 (100 nM) with or without 10 µM CC1 or CC2. SDS-resistant complex formation was monitored as in Otto et al. (1997).
Golgi reassembly assay
Golgi reassembly was performed as in Shorter and Warren (1999). Treatments were as indicated in the figure legends. Tubule/vesicle density was determined as in Nagahama et al. (1996).
SNAREp115 binding reactions
p115 was incubated for 1 h on ice with SNARE(s) in binding buffer (BB; 20 mM Hepes-KOH, pH 7.3, 150 mM KCl, 2 mM MgCl2, 30 mM histidine, 5% glycerol, 0.5% Triton X-100, 0.1 mM DTT, 0.5 mg/ml STI) as indicated in the figure legends. SNAREs were recovered via their tags, and beads were washed with BB and processed for immunoblot. In some reactions, beads were washed with BB plus either 1 M KCl, 2 M urea, or 20 µM CC1 or CC2. In kinetic experiments, GSTsyntaxin-5 was coupled to glutathione-sepharose before addition to the reaction. GSTsyntaxin-5 beads were incubated with HisGOS-28 and retrieved at various times (2 min to 18 h).
To estimate the apparent Kd of p115SNARE interactions, the amounts of p115 or HisGOS-28 bound (pmol) were determined by densitometry with reference to p115 or HisGOS-28 standard curves (0.120 pmol) using NIH image. Means (n = 3) were fitted with binding isotherms to obtain apparent Kd estimates using Prism 3 software (Graphpad).
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Footnotes |
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Acknowledgments |
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This work was funded by National Institutes of Health grant GM60478-01.
Submitted: 24 December 2001
Revised: 21 February 2002
Accepted: 22 February 2002
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References |
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