Article |
Address correspondence to Daniel J. Klionsky, University of Michigan, Life Sciences Institute, Ann Arbor, MI 48109-2216. Tel.: (734) 615-6556. Fax: (734) 647-0884. email: klionsky{at}umich.edu
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Abstract |
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Key Words: class C Vps; HOPS; membrane fusion; Rab; SNAREs
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Introduction |
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In the budding yeast Saccharomyces cerevisiae, the analysis of homotypic vacuole fusion has expanded our understanding of membrane fusion events. A biochemical dissection of this process has identified four distinct stages (Fig. 1; for reviews see Wickner and Haas, 2000; Wickner, 2002). During the first step, priming, Sec18 (yeast NSF) utilizes ATP to drive the release of Sec17 (-SNAP) from a cis-SNARE complex and the transfer of a novel chaperone, LMA1, from Sec18 to the t-SNARE Vam3. Priming also results in the disassembly of the cis-SNARE complex and release of the C-Vps/HOPS complex from the SNAREs. The primed vacuoles then come into contact in the tethering/docking steps. Docking begins with tethering, which requires the interaction of the C-Vps/HOPS complex with the rab GTPase Ypt7. Stable docking requires a membrane electrochemical potential, Rho GTPases, phosphoinositides, and trans-SNARE pairing. The precise events that occur during fusion are still an open question, however, the release of Ca2+ at the conclusion of docking causes calmodulin binding to the vacuole membrane and the interaction of V0-V0 domains of the vacuolar ATPase in trans. Protein phosphatase 1 activity and release of LMA1 and Vac8 are also involved at a very late stage of fusion. A recent report analyzed vertex membranes at the rim of the connected vacuoles where membrane fusion and fusion pore expansion occurs. Proteins needed for docking and fusion are not evenly distributed on the vacuole but accumulate at the vertices.
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Results |
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One strategy to demonstrate that a protein directly participates in homotypic vacuole fusion is to inhibit the in vitro fusion reaction by the addition of affinity-purified antiserum against the protein of interest. However, the success of this approach depends on the affinity of the particular antibody. We found that affinity-purified antiserum against Ccz1 or Mon1 was not able to inhibit the reaction (unpublished data). Accordingly, we adopted an alternative approach. We generated strains with protein A (PA) fusion to Ccz1 and Mon1 to take advantage of the specific affinity between PA and the Fc region of IgG. Fig. 3 A shows a Western blot against vacuole proteins from two fused partner strains, the pho8 (DKY6281, DK) and pep4
(BJ3505, BJ) strains, containing integrated copies of Ccz1-PA and Mon1-PA. The COOH-terminal PA fusion forms of Ccz1 and Mon1 are stable proteins, are both associated with the vacuole, and are maintained at similar levels to their corresponding wild-type proteins. PA tagging did not affect Ccz1 and Mon1 function because all of the tagged strains exhibited normal vacuole morphology in vivo (unpublished data). Furthermore, the purified vacuoles showed fusion activities that were comparable to wild-type vacuoles (Fig. 3 B). As a control, we treated the vacuoles with an antiserum against a protein that is not localized to the vacuole and does not play a role in fusion. We found that treating with this antiserum did not affect wild-type vacuole fusion activity in vitro. However, the fusion activity decreased to 80% when Ccz1-PA was present on both of the fusion partners. Furthermore, when both vacuole partners harboring Mon1-PA were tested, the fusion activity was reduced to 26%. This reduction is similar to inhibition of vacuole fusion with affinity-purified antiserum to Sec18 (Kato and Wickner, 2003). Fusion activities in the presence of antiserum were substantially decreased if either one of the vacuoles contained Mon1-PA. These data suggest that at least Mon1 has a direct role in homotypic vacuole fusion. Vacuoles with Ccz1-PA treated with antibody displayed a smaller defect in fusion (20%). This reduction in fusion activity may be indirect and reflect the requirement for Ccz1 in vacuolar recruitment of Mon1. Alternatively, the difference in inhibition may reflect steric differences resulting from Fc binding to the particular PA construct.
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According to the working model for homotypic vacuole fusion, the major event after Sec18-dependent priming is the disassembly of the cis-SNARE complex that allows the formation of SNARE pairs in trans through the function of a Rab protein (Wickner and Haas, 2000). Ypt7 interacts with the C-Vps/HOPS complex comprised of Vps11, Vps16, Vps18, Vps33, Vps39, and Vps41 and is required for the vacuoles to dock (Sato et al., 2000; Wurmser et al., 2000). A critical event during the docking stage is the interaction between the C-Vps complex and the unassembled t-SNARE Vam3 to achieve trans-SNARE pairing. To monitor this event, we performed a Vam3 coimmunoprecipitation using a strain with HA-tagged Vps18 to see if the unassembled Vam3 on the mon1 and ccz1
vacuoles was able to interact with the C-Vps/HOPS complex. Vps18-HA is present on the purified vacuoles of the wild-type, ccz1
, and mon1
strains (Fig. 4 C). Vam3 on wild-type vacuoles coprecipitated Vps18-HA and Sec17 in the presence and absence of ATP (Fig. 4 C). The Vps18-HA levels appeared to increase slightly after 15 min in the presence of ATP, whereas Sec17 levels were reduced following ATP-dependent priming. In contrast, Vam3 did not coprecipitate Vps18-HA in either the mon1
or ccz1
strain. In addition, we performed the same reaction conditions using anti-HA antibody for the immunoprecipitation to pull-down proteins that are in a complex with Vps18-HA. We found that the SNARE proteins including Vam3 and Vti1 are present in the immune complex with Vps18-HA on the wild-type vacuoles, whereas Sec17, Nyv1, and Vam7 were absent from the same immune complex (Fig. 4 C; data not shown). These data support the previous findings that the C-Vps/HOPS proteins interact with the "unpaired" Vam3 that should be observed only when Sec17 is released from the cis-SNARE complex. With both ccz1
and mon1
vacuoles, we did not detect any Vam3 or Vti1 in the immune complex with Vps18-HA, although these proteins were present at levels similar to those of wild-type vacuoles. Therefore, we conclude that the tethering/docking stage was completely blocked when the Ccz1Mon1 complex was omitted. Because the proposed role of the C-Vps/HOPS complex is to mediate SNARE pairing, we next checked whether SNARE pairs formed on these two mutant vacuoles. To investigate SNARE pairing, we set up two identical reactions in the absence or presence of ATP for 15 min followed by immunoprecipitation using anti-Vam3 antiserum. In the absence of ATP, cis-SNARE complexes were identified from the wild-type vacuole based on the coprecipitation of Sec17, Nyv1, Vam7, and Vti1 with Vam3 (Fig. 4 D). The cis-SNARE complex is disassembled in the presence of ATP and reduced levels of these proteins were coprecipitated with Vam3. We could not detect Nyv1 and Vam7 being pulled down by anti-Vam3 from the ccz1
and mon1
vacuoles even though all of these proteins were present (Figs. 2 B and 4 D). Furthermore, we found that Sec17 was also absent from the Vam3 complex even though Sec18-dependent Sec17 release was functional on the ccz1
and mon1
vacuoles (Fig. 4 B). We found that Vti1 was the only SNARE that was in a complex with Vam3 on the ccz1
and mon1
vacuoles, and the disassembly of this complex appeared to be similar to that on the wild-type vacuoles. We conclude that the absence of Ccz1 or Mon1 affects vacuolar SNARE pair association. Together, we propose that the Ccz1Mon1 complex is required for vacuole fusion by regulating the coordinated priming and tethering/docking stages.
The Ccz1Mon1complex on the vacuole is assembled as part of the cis-SNARE complex
Many proteins known to be needed for homotypic vacuole fusion are found on the vacuole as a cis-SNARE complex, which represents the end product of a previous round of fusion (Wickner and Haas, 2000; Wickner, 2002). We have shown that both Ccz1 and Mon1 are localized on the purified vacuoles and they appear to tightly associate with each other (Fig. 2 D). Next, we examined if Ccz1 and Mon1 are assembled inside the vacuolar cis-SNARE complex. We performed a sucrose density gradient analysis by loading detergent-solublized vacuoles on the top of the gradient. We found that identified fusion components such as Sec18, Sec17, and SNAREs copeaked in fractions 7 to 9 (Fig. 5 A). The majority of Ypt7 appeared to float in the top fraction of this gradient; however, a minor peak of Ypt7 was also present in fractions 7 to 9. Accordingly, we refer to this peak as the previously identified cis-SNARE complex. Furthermore, we found that the C-Vps protein Vps18-HA colocalized with Ccz1 and Mon1 at this cis-SNARE complex peak (Fig. 5 A). The finding that these proteins were assembled into the end product of fusion, the cis-SNARE complex, supports the hypothesis that the Ccz1Mon1 complex directly participates in fusion.
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Regulation of the Ccz1Mon1 complex through the C-Vps/HOPS complex
The interaction between Vps18-HA and unpaired Vam3 was impaired when Ccz1 or Mon1 was not present on the vacuole (Fig. 4 C). Ypt7 coprecipitates with Ccz1-HA, and Ypt7 overexpression rescues the ccz1 vacuole fragmentation phenotype (Kucharczyk et al., 2000, 2001). Therefore, we tested if the Ccz1Mon1 complex participates in the Ypt7-dependent tethering/docking stage through its interaction with the C-Vps/HOPS complex. A report by Wang et al. has shown that SNAREs, Ypt7, and the C-Vps/HOPS complex were enriched at vacuolevacuole junctions (Wang et al., 2002b). We have previously published that Ccz1-GFP and Mon1-GFP localize to punctate structures on the vacuole membrane similar to C-Vps/HOPS complex proteins (Wang et al., 2002a). Accordingly, we decided to test whether the Ccz1-GFP signal was enriched at the vacuolevacuole junctions. We used Atg11-GFP as a control. Atg11 is a protein required for the Cvt pathway and it has been shown to localize at a structure tightly associated with the vacuole (Kim et al., 2001), yet has no known role in homotypic vacuole fusion. We stained cells with the dye FM 4-64 to allow a clear observation of vacuole morphology. From multiple fields we counted 250 "docked" vacuoles and quantified the spatial localization of GFP as shown in Fig. 6 A. We found that 96% of the Ccz1-GFP signal was associated with the docked vacuole, whereas Atg11-GFP only displayed 26% association. Therefore, we concluded that the Ccz1-GFP signals were enriched at the vacuolevacuole junctions reminiscent of the results seen with the HOPS proteins. To see if the Ccz1Mon1 complex colocalizes with the C-Vps/HOPS complex, we generated a strain expressing both Ccz1-YFP and Vps18-CFP under their endogenous promoters. The Vps18-CFP signal was detected on the vacuole membrane as well as on punctate dots adjacent to the vacuoles. The dot structures colocalized with the Ccz1-YFP signal (Fig. 6 B). Using an OptiPrepTM gradient as described in Materials and methods, we tested whether Ccz1 and Mon1 colocalized with the C-Vps/HOPS complex proteins. Consistent with the Vps18-CFP localization in vivo, we detected a peak of Vps18-HA and Vps39-HA in the top few fractions that colocalized with most of the Vam3 and Ypt7, indicating vacuolar localization (Fig. 6 C). In addition to the top vacuole peak, the majority of the Vps18-HA and Vps39-HA (as well as all other C-Vps/HOPS members; data not shown) cofractionated with Ccz1 and Mon1 to a dense part of the gradient, suggesting that they colocalize to a higher density compartment. Together, the fluorescence and fractionation data suggest that the Ccz1Mon1 complex colocalizes with a portion of the HOPS complex that is located in part at vacuolevacuole junctions.
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We have shown that Ccz1 and Mon1 are found on the vacuole together. To see how this binding is regulated, we looked at several vacuole fusion deficient mutants, including the individual C-Vps/HOPS null strains (Fig. 6 F). We found that the absence of C-Vps/HOPS components did not affect the level of Mon1 or Ccz1 under steady-state conditions. We then examined purified vacuoles from these strains. Although pbi2 and vam7
vacuoles, which lack components needed for fusion, accumulated wild-type levels of Ccz1 and Mon1, the vacuoles from the C-Vps/HOPS mutants exhibited two distinct profiles in terms of Mon1 and Ccz1 binding (Fig. 6 F, vacuole). Vacuoles from the vps11
, vps16
, and vps18
strains were almost completely devoid of Mon1 and Ccz1. In contrast, vacuoles from the vps39
and vps41
strains accumulated at least three times more Mon1 and Ccz1 than did wild-type vacuoles. These results indicated that the C-Vps/HOPS complex regulates the association of the Ccz1Mon1 complex with the vacuole. Apparently one subcomplex of C-Vps/HOPS is required for stabilizing vacuolar Ccz1Mon1, whereas the other subcomplex is involved in down-regulation of this complex from the vacuole. Furthermore, this result also supports our finding that one of the functions of the Ccz1Mon1 complex is mediated by its interaction with Ypt7 and the C-Vps/HOPS complex and that these proteins are required for vacuole fusion at the tethering/docking stage.
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Discussion |
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Ccz1 and Mon1 are required for C-Vps/HOPS to bind a Vam3-Vti1 complex
Various models have been proposed to explain the function of the different proteins that are required for the fusion process. According to Rothman's tetrameric vacuole SNARE model, four helices, three "Q"-SNAREs, and one "R"-SNARE are required to form the functional trans-SNARE complex (McNew et al., 2000). However, it was recently proposed that a pentameric SNARE complex is required for yeast homotypic vacuole fusion (Ungermann et al., 1999). The cis-SNARE complex that results from fusion is thought to be thermodynamically stable (Fasshauer et al., 1997) and must be dissociated in an energy-requiring process. However, we observed that the cis-SNARE complex was not properly assembled on the ccz1 and mon1
vacuoles (Fig. 3 D). We found that, in the ccz1
and mon1
strains, the syntaxin heavy chain Vam3 formed a complex only with one of the light chains, Vti1. The absence of proper cis-SNARE assembly might account for the in vitro homotypic vacuole fusion defect observed in the two mutants. Analyses of priming and docking by morphological assay suggested that the primary defect of the ccz1
and mon1
vacuoles is in the tethering/docking stage of the reaction (Fig. 4). In the priming step of homotypic fusion, Sec18-dependent hydrolysis of ATP releases Sec17 and disassembles Nyv1 and Vam7 from the cis-SNARE complex (Ungermann and Wickner, 1998; Ungermann et al., 1999). A second event may be needed to dissociate Vti1 from Vam3 (Boeddinghaus et al., 2002), or these two proteins may remain assembled. Alternatively, Vti1 might have a high affinity for Vam3 resulting in rapid reassociation with unpaired Vam3 after cis-SNARE complex dissociation. We found that Sec17 was quickly released from the ccz1
and mon1
vacuole membranes as an early event of priming (Fig. 4 B). However, we could not observe Sec17 in a complex with Vam3 in the mutant cells (Fig. 4, C and D). The binding of Sec17 to the vacuole membrane was also observed with strains lacking SNAREs (Ungermann and Wickner, 1998; Ungermann et al., 1998, 1999), suggesting that Sec17 binding and release is accomplished through the action of an as yet unidentified non-SNARE component. Thus, we suspect the Sec17 binding and release on the mutant vacuoles was accomplished through this unidentified membrane receptor(s). However, Sec17 release from the ccz1
and mon1
mutant vacuoles may not reflect a fully functional priming step.
The C-Vps/HOPS complex interacts with Ypt7 and may also function as an Ypt7 effector to achieve docking between two membranes (Sato et al., 2000; Wurmser et al., 2000). The completion of the docking stage joins the t-SNAREs, offering three "Q" -helices, and the v-SNARE, providing one "R"
-helix based on Rothman's model, for trans-SNARE pairing. The fusion process joins the two membranes resulting in the cis-SNARE complex (Fig. 1). None of the C-Vps/HOPS complex proteins interacted with Vam3 in the ccz1
or mon1
strains, suggesting that the tethering/docking stage was impaired (Fig. 4 C). This result fits with our morphological data that indicated that the defect in the ccz1
and mon1
strains prevented the vacuoles from tethering/docking (Fig. 4 A; Table I). We found that Vps18-HA coprecipitated both Vam3 and Vti1, leading us to propose that the C-Vps/HOPS complex interacts with a Vam3-Vti1 complex rather than unpaired Vam3 (Fig. 4 C). Therefore, we propose that the Ccz1Mon1 complex is required for regulating the SNARE complex during the coordinated priming and docking stages of fusion, and particularly at the stage of tethering/docking.
The C-Vps/HOPS complex and the Ccz1Mon1 complex are key players in the Ypt7-dependent tethering/docking stage
The C-Vps/HOPS complex is required for regulating the SNARE complex during the docking stage of fusion. The finding that the cis-SNARE complex was not properly assembled on the ccz1 and mon1
vacuoles along with the previous report revealing the association between Ccz1 and Ypt7 (Kucharczyk et al., 2001) led us to check if the Ccz1Mon1 complex is part of the C-Vps/HOPS complex. We found that the two protein complexes colocalize, however, based on PA affinity purification they are not simply assembled into one higher order protein complex (Fig. 6 D). The Ccz1Mon1 complex and the C-Vps/HOPS proteins appeared to interact weakly or transiently based on affinity isolation and yeast two-hybrid analyses (Fig. 6, D and E). Interestingly, the C-Vps/HOPS complex may be involved in two levels of regulation of the Ccz1Mon1 complex; vacuolar localization of the Ccz1Mon1 complex was lost from strains that delete one set of the C-Vps/HOPS complex (Vps11, Vps16, and Vps18), however, removing two other C-Vps/HOPS proteins (Vps39 and Vps41) in turn enriched the association of Ccz1 and Mon1 with the vacuole. Because the C-Vps/HOPS complex functions as the Ypt7 effector as well as exchange factor, it is possible that the differences in binding are due to the different Ypt7 stage that is regulated. Further data are required to resolve this speculation.
Our data and previous findings have predicted that two functionally separated complexes are required for the formation of the C-Vps complex (Wurmser et al., 2000). To further investigate C-Vps complex assembly, we checked the two-hybrid interaction of the C-Vps components in several combinations. We identified three strong two-hybrid interactions (unpublished data). In addition to the published two-hybrid interaction between binding domain (BD)-Vps11 and AD-Vps39 (Wurmser et al., 2000), we found that BD-Vps18 and AD-Vps11, and BD-Vps18 and AD-Vps39, also showed good growth on the selective plate. We then tested these three interactions in strains carrying deletions of the individual C-Vps components. We found that the interaction between BD-Vps18 and AD-Vps11 remained unaffected when other C-Vps components were deleted, indicating that the two proteins directly interact. Interestingly, the interaction between BD-Vps18 and AD-Vps39 was impaired in the vps11, vps16, and vps33 deletion backgrounds but not in the vps18, vps39, and vps41 deletion strain. This result supports the two subcomplex model for the assembly of the C-Vps complex. Specifically, Vps11, Vps16, Vps18, and Vps33 appear to be assembled first before the other subcomplex of Vps39-Vps41 can be recruited. In contrast, the interaction between BD-Vps11 and AD-Vps39 remained unaffected in the mutant backgrounds. This result supports the previously published data for a direct association between Vps11 and Vps39 that is required for C-Vps protein assembly. Together, these results suggest that after the Vps11-16-18-33 complex has formed, it recruits the Vps39-Vps41 complex through direct binding between Vps11 and Vps39 to form a C-Vps/HOPS complex.
Based on these data, we propose a model for how tethering and docking could be regulated. We suggest that the subC-Vps complex (Vps11, Vps16, Vps18, and Vps33) is required to recruit two complexes, one composed of Vps39-Vps41, and the other of Ccz1Mon1. The recruitment of these two complexes appears to be two independent events, and specifically, both may not require Ypt7. It has been suggested that binding of Vps39-Vps41 to the rest of the C-Vps components is membrane dependent, although the association with the Vps39-Vps41 complex may not necessarily occur on the vacuole. However, we propose that the recruitment of the Ccz1Mon1 complex is regulated on the vacuole membrane. Specifically, we propose that recruitment of Ccz1Mon1 is regulated by other fusion components at a specific stage during the fusion process; one possibility is that the Ccz1Mon1 proteins might be recruited through the C-Vps interaction with unpaired Vam3. It has been proposed by Sato et al. (2000) that the C-Vps complex functions in promoting the assembly of Vam3 into trans-SNARE complexes. In particular, they suggest that the C-Vps complex may maintain Vam3 in an unpaired stage by preventing nonproductive cis-SNARE associations. However, we found that Vam3 and Vti1 are affinity isolated together with Vps18-HA. We have demonstrated that the Ccz1Mon1 complex is required for the interaction between the C-Vps proteins and Vam3. Accordingly, the recruitment of the Ccz1Mon1 complex will stabilize the interaction between C-Vps and Vam3. Alternatively, the association of Vti1 with Vam3 in the ccz1 and mon1
background may reflect a nonproductive cis-SNARE association. Furthermore, it has been reported that Ccz1 preferentially binds to the nucleotide-free form of Ypt7, similar to the result seen with the two-hybrid interaction between Ypt7 and Vps39 (Wurmser et al., 2000; Kucharczyk et al., 2001). This suggests that the Ccz1Mon1 complex may interact with Ypt7 and bring Ypt7 to its guanine nucleotide exchange factor, Vps39. It is still not known, however, if the Ccz1Mon1 complex is required as part of the Ypt7 GDP/GTP cycle. Overexpression of Ypt7 or expression of an Ypt7 mutant that cannot bind nucleotide suppresses the Ccz1 deletion phenotype (Kucharczyk et al., 2001). However, the Ccz1Mon1 complex might have another role after GDP/GTP exchange. Expression of Ypt7Q68L, a mutant form of Ypt7 that remains in the GTP-bound state and that has a wild-type vacuole phenotype, could not prevent vacuole fragmentation in strains deleted for CCZ1 or MON1 (unpublished data). Hence, we suggest that the Ccz1Mon1 complex may function in part as the Ypt7 downstream effector during the tethering/docking stage. Our data suggest that the Ccz1Mon1 complex is down-regulated from the vacuole by Vps39-Vps41. The particular timing that is needed for this down-regulation may require activation of the Vps39-Vps41 complex. It is possible that down-regulation occurs before or during trans-SNARE pairing, although further data are required to carefully dissect this part of the fusion process.
Overall, we propose that the Ccz1Mon1 complex is required for regulating the SNARE complex during the coordinated priming and docking stages of fusion, and particularly at the stage of tethering/docking. Accordingly, the absence of Ccz1 or Mon1 prevents subsequent fusion. Further studies of the C-Vps/HOPS proteins and their interaction with the Ccz1Mon1 complex and Ypt7 and with SNARE proteins will be necessary to understand how the priming and docking steps are regulated to achieve functional SNARE association.
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Materials and methods |
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Vacuole preparation and in vitro vacuole fusion assay
Preparation of cytosol and vacuoles was described previously (Haas, 1995). To compare the in vitro fusion activity, vacuoles were prepared separately from the pep4 and pho8
backgrounds. The standard 30-µl reaction contained 3-µg vacuoles prepared from the pep4
(BJ3505) and pho8
(DKY6281) strains in physiological salts (Haas, 1995). To examine deletion strain vacuole fusion, every set of experiments was assayed with duplicate samples, and the data from three independent experiments were averaged. To examine PA-Ccz1 and/or PA-Mon1 vacuole fusion activity, every set of experiments was assayed and averaged from six independent experiments with or without the addition of 0.5 µl of antiserum against Atg2. To examine Sec17 release, 90-µl standard reactions without the addition of cytosol were aliquoted for each time point. Reactions were stopped by transferring to ice for 12 min and spun at 10,000 g for 4 min. Supernatant and pellet fractions were TCA precipitated separately followed by Western blot analysis. A similar approach was used for examining release of Mon1. The microscopy docking/fusion assay was performed as described (Wang et al., 2001b).
OptiPrepTM density gradient and sucrose velocity gradient analyses
To examine the localization of Ccz1Mon1 and the C-Vps complex, we performed OptiPrepTM density gradient analysis using a modification of a previously described procedure (Wang et al., 2002a). The gradient was 1055% OptiPrepTM and centrifugation was performed for 12 h.
To examine the vacuolar SNARE complex, we performed sucrose gradient analysis. Purified vacuoles (0.5 mg) were pelleted and resuspended in 300-µl PS200 buffer containing 1% Triton X-100. Vacuoles were solublized on ice for 20 min and loaded on the top of a 12-ml 1060% linear sucrose gradient (PS200). The gradient was centrifuged at 4°C for 14 h. A total of 13 fractions were collected from the top of the gradient and TCA precipitated followed by Western blot analysis.
Immunoprecipitation and PA affinity purification
To analyze the association with the C-Vps and SNARE complexes, a 750-µl standard fusion reaction was set up in the presence of an ATP regeneration system (ARS; +ATP) or the absence of ARS with the addition of 30 U/ml apyrase (-ATP). All reactions were incubated at 27°C for 15 min and chilled on ice for 2 min. Vacuoles were reisolated by spinning at 10,000 g for 5 min, followed by coimmunoprecipitation as described (Wang et al., 2002a). The protocol for coimmunoprecipitation with Vps18-HA and Vam3 was as described previously (Wang et al., 2002a). The PA affinity purification procedure using IgG sepharose was modified from Sato et al. (2000). Spheroplasts were osmotically lysed on ice in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1x complete protease inhibitors (Roche Molecular Biochemicals), and 1 mM PMSF). After 20 min solubilization on ice, total cell lysates were centrifuged at 10,000 rpm for 5 min at 4°C. To the resulting supernatant, 25 µl of IgG-Sepharose 6 Fast Flow was added and incubated at 4°C for 2 h. Sepharose beads were washed eight times with lysis buffer. Bound proteins were eluted in MURB (Wang et al., 2002a), followed by SDS-PAGE and Western blot analysis.
Microscopy
All strains used for microscopy were grown in synthetic minimal medium with dextrose (SMD) to mid-log phase. In vivo FM 4-64 staining and microscopy analyses were performed as described previously (Wang et al., 2002a).
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Acknowledgments |
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This work was supported by Public Health Service grants GM53396 and GM50403 from the National Institutes of Health to D.J. Klionsky and L.S. Weisman, respectively.
Submitted: 13 August 2003
Accepted: 23 October 2003
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