Article |
2 Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510
Correspondence to Peter Novick: peter.novick{at}yale.edu
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Abstract |
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Introduction |
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The initial recognition and binding of the two membrane compartments has been termed tethering. Tethering contributes to the specificity of intracellular transport by linking only appropriate membranes to initiate their docking and fusion. In contrast to the Rabs, SM proteins, and SNAREs, the various tethering proteins that act at different stages of transport share little or no sequence homology. Each stage of transport thus appears to rely on a somewhat different molecular mechanism for tethering (Guo et al., 2000; Whyte and Munro, 2002).
The exocyst was first identified as a complex composed of eight different subunits required for exocytosis in yeast (TerBush and Novick, 1995; TerBush et al., 1996). One subunit, Sec3p, stably associates with the plasma membrane at specialized exocytic sites, whereas other subunits become localized only while secretory vesicles are delivered to these sites (Salminen and Novick, 1989; Ayscough et al., 1997; Finger et al., 1998). Therefore, we proposed that the exocyst complex assembles as vesicles arrive at the exocytic sites, and thereby establishes an initial connection between the plasma membrane and the secretory vesicles. Tethering may be regulated by Sec4p, a member of the Rab family of small GTPases. Rab GTPases play important roles in many aspects of membrane trafficking. In their GTP-bound form, Rabs bind to downstream effectors to regulate their function. Sec4p is present on the secretory vesicles and it binds to the Sec15p subunit of the exocyst in its GTP-bound form, and may thereby promote exocyst function (Walch-Solimena et al., 1997; Guo et al., 1999).
Productive membrane tethering is followed by membrane docking; a stronger, less reversible interaction of the two membrane bilayers engaged in fusion. Central to membrane docking is the function of the SNARE proteins (Ungar and Hughson, 2003). For each membrane fusion step at least one SNARE protein is embedded in the membrane of the transport vesicle (v-SNARE) and in the target membrane (t-SNARE). These SNARE proteins engage each other in a highly stable SNARE complex via their coiled-coil domains. As SNARE complexes form, the two membranes are pulled into very close proximity, which is necessary to initiate membrane fusion. In the yeast exocytic reaction, the v-SNARE Snc forms a complex with the two t-SNAREs, Sso and Sec9p (Rossi et al., 1997). In vitro, SNAREs alone are capable of mediating the fusion of liposomes, albeit at a nonphysiologically slow rate (Weber et al., 1998). However, in vivo, SNAREs are likely to work together with other essential factors to promote membrane fusion.
The closest functional link to SNARE-mediated docking and membrane fusion has so far been demonstrated for the SM family of proteins (Toonen and Verhage, 2003). SM family proteins are essential for membrane fusion and, like SNAREs, act downstream of the tethering reaction. Furthermore, they bind to the SNARE protein(s) that act at the corresponding stage of membrane traffic (Carr et al., 1999; Sato et al., 2000; Yamaguchi et al., 2002). Sec1p is essential for yeast exocytosis and binds to the Sso/Sec9p t-SNARE complex (Scott et al., 2004) as well as the fully assembled Snc/Sso/Sec9p SNARE complex (Carr et al., 1999). However, unlike several other SM family proteins, Sec1p does not bind directly to the unassembled syntaxin-like t-SNARE, Sso. The role of Sec1p and other SM family proteins in SNARE function remains incompletely understood; however, stimulation of SNARE-mediated liposome fusion by Sec1p was recently demonstrated (Scott et al., 2004).
In addition to its essential role in vesicle fusion, the yeast exocytic apparatus is also required for the proper targeting of secretory vesicles to specific subdomains of the plasma membrane. As the sites of exocytosis shift with the cell cycle, so too must the localization of the exocytic machinery. Polarized targeting of yeast secretory vesicles is a two-step process. Secretory vesicles bearing activated Sec4p are transported along actin cables toward regions of active surface growth, such as the bud early in the cell cycle or the neck separating the mother and daughter cell late in the cycle (Walch-Solimena et al., 1997; Pruyne et al., 1998). The exocyst proteins then further restrict the tethering of these vesicles to small subdomains of the plasma membrane; for example to the apical bud tip during early bud formation (Finger et al., 1998; Wiederkehr et al., 2003). The localization of Sec4p, most exocyst subunits, and Sec1p to sites of polarized secretion depends on each other in a hierarchical manner that reflects their order of function in the fusion reaction. Thus, inhibition of Sec4p function leads to a loss of polarized localization of secretory vesicles, the exocyst subunit Sec8p and Sec1p, whereas loss of exocyst function blocks proper localization of Sec1p (Ayscough et al., 1997; Walch-Solimena et al., 1997; Finger et al., 1998; Carr et al., 1999; Grote et al., 2000). The exception to this rule is the exocyst subunit Sec3p. Its localization to sites of exocytosis is independent of the function of both the secretory pathway and the actin cytoskeleton, and Sec3p was therefore proposed to act as a spatial landmark, defining exocytic sites (Finger et al., 1998). SEC3 is also unique among the structural genes encoding exocyst subunits in that, under certain conditions, it is not essential for growth and secretion (Wiederkehr et al., 2003). Consistent with its role as a spatial landmark, Sec3p is required for the correct targeting of the exocyst and secretory vesicles during polarized secretion.
Surprisingly, the absence of the Sec3p protein also leads to a defect in the inheritance of the ER into the yeast bud (Wiederkehr et al., 2003). ER tubules form and are delivered into the bud, but fail to be anchored at the tip and ultimately recede back into the mother cell. Although the molecular details of the connection between Sec3p and the ER are still unclear, the results suggest that this role of Sec3p is not directly connected to its role in exocytosis.
Here, we find a close functional connection between Sec1p, Sec4p, and the exocyst in yeast exocytosis. Overproduction of Sec1p or Sec4p not only rescues the partial secretion defect of the sec3 mutant, but also bypasses the need for the otherwise essential exocyst genes, SEC5 and EXO70. The sec3
, sec5
, and exo70
mutants differ with respect to their phenotypes, suggesting subunit-specific roles in vesicle targeting and ER inheritance. Sec1p overproduction increases the levels of SNARE complexes in vivo, which could explain mechanistically how Sec1p is able to promote exocytosis downstream of a partially defective exocyst. We also find that Sec1p binds to the exocyst and may thus establish a functional link between membrane tethering and SNARE-mediated vesicle docking.
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Results |
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Multi-copy plasmids used to overexpress the genes of interest were introduced into a sec3/SEC3 heterozygous diploid strain. The transformants were then sporulated and dissected. After dissection and marker analysis, wild-type and sec3
mutant haploids that retained the URA3 based multi-copy plasmid were struck out for single colonies on synthetic complete (SC)-Ura plates at 25°C. Overproduction of Sec1p or Sec4p clearly suppressed the growth defect of sec3
cells (Fig. 1 A and Table I). However, the suppressed sec3
strains remain temperature-sensitive at 37°C (unpublished data and Table I). As expected, a control strain overproducing Sec3p restored growth at all temperatures. An empty plasmid control (Fig. 1 A) or multi-copy plasmids carrying SEC5 or SEC6, encoding two other subunits of the exocyst, had no effect on sec3
growth. Interestingly, the multi-copy SSO2 or SEC9 plasmids also improved sec3
growth but less strikingly than either SEC1 or SEC4 (Fig. 1 A). These genetic results show that Sec1p, Sec4p and, to a lesser extent SNAREs, can compensate for the absence of Sec3p from the exocyst complex suggesting a functional connection between the exocyst, Sec1p, Sec4p, and SNARE proteins.
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In an earlier study we found several phenotypes of sec3 cells suggesting a defect in polarized secretion. Although Sec4p is concentrated in a very small area at the bud tip of wild-type cells, it is broadly distributed in the buds of sec3
cells. Unlike the elongated wild-type cells, sec3
cells are also round and are unable to extend normal mating projections (Wiederkehr et al., 2003). Therefore, we tested whether overproduction of Sec1p or Sec4p, in addition to stimulating secretion, would also restore the polarity of sec3
cells. The sec3
cells overproducing Sec4p were round and showed defects in mating projection formation similar to sec3
cells (Fig. 2, AC). As Sec4p was overexpressed the Sec4p staining was stronger, but was still distributed broadly in the bud as in sec3
cells (Fig. 2, D and E). In a wild-type background, Sec4p overexpression did not significantly affect the focal localization of Sec4p in the bud, although a fraction of the cells expressing very high levels of Sec4p showed additional cytoplasmic Sec4p staining. Surprisingly, overproduction of Sec1p led to a partial restoration of these sec3
defects. A much larger fraction of sec3
cells overproducing Sec1p were elongated, similar to the morphology of wild-type cells (Fig. 2 A). The sec3
cells carrying the SEC1 multi-copy plasmid were also better at forming mating projections than sec3
cells, although quite a few cells in the culture still showed aberrant, rounded projections (Fig. 2, B and C). Sec4p localization remained partially delocalized in sec3
cells overexpressing Sec1p, but was more restricted at sites of polarized secretion than in the corresponding sec3
strain (Fig. 2, D and E). It was surprising to find that overproduction of Sec1p restored secretion to a similar extent as Sec4p, yet unlike Sec4p also partially restored polarity. The final parameter we examined was the inheritance of cortical ER into the yeast bud. The sec3
cells extend ER tubules into the bud, but the cortical ER fails to be established in the daughter cells. Overproduction of either Sec1p or Sec4p in the sec3
cells failed to completely restore inheritance of the ER into the bud. Although most small buds still lacked cortical ER, in both cases overproduction did improve ER inheritance, as a significant fraction of the cells were able to establish cortical ER by the time the cells were large budded (Fig. 3, A and B). Tubule number, dynamics, and orientation appeared normal (Table II).
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Phenotypic analysis of sec5 and exo70
mutant strains
We used invertase as a marker to measure the secretory defects of these strains. All of the suppressed mutants had only a weak secretory defect, accumulating 2030% of the derepressed invertase in an intracellular pool (Fig. 4 B). Improvement of secretion by Sec1p or Sec4p is a likely explanation for the restoration of viability of the sec5 and exo70
mutants.
Although the exocyst works as a complex in secretion, specific subunits might confer different aspects of exocyst function. Therefore, we tested whether Sec5p and Exo70p, like Sec3p, are required for polarized secretion and ER inheritance. Cells lacking SEC5 have the broad Sec4p distribution and morphology defects observed for the sec3 cells (Fig. 5). The sec5
mutants also have a severe ER inheritance defect, similar to the sec3
strain. At each stage during bud growth, a large fraction of the sec5
cells have little or no cortical ER (Fig. 6, A and B), although the number of tubules is equal or higher than in the wild-type cells and tubule dynamics and orientation appear normal (Table II). In summary, Sec5p appears to be as important for polarized secretion and ER inheritance as Sec3p. With regard to its Sec4p localization and morphology phenotypes, the exo70
strain overproducing Sec4p is similar to the sec3
and the suppressed sec5
mutant strains (Fig. 5). However, ER inheritance is only delayed in this mutant strain, as the defect is restricted to small budded cells (Fig. 6, A and B). By the time larger buds have formed, most exo70
cells have inherited cortical ER. Tubule number, dynamics, and orientation appear normal (Table II). This distinction from the sec3
and sec5
mutants is even more striking in an exo70
mutant overproducing Sec1p, where ER inheritance is close to normal even in small budded cells (Fig. 6, A and B). In contrast, the sec3
and sec5
mutant overproducing Sec1p have very dramatic defects in ER inheritance, suggesting that the function of the Exo70p is less directly linked to ER inheritance than Sec3p and Sec5p. In addition, exo70
cells overproducing Sec1p are mostly elongated, similar to wild-type yeast cells (Fig. 5 A). Furthermore, the mating projections of the exo70
strain overproducing Sec1p are even more pronounced than those of the wild-type cells or wild-type cells overproducing Sec1p. Of all the mutants analyzed here, Sec4p localization was most highly polarized in the exo70
2µSEC1 cells, although compared with wild-type cells, Sec4p was still partially delocalized (Fig. 5, D and E). Sec1p overproduction appears to improve polarized secretion, as in both the sec3
and exo70
mutant backgrounds Sec1p, but not Sec4p, clearly improves the morphology of the cells. The differences observed for the various strains, especially when overproducing Sec1p, show that Exo70p contributes differently to polarized secretion and ER inheritance than do Sec3p or Sec5p (Table III). In summary, Sec5p and Exo70p carry out essential functions in the exocyst, but their function can be bypassed when secretion is stimulated by the overproduction of either Sec1p or Sec4p.
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Prior results from our laboratory showed that Sec1p binds to SNARE complexes (Carr et al., 1999). Therefore, we tested whether the exocyst, possibly via its interaction with Sec1p, can associate with SNARE proteins. However, we could not detect any of the syntaxin-like SNARE Sso in the exocyst immunoprecipitates (Fig. 7 and Fig. 8). These results imply that Sec1p can bind to the exocyst independent of assembled SNARE complexes.
Sec1p increases the levels of SNARE complexes
Members of the SM family bind to SNARE proteins and may regulate SNARE complex assembly, stability, or function (Kosodo et al., 2002; Peng and Gallwitz, 2002; Toonen and Verhage, 2003; Scott et al., 2004). Ongoing membrane traffic is essential for SNARE complex formation, and temperature-sensitive sec4 and exocyst mutants lead to the rapid loss of exocytic SNARE complexes after a shift to the restrictive temperature (Carr et al., 1999; Grote et al., 2000). Therefore, we tested how SNARE complex levels are affected by the absence of Sec3p, Sec5p, or Exo70p, as well as by Sec1p or Sec4p overproduction. For this analysis, steady-state levels of SNARE complexes were measured in the different mutants (Fig. 9). The SNARE complexes were isolated using an antibody against the v-SNARE Snc, and the relative amount of Sso in the immunoprecipitates was determined. Consistent with our earlier results, 1% of Sso was co-isolated with Snc from a wild-type strain (Grote et al., 2000). Upstream inhibition of membrane traffic and the concomitant slowed formation of SNARE complexes leads to a decrease in steady-state levels as Sec18p-mediated disassembly of SNARE complexes continues. As secretory function is only partially affected in the sec3
mutant, SNARE complex levels were only reduced in this strain (Fig. 9 B, lane 6). In a sec3
strain about half as much Sso was isolated in SNARE complexes together with Snc (40% ± 10; n = 5), compared with a wild-type strain. Consistent with its ability to restore secretion, Sec4p overproduction also restored the amount of SNARE complexes that can be isolated from a sec3
mutant background (Fig. 9 B, lane 8).
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In summary, the results with the sec3 strain show that overproduction of Sec4p can increase SNARE complex levels in a strain partially defective for tethering, presumably by restoring the flux of membrane through the pathway. However, Sec1p is more likely to play a direct role in SNARE function. The unexpected finding that SNARE complex levels are actually higher than normal in both mutant and wild-type strains overproducing Sec1p indicates that Sec1p can either increase assembly or slow disassembly of SNARE complexes. The bypass of the exocyst mutants sec3
, sec5
, and exo70
by Sec1p could be due to the increased SNARE complex levels under conditions where tethering is partially inhibited.
The Sec3, Sec5, and Exo70 proteins are apparently less essential for membrane traffic than the other five exocyst subunits. These results and the phenotypic analysis of the mutants described here show that different subunits are preferentially important for different aspects of exocyst function. As Sec1p binds to both the exocyst and SNARE complexes and can increase SNARE complex levels in vivo, we propose that Sec1p creates a functional link between exocyst-mediated vesicle tethering and SNARE complexmediated vesicle docking and fusion.
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Discussion |
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In all the deletion mutants analyzed here, the exocyst is largely disassembled. This assembly defect may reflect a reduced stability of the protein complex in the absence of Sec3p, Sec5p, or Exo70p. Complex assembly and stability may play an important role in exocyst function. Our working model is that the exocyst assembles as secretory vesicles arrive at exocytic sites on the plasma membrane. Formation of the exocyst complex thereby creates a link between secretory vesicles and the plasma membrane. This exocyst-dependent vesicle tether must then keep the vesicle in place until it is docked and committed to fusion. As exocyst assembly is partially defective in these mutants, the connection between the secretory vesicle and the plasma membrane may be unstable. The mutant exocyst complexes may frequently disassemble before the SNARE complex is able to complete vesicle docking. As a result, a significant fraction of the secretory vesicles fail to fuse, leading to the observed accumulation of secretory cargo in the mutant strains.
How does the function of Sec3p, Sec5p, and Exo70p differ from that of the other five subunits, which cannot be deleted? One possibility is that these three subunits may be largely regulatory in nature, whereas the other five subunits fulfill a "core" function of the exocyst that cannot be bypassed. In this regard it is interesting to note that in mammalian cells, Sec5 binds to the Ras family GTPase Ral, and this interaction may regulate exocyst assembly (Moskalenko et al., 2002). This is consistent with our conclusion that Sec5p is important for exocyst stability, although in yeast no upstream regulators of Sec5p function are currently known. Sec3p and Exo70p are known to bind to small GTPases of the Rho family and could therefore also be regulatory subunits of the exocyst (Adamo et al., 1999; Guo et al., 2001). By analogy to the regulation of Sec5 by Ral, Rho GTPases may bind to Sec3p or Exo70p to regulate the assembly or stability of the exocyst complex. Rho proteins may increase exocyst stability preferentially at the tip of small buds and at the mother-bud neck toward the end of the cell cycle to ensure localized cell surface expansion at these specific sites.
In the sec3, sec5
, and exo70
mutant backgrounds the secretion and growth defects can be fully or partially suppressed by overproduction of Sec1p or Sec4p. Therefore, both proteins act as positive regulators of exocytosis. Although Sec4p was already known to promote exocyst function, we have obtained several results that give new insights into the molecular mechanism by which Sec1p acts to facilitate membrane fusion. Previously we demonstrated that Sec1p binds to exocytic SNARE complexes (Carr et al., 1999). Here, we find that overproduction of Sec1p increases SNARE complex levels several fold over those observed in control wild-type cells. Increased SNARE complex levels could be important in exocyst mutants where tethering may be short lived due to defects in complex stability.
In addition to finding that Sec1p can promote an increase of SNARE complex levels, we also find that a fraction of Sec1p can be coprecipitated with the exocyst. These results lead us to speculate that Sec1p forms a link between exocyst-mediated tethering and SNARE complex formation or stabilization. Some SM family proteins have been shown to bind to the corresponding syntaxin-type SNARE (Sato et al., 2000; Yamaguchi et al., 2002; Toonen and Verhage, 2003). These interactions may be important to localize SM function to the correct target membrane. In contrast, yeast Sec1p binds to the assembled t-SNARE complex or the fully assembled SNARE complex (Carr et al., 1999; Scott et al., 2004). We propose that it is the interaction between Sec1p and the exocyst that serves to localize and possibly activate Sec1p at appropriate exocytic sites on the plasma membrane. This proposal is supported by our earlier results showing that the normally polarized distribution of Sec1p is lost when exocyst function is inhibited (Carr et al., 1999; Grote et al., 2000).
In conjunction with its role in membrane tethering, the exocyst is also involved in polarized vesicle targeting. Furthermore, Sec3p is required for ER inheritance, a biological process not directly connected to exocytosis (Wiederkehr et al., 2003). Therefore, we have used a basic phenotypic analysis of the suppressed sec3, sec5
, and exo70
mutants to establish how these three exocyst subunits contribute to the different aspects of exocyst function (Table III). We find that Sec5p, in addition to Sec3p, is required for ER inheritance. By extension, other exocyst subunits may play a role in ER inheritance as well. In contrast, Exo70p appears to have a function that is more restricted to exocytosis. The loss of Sec3p, Sec5p, or Exo70p from the complex leads to changes linked to defects in polarized cell surface expansion. In the sec3
and exo70
mutant, Sec1p (but not Sec4p) overproduction partially restores the polarity defects. The results show that in these two mutant backgrounds, Sec1p may be able to work together with the partially defective exocyst to restore polarized secretion.
In general, our analysis shows that there is no clear correlation between the secretion, vesicle targeting, and ER inheritance defects, but that different subunits are more or less important for different aspects of exocyst function (Table III). Our findings also show that different subunits of the exocyst are more or less essential for exocytosis. Thus, the exocyst should not be regarded as a large molecular structure with one function, but rather as a complex composed of different proteins that contribute differentially to the multiple functions of the complex. Based on our results, we also propose that one important function of the exocyst is to recruit Sec1p to exocytic sites and thereby promote SNARE-mediated vesicle docking and fusion.
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Materials and methods |
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Plasmids and DNA manipulations
SEC1 was cloned as a 3.4-kb HindIIISphI fragment into YEp24 (pNB680). SEC4 was cloned as a 1.4-kb EcoRIBamHI fragment into a multi-copy vector as described previously (Salminen and Novick, 1987). Overexpression of Sec1p and Sec4p was confirmed by Western blotting. Description of the construct used for the in vivo localization of the ER marker HMG1-GFP can be found elsewhere (Wiederkehr et al., 2003). For the integration of the HMG1-GFP marker at the LEU2 locus, the vector pSFN1015 was linearized with BstEII.
For the myc tagging of SEC8 or SEC10, long oligonucleotide primers were designed to remove the stop codon and fuse these genes in frame with multiple myc tags as described previously (Longtine et al., 1998).
Invertase secretion
The invertase secretion and activity assay was performed as described previously (Wiederkehr et al., 2003).
Morphological analysis
The length and width of yeast cells was measured on differential interference contrast (DIC) pictures using the NIH Image 1.62 program. The ratio of length to width is a measure for the elongated shape of the yeast mother cells, and therefore the buds were not included in the measurements. Cells with an axial ratio <1.1 were considered round. The percentage of cells with an axial ratio >1.1 is shown in the graphs. For the shmooing reaction, the density of the yeast cultures was adjusted to equal density (OD600 = 0.25) and 10 µg/ml -factor was added to the Mata yeast strains. DIC pictures were taken 6 h after
-factor addition. The radius of curvature of the shmoo tips was analyzed using the OpenLab 3.1.4 program.
Fluorescence microscopy
Cells were examined on an Axioplan2 microscope (Carl Zeiss MicroImaging, Inc.) with a 63x Plan Neofluor apochromatic oil-immersion objective lens (N.A. 1.4). Pictures and videos of cells were taken with a cooled CCD camera (ORCA ER; Hamamatsu Corporation) at RT (2123°C). The images were analyzed with OpenLab software. To visualize the ER, yeast strains expressing the marker Hmg1-GFP were grown to an OD600 of 0.20.3. The cell suspension was mixed with an equal volume of 0.6% NuSieve GTG low melting temperature agarose (FMC BioProducts) and mounted on a glass slide. Hmg1-GFP was visualized using the FITC filter set. For quantitation of the ER inheritance defect, cells at different stages of bud growth were grouped together. The three different categories were defined as described in the legend for Fig. 3 (A and B). For the purpose of quantitation of cortical ER inheritance, we only considered the ER apposed to the plasma membrane in the bud and not cytoplasmic tubules or diffuse fluorescence in the bud. Sec4p immunofluorescence labeling and quantification was performed as described in Wiederkehr et al. (2003).
Exocyst isolation
The exocyst was isolated by immunoprecipitation from yeast strains expressing myc-tagged Sec8p or Sec10p from their endogenous promoter. After growth in SC medium, 50 OD600 units of cells were harvested and washed in 50 ml ice-cold washing buffer (10 mM Tris, pH 7.4, and 10 mM NaN3). The washed cells were then resuspended in 5 ml spheroblasting buffer (50 mM NaPO4, pH 7.4, 1.4 M sorbitol, 35 mM ß-mercaptoethanol, 10 mM NaN3, and 100 µg/ml Zymolyase [Seikagaku Corporation]) and incubated at 30°C for 30 min. The cell suspension was then layered on top of a 5-ml 1.5 M sorbitol solution (50 mM NaPO4, pH 7.4, 1.5 M sorbitol, 10 mM NaN3, and protease inhibitors). The cells were spun through the sorbitol cushion for 10 min at 1,000 g. The pellets were resuspended in cold lysis buffer (20 mM Pipes, pH 6.8, 100 mM NaCl, 1 mM EDTA, 1% Nonidet-P40 (IGEPAL CA 630), 1 mM DTT, and protease inhibitors). All further steps were performed on ice or at 4°C. The lysates were cleared by centrifugation for 10 min at 10,000 g and were adjusted for equal protein concentrations (4 mg/ml). 1 ml of lysate was used per immunoprecipitation. After adding 3 µl of an anti-myc mAb (Babco), the lysates were incubated for 3 h at 4°C. Finally, protein ASepharose (45 µl 50% slurry; Sigma-Aldrich) was added, followed by another hour of incubation at 4°C. After washing the beads, the bound proteins were separated on 8% SDS-PAGE protein gels.
SNARE complex isolation
SNARE complexes were isolated by immunoprecipitation as described previously (Carr et al., 1999). Where mentioned, protease inhibitors were used at the following concentrations: 6 µg/ml antipain, 2 µg/ml aprotinin, 2 µg/ml chymostatin, 8 µg/ml leupeptin, 12 µg/ml pepstatin A, and 1 mM PMSF (Sigma-Aldrich).
Online supplemental material
Videos show ER tubule dynamics in the indicated mutant strains. For all videos, Hmg1-GFP was used as marker to follow ER dynamics. Videos were captured at one frame per 3 s and are played at 10 frames/s. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200408001/DC1.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health to P. Novick (GM35370) and S. Ferro-Novick (CA 46128). A. Wiederkehr was supported by a fellowship from the Swiss National Science Foundation.
Submitted: 2 August 2004
Accepted: 22 October 2004
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