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. 2024 Jan 29;35(3):br8. doi: 10.1091/mbc.E23-08-0311

Asymmetric tethering by exocyst in vitro requires a Rab GTPase, an R-SNARE and a Sac1-sensitive phosphoinositide lipid

Guendalina Rossi a, Gabrielle C Puller a,b, Patrick Brennwald a,b,*
Editor: Mary Munsonc
PMCID: PMC10916868  PMID: 38198574

Abstract

Tethering factors play a critical role in deciphering the correct combination of vesicle and target membrane, before SNARE complex formation and membrane fusion. The exocyst plays a central role in tethering post-Golgi vesicles to the plasma membrane, although the mechanism by which this occurs is poorly understood. We recently established an assay for measuring exocyst-mediated vesicle tethering in vitro and we have adapted this assay to examine the ability of exocyst to tether vesicles in an asymmetric manner. We demonstrate that exocyst differs from another post-Golgi vesicle tethering protein, Sro7, in that it is fully capable of tethering vesicles with a functional Rab GTPase, Sec4, to vesicles lacking a functional Rab GTPase. Using this assay, we show that exocyst requires both the Rab and R-SNARE, Snc1, to be present on the same membrane surface. Using Sac1 phosphatase treatment, we demonstrate a likely role for phosphoinositides on the opposing Rab-deficient membrane. This suggests a specific model for exocyst orientation and its points of contact between membranes during heterotypic tethering of post-Golgi vesicles with the plasma membrane.

  • Although the structure of the exocyst tethering complex has been determined, how this structure acts to tether two distinct membranes together is unknown.

  • The authors demonstrate that the exocyst complex is able to tether membranes in an asymmetric fashion. The asymmetry involves the ability to tether membranes containing both Rab GTPase and v-SNARE on the same membrane to vesicles containing a phosphoinositide lipid.

  • This work suggests a specific model for the exocyst orientation and points of contact between the two membranes during heterotypic tethering of post-Golgi vesicles with the plasma membrane.

INTRODUCTION

Tethering of transport vesicles to the appropriate membrane at the correct time and space is a critical component of generating and maintaining organelle and plasma membrane identity (Yu and Hughson, 2010). An important model organism for understanding the process of exocytic transport is the budding yeast, Saccharomyces cerevisiae. In particular, it has been shown that vesicle tethering of post-Golgi vesicles at the plasma membrane requires the Rab GTPase Sec4 and two direct effectors: the multisubunit exocyst complex and the tomosyn/Lgl homologues Sro7/Sro77 (Guo et al., 1999b; Grosshans et al., 2006, Watson et al., 2015).

Exocyst is a member of the CATCHR family of multisubunit tethering complexes and consists of eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (Terbush et al.,1996, Stanton and Hughson, 2023). The cryo-EM structure of the yeast exocyst has shown that all the subunits have a similar structural organization and interact with one another to form an elongated structure 32 nm long and 13 nm wide (Mei et al., 2018). Initial contact between the exocyst and the vesicle surface is thought to involve the direct interaction between Sec4-GTP and the C-terminus of the Sec15 subunit of the exocyst which is present at one end of the elongated structure (Guo et al., 1999b). The exocyst also interacts with the R-SNARE Snc1/2 through its Sec6 component (Shen et al., 2013) at a site which is located at the opposite end of the complex from Sec15. The dual recognition of both Sec4 and Snc1/2 places additional specificity onto post-Golgi vesicle tethering by the exocyst. An important component of exocyst tethering with the plasma membrane is the phosphoinositide PI(4,5)P2 which has been shown to bind to both the Sec3 and Exo70 subunits of the exocyst complex (He et al., 2007).

Sro7 is a member of the tomosyn/Lgl family of tumor suppressors. The crystal structure shows it is composed of two adjacent beta propellers which are conserved amongst all family members (Hattendorf et al., 2007). There is one binding site for Sec4 on the surface of Sro7 which maps to a conserved cleft between the two beta propellers (Watson et al., 2015). Homo-oligomerization of Sro7 has been shown to occur during vesicle tethering in vitro suggesting this is a mechanism by which Sro7 binds to Sec4 at the vesicle surface, bringing the separate membranes into proximity (Rossi et al., 2018).

Exocyst and Sro7 both work in parallel and interact directly with one another though the Exo84 subunit of the exocyst (Zhang et al., 2005). Additionally, Sro7 and Rho GTPases, concentrated at sites of polarized growth, can activate exocyst tethering by increasing the strength of exocyst binding to Sec4 and Snc1/2 at the vesicle surface, respectively (Miller et al., 2023). For both exocyst and Sro7, the initial docking event is thought to be followed by SNARE-mediated fusion by promoting the localized assembly of SNARE monomers into fusion complexes at sites of polarized growth (Hattendorf et al., 2007; Wu et al., 2008).

We recently reconstituted an in vitro vesicle:vesicle tethering assay using purified Sro7 and exocyst protein and fluorescently labeled post-Golgi vesicles isolated from yeast (Rossi et al., 2020). The in vitro assay closely mirrored tethering in vivo as both tethers relied on the presence of a functional Sec4 for vesicle tethering to occur. Additionally, the exocyst-mediated in vitro tethering also demonstrated a requirement for the R-SNARE Snc1/2 on the vesicle surface. When we modified the assay to include both Rab-proficient and Rab-deficient vesicles, we observed that Sro7 mediated symmetric vesicle tethering and functioned as a tether only when both opposing membranes contained Sec4 (Rossi et al., 2018). This suggests Sro7 could function to help cluster, retain, and concentrate post-Golgi vesicles at sites of polarized growth in the cell. Here we show that unlike Sro7, the exocyst mediates asymmetric vesicle tethering between a Rab-proficient and a Rab-deficient surface. We have used this behavior to test several fundamental properties about how the exocyst recognizes each of these two membranes and how it is likely to be aligned during the tethering process. We demonstrate that the Rab and SNARE requirements for exocyst tethering in yeast are from the same membrane and not from opposing membranes, while a Sac1-sensitive phosphoinositide, likely PI4P, is important solely on the opposing membrane. These results suggest exocyst is likely to adopt an alignment parallel to the surface of the vesicle during vesicle tethering in a manner that would be distinct from that suggested for the structurally related CATCHR family member Dsl1 (DAmico et al., 2023; Stanton and Hughson, 2023).

RESULTS AND DISCUSSION

Assaying the exocyst for asymmetric tethering activity

The membrane tethering process involves bringing two membranes into close proximity without fusion. To study post-Golgi vesicle tethering biochemically, we developed a reconstitution assay using post-Golgi vesicles isolated from yeast and purified forms of either of two yeast vesicle tethering factors, Sro7 or the multisubunit exocyst complex (Rossi et al., 2015, 2020). Our initial assays for both Sro7 and exocyst mediated tethering made use of a single population of post-Golgi vesicles in each assay. To determine whether Sro7-mediated tethering assays required a functional Sec4 protein on both membranes being tethered, we modified the Sro7 tethering assay to include two different populations of vesicles (Rossi et al., 2018). One population of vesicles contained a functional GFP-Sec4 (green) and was isolated from a sec6-4 mutant, while the other population of vesicles, labeled with FM4-64 (red), lacked a functional Sec4, as it was derived from a sec4-8 strain (Goud et al., 1988). Using this assay, we demonstrated that Sro7 was only able to tether vesicles containing a functional Sec4 on both populations of vesicles used in the assay (Rossi et al., 2018). To examine whether the exocyst demonstrates a similar requirement for Rab GTPase on both vesicle populations, we developed an assay similar to that used for Sro7 (Figure 1). As expected from our previous work, Sro7, by itself is unable to tether Rab-proficient GFP-Sec4 (green) vesicles to Rab-deficient, sec4-8 (red) vesicles but was fully capable of tethering with control Rab-proficient sec6-4 (red) vesicles. In contrast, when purified exocyst is included in the asymmetry assay, we observe quite robust and significant coclustering of Rab-deficient, sec4-8 vesicles (red) with GFP-Sec4 labeled (green) vesicles (Figure 1, A and C). The vesicles used in this assay were normalized by immunoblot analysis of vesicle marker proteins Snc1/2, Sso1/2, and Sec4 to make sure they were present in equal amounts in the tethering assay (Figure 1B). Vesicle tethering was quantitated using automated detection by commercially available software with a slight modification to the method we recently described (Miller et al., 2023; Materials and Methods). This striking result may reflect an inherent property of the exocyst that is important for heterotypic tethering of post-Golgi vesicles to the plasma membrane within the cell. This novel property of the exocyst in the in vitro tethering assay allowed us to test other fundamental aspects of how the exocyst might recognize and tether two opposing yet distinct membranes.

FIGURE 1:

FIGURE 1:

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Asymmetric vesicle tethering by the exocyst. (A) Red vesicles labeled with the lipid dye FM4-64 were isolated from both sec6-4 and sec4-8 mutant strains and mixed with green vesicles isolated from a sec6-4 mutant strain expressing GFP-Sec4 (CEN) in an in vitro tethering assay in the presence of Sro7 alone or Sro7 and wild type exocyst. Scale bar: 5 μm. (B) Immunoblot analysis of vesicle fractions used in the assay with vesicle marker proteins: Sso1/2, Snc1/2, and Sec4. We estimate that the amount of Sec4-8 protein on vesicles from the sec4-8 strain is ∼2% of the amount present on vesicles from the sec6-4 strain. (C) Quantitation of “mixed” vesicle clusters defined as vesicle clusters in the TRITC channel which show at least 40% overlap with clusters in the FITC channel. N is the number of independent data points. Error bars represents SD obtained from counting images at 60x magnification. P values were obtained using a two-tailed Student’s t test.

Snc1/2 is required on the Sec4-proficient but not the Sec4-deficient vesicles in exocyst-mediated asymmetric tethering

We have previously shown that exocyst-mediated tethering requires both the Rab GTPase, Sec4, and the R-SNARE, Snc1/2 (Rossi et al., 2020). The asymmetric tethering assay described above allowed us to ask whether the requirement for Snc1/2 is on the same membrane as that containing Sec4, on the membrane deficient for Sec4 (sec4-8), or both. To answer this question, we constructed modified versions of the yeast strains used above, where the only source of the R-SNARE, Snc1/2, was under a regulatable GAL promoter (pGAL-SNC1). Using these strains, we were able to generate both green Sec4-proficient vesicles that were depleted of Snc1 and red Sec4-deficient vesicles that were depleted of Snc1 (see Materials and Methods). The vesicles were normalized following immunoblot analysis and then mixed in a tethering assay with the respective red Sec4-deficient or green Sec4-proficient vesicles in the presence of both Sro7 and exocyst (Figure 2). This experiment demonstrates that when GFP-Sec4 vesicles are depleted of Snc1 we observe a near total loss of asymmetric tethering to sec4-8 vesicles (Figure 2C, left graph). In contrast, when sec4-8 vesicles are depleted of Snc1, we observe robust tethering with GFP-Sec4 vesicles. Quantitation of these results demonstrated a significant, nearly threefold, increase in asymmetric tethering of the Snc1-depleted, sec4-8 vesicles compared with the undepleted sec4-8 control vesicles (Figure 2C, right graph). These results demonstrate that during asymmetric tethering the recognition of the R-SNARE and the Rab GTPase by exocyst occurs on the same membrane. The requirement for Sec4 and Snc1/2 on the same vesicle suggests a mechanism for how the tethering reaction might act as a proofreading mechanism to ensure that only vesicles with the appropriate combination of both Rabs and R-SNAREs are presented for tethering to the plasma membrane. The surprising increase in tethering when the R-SNARE was removed from the Rab-deficient membrane might reflect either the removal of a steric inhibition or an increase in properly oriented tethering complexes in the absence of any competing signals.

FIGURE 2:

FIGURE 2:

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The R-SNARE Snc1/2 is required on the same membrane as GFP-Sec4 in asymmetric vesicle tethering by the exocyst. (A) Snc1/2 was depleted from green “Sec4-proficient” vesicles expressing GFP-Sec4 or from red “Sec4-deficient” vesicles containing the sec4-8 mutation by genetic manipulation which involved placing Snc1 under a GAL promoter in strains expressing GFP-Sec4 (snc1Δ; snc2Δ + pGAL-SNC1) or strains containing a sec4-8 mutation (sec4-8, snc1Δ; snc2Δ +pGAL-SNC1). Vesicles were then isolated, normalized and used in an in vitro tethering assay in the presence of Sro7 alone or Sro7 and wild type exocyst complex. Scale bar: 5 μm. (B) Immunoblot analysis of vesicle fractions used in the assay with vesicle marker proteins: Sso1/2, Snc1/2, and Sec4. We estimate that the amount of Snc1 protein on vesicles isolated following GAL-depletion is ∼5% of the amount present on vesicles from the control sec6-4 strain. (C) Quantitation of “mixed” vesicle clusters defined as vesicle clusters in the TRITC channel which show at least 40% overlap with clusters in the FITC channel. Error bars represents SD obtained from counting images at 60x magnification. P values were obtained using a two-tailed Student’s t test.

A role for PI4-phosphorylated lipids in asymmetric tethering by the exocyst

The results described above leave an open question of how exocyst is able to recognize and bind to the Sec4 and Snc1/2-deficient vesicles during asymmetric tethering. Phosphoinositide lipids are attractive candidates to perform this function. Phosphinositides have been implicated in exocyst function in post-Golgi transport; the Exo70 subunit has been shown to bind both PI(4,5)P2 and PI4P (He et al., 2007) while Sec3 has also been shown to interact with PI(4,5,)P2 (Zhang et al., 2008). Although PI(4,5)P2 is enriched at sites of polarized growth on the plasma membrane, only PI4P is thought to be present on the surface of post-Golgi vesicles (Mizuno-Yamasaki et al., 2010; Santiago-Tirado et al., 2011). To examine the role of phosphoinositides in our assay, we made use of the Sac1 enzyme, which has a well described phosphoinositide phosphatase activity. In vivo this enzyme is thought to act primarily on PI4P, but in vitro also shows activity on the monophosporylated phosphoinositides PI3P and PI5P (Guo et al., 1999a, Zhong et al., 2012).

We treated fluorescently-labeled sec4-8 and GFP-Sec4 vesicles with purified recombinant catalytically active Sac12-511 or the catalytically inactive Sac12-460 (Cai et al., 2014) as described in Materials and Methods. Following Sac1 treatment, vesicles were subjected to a final purification by pelleting though a sorbitol cushion to remove excess enzyme. We first analyzed the effect of Sac1 treatment on the red Sec4-deficient sec4-8 vesicles in an assay mix with green (GFP) Sec4-proficient vesicles, Sro7 and exocyst. The results of this experiment (Figure 3) demonstrate clearly that treatment of the red Sec4-deficient vesicles with the catalytically active Sac12-511 dramatically inhibit the ability of the treated vesicles to tether to green untreated Sec4-proficient vesicles. In contrast, both the sec4-8 vesicles treated with catalytically inactive Sac12-460 and mock-treated vesicles demonstrate comparable tethering activity to each other (Figure 3, A and C). Immunoblot analysis of both the vesicle marker proteins and cargo protein, Bgl2, (Harsay and Bretscher, 1995) confirmed the integrity and normalization of vesicles following Sac1 treatment (Figure 3B, Supplemental Figure S1).

FIGURE 3:

FIGURE 3:

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Sac1 treatment of the sec4-8 vesicles disrupts asymmetric tethering by the exocyst. (A) Red “Sec4-deficient” vesicles generated from a sec4-8 mutant strain were treated with active Sac1 (aa2-511), inactive Sac1 (aa2-460), or buffer only before using in an asymmetric assay with green “Sec4-proficient” vesicles, Sro7, and exocyst. Scale bar: 5 μm. (B) Immunoblot showing the normalization of the vesicles used in the assay with vesicle marker proteins: Sso1/2, Snc1/2, and Sec4. Coomassie of the purified Sac1 enzymes used in the assay is shown to the left. (C) Automated quantitation of “mixed” vesicle clusters defined as vesicle clusters in the TRITC channel which show at least 40% overlap with clusters in the FITC channel. Error bars represents SD obtained from counting images at 60x magnification. P values were obtained using a two-tailed Student’s t test.

To determine whether phosphoinositides have a role in the activity of the Sec4-proficient vesicles used in the asymmetric tethering assay, we carried out a set of Sac1 treatments on the GFP-Sec4 vesicles identical to those used with the sec4-8 vesicles above. In contrast to the results for the sec4-8 vesicles, treatment of GFP-Sec4 vesicles with active Sac12-511 caused an apparent increase – rather than a decrease– in tethering activity when compared with catalytically inactive Sac12-460 or buffer controls (Figure 4A). In fact, quantitation of these results demonstrated a significant, roughly twofold, increase in asymmetric tethering of the Sac1 treated GFP-Sec4 vesicles compared with the two control-treated GFP-Sec4 vesicles (Figure 4C). This surprising increase in tethering when PI4P was removed from the Rab-proficient vesicles is remarkably similar to the effect of Snc1 depletion we saw in Figure 2. Likewise, this may also reflect an increase in properly oriented tethering complexes via a reduction of any competing signals from the GFP-Sec4 vesicles.

FIGURE 4:

FIGURE 4:

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Sac1 treatment of GFP-Sec4 vesicles promotes asymmetric tethering by the exocyst. (A) Green “Sec4-proficient” vesicles generated from a GFP-Sec4/sec6-4 mutant strain were treated with active Sac1 (aa2-511), inactive Sac1 (aa2-460) or buffer only before being used in an asymmetric assay with red “Sec4-deficient” sec4-8 vesicles, Sro7, and exocyst. Scale bar: 5 μm. (B) Immunoblot showing the normalization of the vesicles used in the assay with vesicle marker proteins: Sso1/2, Snc1/2, and Sec4. (C) Automated quantitation of “mixed” vesicle clusters defined as vesicle clusters in the TRITC channel which show at least 40% overlap with clusters in the FITC channel. Error bars represents SD obtained from counting images at 60x magnification. P values were obtained using a two-tailed Student’s t test.

To understand whether the increased tethering associated with the sec4-8, Snc1-depleted vesicles seen in Figure 2, is also through a phosphoinositide lipid dependent process, we repeated the Sac1 treatment protocol on these vesicles (Supplemental Figure 2). The result clearly demonstrates that sec4-8 vesicles depleted of Snc1 are just as sensitive to Sac1-treatment as sec4-8 vesicles. This demonstrates that the increase in tethering observed with sec4-8 vesicles depleted of Snc1 occurs through the same PI4P-dependent, exocyst-dependent pathway used by the sec4-8 vesicles with the normal complement of R-SNARE.

Taken together these experiments demonstrate a role for a phosphorylated phosphoinositide lipid species in exocyst-mediated tethering in vitro. PI4P, generated by Pik1 kinase in the Golgi, has been shown to be present on post-Golgi vesicles in vivo (Mizuno-Yamasaki et al., 2010; Santiago-Tirado et al., 2011) and is likely to represent the functional target of Sac1 treatment of post-Golgi vesicles in the assay. Recently, PI4P has been found associated with post-Golgi secretory vesicles throughout their transport from within mother cells to sites of fusion within the bud (Gingras et al., 2022). Furthermore, the specificity and asymmetry of the inhibitory and stimulatory effects of Sac1 treatment strongly support a role of phosphoinositide recognition in mediating the inherent asymmetry of post-Golgi vesicle tethering with the plasma membrane. While PI4P appears to be the relevant lipid in the asymmetry assay in vitro, PI(4,5)P2 is the likely target of exocyst recognition of the plasma membrane in heterotypic tethering of vesicles in vivo (Figure 5A).

FIGURE 5:

FIGURE 5:

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Models for exocyst-mediated tethering to the plasma membrane. (A) The cryo-EM structure of the exocyst places the binding sites for Sec4 and Snc1/2 on the opposite ends of the elongated exocyst complex. This allows for two distinct arrangements of the complex during tethering in the presence of Sro7. The left shows what we refer to as “extended tethering” model where the Rab GTPase (Sec4) binding and R-SNARE (Snc1) binding occur on two distinct vesicle surfaces during tethering. The right shows “parallel tethering” where Rab and R-SNARE binding to the exocyst are provided from the same vesicle surface. (B) Comparison of asymmetric tethering of post-Golgi vesicles in the in vitro assay to heterotypic tethering of post-Golgi vesicles to the plasma membrane in the cell.

While PI4P is the primary target of Sac1 phosphatase in vivo, in vitro Sac1 has been shown to also act on PI3P and PI5P species (Zhong et al., 2012). PI5P is thought to be a short-lived species and not commonly detected in yeast (Foti et al., 2001) and PI3P is found on endosomal membranes (Hasegawa et al., 2017). Endosomal conversion of PI3P to PI4P and its role in exocyst-mediated MVB exocytosis has been described in mammalian cells – suggesting a direct role of PI4P in exocyst recruitment to endosomal membranes in these cells (Ketel et al., 2016; Liu et al., 2023). However, the relevance of these findings to yeast exocyst function are, as yet, unknown. Taken together it is highly likely that the effects we observe of Sac1 inhibition in the asymmetric tethering assay are mediated by the loss of PI4P (conversion of PI4P to PI) from the vesicle surface.

Models for exocyst-mediated membrane tethering

The results described in the previous sections have important mechanistic implications for models of how exocyst acts to tether two distinct membranes in the presence of Sro7. Our results also suggest a model for the relationship between asymmetric tethering in this assay in vitro and heterotypic tethering of post-Golgi vesicles to the plasma membrane in vivo. First, the finding that the R-SNARE, Snc1/2, is required on the same membrane as the Rab GTPase, Sec4, (Figure 2) has very important implications for structure-based models on how the exocyst complex is likely to be arranged during tethering. This is due to the fact that the binding sites for Snc1/2 and Sec4 have been shown to reside on distinct ends of the exocyst complex (Mei et al., 2018; Figure 5A). The site of Snc1/2 binding is known to reside within the Sec6 subunit or the “Sec6 cap” present at one end of the complex (Shen et al., 2013), while the binding site for Sec4 GTPase is known to reside in the Sec15 subunit or “Sec15 pole” at the opposite end of the complex (Wu et al., 2005; Jin et al., 2011; Lepore et al., 2018). Simple modeling of the two possible arrangements of the exocyst cryo-EM structure with the Snc1-depletion data demonstrate that the “extended tethering” model is inconsistent with our analyses. Rather, the “parallel tethering” model is in strong agreement with all of our experimental results. This immediately poses a new question. How does the exocyst recognize and physically interact with the Sec4-deficient sec4-8 vesicles during asymmetric tethering? An attractive answer comes from our studies with the Sac1 phosphoinositide phosphatase.

The demonstration that Sac1 treatment of post-Golgi vesicles has a potent inhibitory effect in exocyst-mediated asymmetric tethering suggests a role for phosphoinositides in this process. This fits nicely with the identification of PI(4,5)P and PI4P binding to the Exo70 subunit of the exocyst and the importance of this binding to the function of the exocyst in vivo (He et al., 2007). It is worth noting however that the reported binding of Exo70 to PI4P in the context of synthetic liposomes in this study was 20-fold weaker than to PI(4,5)P2 (He et al., 2007). The yeast Sec3 subunit of exocyst also has been shown to bind to PI(4,5)P2 in vitro (Zhang et al., 2008). However, the interaction of Sec3 with PI4P has not as yet been reported. Therefore, it is currently unclear whether the Sac1 sensitivity in the assay is mediated through Exo70 alone or a combination of the Exo70 and Sec3 subunits of the exocyst in yeast. Future studies using mutant forms of the exocyst subunits should clarify how these subunits contribute to the exocyst recognition of phosphoinositides in the asymmetric tethering assay.

The Sac1 phosphatase is well known to have a high degree of specificity for phosphoinositide lipids in vivo and in vitro (Guo et al., 1999a; Foti et al., 2001). Therefore, we suggest that PI4P is likely to be the relevant phosphoinositide present on sec4-8 vesicles in the in vitro system, as this lipid has been shown to be present at least transiently on post-Golgi vesicles, while PI(4,5)P2 presence is thought to be specific to the plasma membrane (Mizuno-Yamasaki et al., 2010; Ling et al., 2012). Importantly, we find that the inhibitory effect of Sac1 phosphatase treatment is quite specific to the Sec4-deficient sec4-8 membranes and has no effect on Sec4-proficient membranes. This suggests a simple model, seen in Figure 5B, where PI4P in the asymmetric tethering system in vitro plays a role analogous to the role of PI(4,5)P2 in heterotypic tethering of post-Golgi vesicles with the plasma membrane in vivo. Moreover, it suggests a simple mechanism where recognition of plasma membrane-specific phosphoinositides would play an important role in providing both the specificity and physical interaction with the “target” membrane during heterotypic vesicle tethering.

Maib and Murray (2022) have recently shown PI(4,5)P2 dependence in exocyst-recruitment to vesicle membranes in vitro using reconstituted mammalian exocyst and synthetic lipid bilayers. Interestingly in this system PI(4,5)P2 appears to be important on both the vesicle and target membrane whereas in our assay phosphoinositides do not appear to play a critical role on the Rab/R-SNARE-containing vesicles. Future studies will be directed at determining whether this represents a difference between the assay systems – the synthetic liposomes used by Maib and Murray (2022) lacked both Rab or R-SNARE components for example– or a difference between how yeast and mammalian forms of the exocyst utilize phosphoinositides in heterotypic vesicle tethering to the plasma membrane.

Recently, Hughson and colleagues (2023) have described an overall structural similarity between the exocyst and another CATCHR family multisubunit tethering complex, the Dsl1 complex that mediates tethering of COP1 vesicles to the endoplasmic reticulum (DAmico et al., 2023; Stanton and Hughson, 2023). This complex is thought to capture vesicles in an extended arrangement with one end binding vesicles and the other end associated with Q-SNAREs on the endoplasmic reticulum. Although the structural similarity of Dsl1 complex with exocyst is striking, the proposed arrangement for tethering appears to be quite distinct from what we have found in the present work. One difference in the systems is that the in vitro assay described here lacks an active Q-SNARE component as the Sec9 Qbc-SNARE is completely absent from the reaction. Therefore, it is possible that other arrangements of the exocyst may become relevant during the trans-SNARE assembly step that may bring this structural similarity into play as the two membranes come into closer proximity. Previous work (Rossi et al., 2020; Miller et al., 2023) has demonstrated allosteric conformational changes to the structure of the exocyst in response to Rho GTPase or Sro7 activation. However, the structural changes in these activation pathways are unlikely to involve large scale rearrangements of the Rab (Sec15) and R-SNARE (Snc) binding sites relative to each other. Therefore, vesicle tethering of the activated exocyst complex would still involve a parallel mechanism similar to that depicted in Figure 5.

MATERIALS AND METHODS

Protein purification

Sro7 and exocyst purification were carried out as described in (Rossi et al., 2020 and Miller et al., 2023). In the case of Sro7, a high copy plasmid expressing N-terminal Protein A tagged Sro7 behind a ADH1 promoter was used to express Sro7 in a yeast pep4Δ strain. Cells were grown overnight in synthetic media at 30°C to an OD599 of 3.0 and then shifted into YPD (2% glucose) for one doubling time. Sodium azide and sodium fluoride were then added to a final concentration of 20 mM before the cells were harvested and washed with ice-cold 10 mM Tris, pH 7.8, 20 mM sodium azide, and 20 mM sodium fluoride followed by freezing on dry ice. Approximately 50 g of yeast were then lysed with a bead beater in ice cold buffer containing Tris, 7.8, 150 mM NaCl, 0.5% Tween-20, 1 mM dithiothreitol (DTT) and protease inhibitors (2 μg/ml leupeptin,2 μg/ml aprotinin, 2 μg/ml antipain, 14 μg/ml pepstatin A, and 2 mM 4-[2-aminoethyl] benzenesulfonyl fluoride hydrochloride). Five 1-min pulses of bead beating were interrupted by five 2 min intervals to yield a yeast lysate that was centrifuged at 17,400xgmax for 10 min in a JA25.5 rotor and then ultracentrifuged at 140,000xgmax for 30 min at 4°C in a 45Ti rotor. The final protein concentration was adjusted to ∼ 25 mg/ml before the lysate was precleared with Sepharose CL-6B beads for 30 min at 4°C. The cleared lysate was then bound for 2 h with IgG Sepharose beads at 4°C. IgG beads bound to ProtA-Sro7 were then washed five times with ice cold lysis buffer, three times with lysis buffer containing 400 mM NaCl and three ties with cleavage buffer (20 mM Tris pH 7.8, 150 mM NaCl, 0.1 mM EDTA and 1 mM DTT) before cleavage with TEV protease for 5 h at 17°C. The supernatant containing the protein was then aliquoted and frozen at –80°C.

For exocyst purification, a yeast strain containing a chromosomal copy of C-terminally-tagged Sec8-3xMYC and a CEN plasmid expressing wild type Exo70 as the sole source of Exo70 was used. Cells were grown overnight in synthetic media at 30°C to mid-log phase of OD599 1.5 before shifting into YPD (2% glucose) to a final OD599 of 3.0. Sodium azide and sodium fluoride were then added to a final concentration of 20 mM before the harvesting and washing with ice cold Tris pH 7.5, 20 mM sodium azide, and 20 mM sodium fluoride and freezing on dry ice. Approximately 50 g of cells were then lysed in a bead beater in ice cold buffer containing 20 mM Pipes pH 6.8, 120 mM NaCl, 1 mM EDTA 1 mM DTT and protease inhibitors (2 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml antipain, 14 μg/ml pepstatin A, and 2 mM 4-[2-aminoethyl] benzenesulfonyl fluoride hydrochloride). Five 1 min pulses of bead beating were interrupted by five 2-min intervals to yield a yeast lysate which was centrifuged at 17,418xgmax for 10 min at 4°C in a JA25.5 rotor before untracentrifugation at 50,000×g for 30 min at 4°C in a 45Ti rotor. The final lysate concentration was then adjusted to 30 mg/ml before preclearing with Sepharose CL-6B beads for 1 h at 4°C to reduce nonspecific binding. The lysate was then incubated overnight on ice with 9E10 monoclonal antimyc antibody. Protein A Sepharose beads were then added for 2 h at 4°C. The beads were then washed three times in ice cold lysis buffer and then two times in cleavage buffer (20 mM Tris pH 7.5, 140 mM NaCl, 0.1 mM EDTA, and 1 mM DTT) before cleaving in cleavage buffer with TEV protease for 4 h at 17°C. Cleaved exocyst complexes were collected, aliquoted and frozen at –80°C.

Active Sac1 (BB2585) was cloned as a BamHI-NdeI fragment (aa 2-511) into the pET15b vector. Inactive Sac1 (BB2603) was subcloned as a NdeI-BamHI fragment (aa2-460) in the pET15b vector. 6xHis tag purification of active and inactive Sac1 enzymes was performed by binding the bacterial lysate to a HIS-select Nickel Affinity Gel (G-BioScience) in buffer containing 50 mM Tris pH 7.5, 350 mM sodium chloride, 10% glycerol, 0.5% Triton X-100, and 20 mM imidazole. Bound material was washed in buffer with no detergent before elution of the His tagged protein with 500 mM imidazole. The 6xHistidine eluates were then pooled and dialyzed for 1 h in 20 mM Tris pH 7.5, 200 mM sodium chloride, 0.1 mM EDTA, and 10% glycerol, before dialyzing for 1.5 h in 20 mM Tris pH 7.5, 150 mM sodium chloride and 10% glycerol. Purified proteins were aliquoted and frozen at minus –80°C.

Protein concentration estimates of purified proteins were obtained by comparison to Precision Plus Protein Standards-unstained (Bio-Rad) following SDS–PAGE and Coomassie stain. Coomassie stained gels were imaged and quantitated using Odyssey Imaging system (LICOR).

Generation of vesicles labeled with FM4-64 or GFP-Sec4 for the in vitro asymmetry tethering assay

Post-Golgi vesicles labeled with FM4-64 were isolated from single sec6-4, sec4-8 mutant strains and a double sec4-8, snc1Δ; snc2Δ+pGAL-SNC1 secretory mutant strain. The sec6-4 mutant strain was grown in YP+2% glucose overnight at the permissive temperature of 25°C to a final OD599of 0.6. Cells were then shifted to 37°C for 2 h to accumulate vesicles. Sodium azide was added to a final concentration of 20 mM and 300 absorbance units were centrifuged and washed with ice-cold 10 mM Tris, pH 7.5, and 20 mM sodium azide. Cells were spheroplasted in 10 ml of buffer (0.1 M Tris, pH 7.5, 1.2 M sorbitol, 10 mM sodium azide, 21 mM β-mercaptoethanol, and 0.05 mg/ml Zymolyse 100T) for 30 min at 37°C and then lysed in 4 ml of ice-cold buffer (10 mM triethanolamine, pH 7.2, and 0.8 M sorbitol) containing protease inhibitors (2 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml antipain, 14 μg/ml pepstatin A and 1 mM phenylmethylsulphonyl fluoride). The lysate was spun at 4°C to preclear unbroken cells and then spun at 30000 x gmax for 15 min in a Sorvall centrifuge to preclear large membranes. Approximately 3 ml of lysate was then labeled on ice for 10 min with FM4-64 at a final concentration of 1 μg/ml. The labeled lysate was the layered over a 2 ml ice cold sorbitol cushion (20% wt/vol in 10 mM triethanolamine, pH 7.2) and spun at 128000gmax for 1 h at 4°C. The final high-speed pellet was resuspended in 600 μl of lysis buffer and frozen at –80°C. For vesicles isolated from the sec4-8 mutant strain the following modifications were made: 600 absorbance units were spheroplasted in 15 ml of buffer before lysis in 4 ml of lysis buffer and final resuspension in 1 ml of lysis buffer. Vesicles isolated from the sec4-8, snc1Δ; snc2Δ deletion strain containing a (CEN) plasmid expressing Snc1 from a GAL promoter were generated by growing cells in YP+3% raffinose and 1% galactose for 5 h to a final OD599 of 1.5 before shifting to YP+2% glucose for 15 h at 25°C to inhibit Snc1 expression from the GAL promoter. Cells were then shifted to the restrictive temperature of 36°C for 2 h. Three-hundred and fifty absorbance units were then spheroplasted in 10 ml of buffer, lysed in 4 ml of lysis buffer and resuspended in final volume of 400 μl before freezing at –80°C.

Post-Golgi vesicles labeled with GFP-Sec4 were generated from both a sec6-4 mutant strain containing a CEN plasmid expressing GFP-Sec4 and from a snc1Δ; snc2Δ secretory mutant strain expressing pGAL-SNC1 (CEN) and GFP-Sec4 (CEN). Vesicles isolated from the sec6-4 mutant strain expressing GFP-Sec4 were obtained by growing the strain overnight at 25°C in synthetic media to an OD599 of 0.6 and then in YP+2% glucose for 1 h at 25°C before shifting the cells for 2 h to 36°C. Three-hundred and fifty absorbance units were then spheroplasted with 10 ml of buffer before lysis with 4 ml of lysis buffer and final resuspension in 1 ml of lysis buffer. Vesicles isolated from a snc1Δ, snc2Δ deletion strain expressing Snc1 under a regulatable GAL promoter (pGAL-SNC1 CEN) and expressing GFP-Sec4 from a CEN plasmid were grown in synthetic media with 3% raffinose and 1% galactose for 5 h to a final OD599 of 1.5 before shifting to YP+2% glucose for 17 h at 25°C to repress the GAL promoter, inhibit Snc1 expression and accumulate vesicles. Three-hundred and fifty absorbance units were then spheroplasted in 10 ml of buffer before lysis in 4 ml of lysis buffer and final resuspension of vesicles in 1 ml of lysis buffer. Vesicles were then frozen at –80°C.

Sac1 treated green sec6-4, and red sec4-8, and sec4-8, snc1Δ; snc2Δ + pGAL-SNC1 vesicles were obtained by generating the vesicles as described above and then adding 50 nM Sac1 (aa2-511), Sac1 (aa2-460), or buffer only (10 mM Tris 7.5, 150 mM sodium chloride) with the vesicles for 1 h at 25°C. The vesicles were then diluted in 2.5 ml of lysis buffer before layering on a 20% (wt/vol) sorbitol cushion in 10 mM triethanolamine, pH 7.2. The samples were then spun at 128000gmax for 1 h at 4°C before resuspending in 450 and 350 μl of lysis buffer, respectively. Vesicle fractions were frozen at –80°C.

All vesicle preparations were normalized to vesicles obtained from a sec6-4 mutant strain following the procedure described above using immunoblot analysis with Sec4, Snc, and Sso antisera. Additional immunoblotting with Bgl2 antisera (Supplemental Figure S1) confirmed normalizations with vesicle markers and demonstrated vesicle integrity was unaffected by Sac1 treatment.

Strain construction

BY3215 (mat a, sec4-8::NatR, snc1Δ:URA3, snc2Δ:ADE8, pGAL-SNC1) was generated by transforming BY101 (mat a, snc1Δ:URA3, snc2Δ:ADE8, pGAL-SNC1) with an integrating form of sec4-8 linked with a NatR gene. Nourseothricin (Nat) resistant transformants were confirmed to be temperature-sensitive and demonstrated a requirement for galactose in growth media. Immunoblot analysis demonstrated loss of both Snc1 and Sec4 following shift to glucose media and a subsequent shift to 36°C (see above). BY3212 (mat a, snc1Δ:URA3, snc2Δ:ADE8, pGAL-SNC1, GFP-SEC4-pRS315) was created by transforming BY101 with a CEN/LEU2 plasmid expressing GFP-SEC4.

In vitro asymmetry tethering assay

Vesicles generated as described above were thawed, spun in a cold microfuge for 2 min and then treated with MgCl2 (3 mM) and GTPγS (1 mM) for 60 min on ice. Vesicles were then mixed in equal amounts in the presence of Sro7 (0.4 μM) or Sro7 and exocyst (0.4 μM and 20 nM, respectively) for 60 min at 30°C. Vesicle–vesicle clustering was detected by taking six images at 60x magnification in both FITC and TRITC channels using a Nikon E600 microscope with a Nikon Planar 60x oil-immersion lens (NA 1.4) and a Photometrics CoolSNAP Dyno-charge-coupled device camera.

Quantification and Statistical analysis

Images in both FITC and TRITC channels were quantitated by Imaris Software v9.9. To count puncta that were defined in both channels, we defined clusters as surfaces with Surface Grain Size = 0.25 μm, and a cluster size ≥ 1 μm in any direction. We set a Manual Threshold Value = 10 for FITC images and a Manual Threshold Value = 50 for TRITC images. Colocalization was obtained using the object-to-object statistics function in the algorithm settings and was defined as fluorescent puncta in the TRITC channel which were above 1 μm in diameter and which showed at least 40% overlap with clusters in the FITC channel.

Statistical analysis was performed using GraphPad Prism (version 9) and statistical significance was determined using a two tailed Student’s t test. Results are represented as the mean ± SD (SD) and p values [gt] 0.05 are considered significant. Data for each figure were pooled from multiple technical replicates of at least three independent biological replicates for each experimental condition. N values are shown underneath each graph.

Supplementary Material

mbc-35-br8-s001.pdf (1.7MB, pdf)

Acknowledgments

We thank Wendy Salmon and the UNC Hooker Imaging Core Facility for help with the use of Imaris software in the automated quantitation of tethering in this study, and Benjamin Twara for technical help with the recombinant Sac1 purifications. This work was supported by National Institutes of Health award R01 GM054712 (to P.B). G.C.P. was supported in part by a grant from National Institute of General Medical Sciences award T32 GM135128. Wendy Salmon and the UNC Hooker Imaging Core Facility are supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.

Abbreviations used:

CATCHR

complexes associated with tethering containing helical rods

FITC

fluorescein isothiocyanate

GFP

green fluorescent protein

GST

glutathione S-transferase

GTPase

guanosine triphosphatase

GTPγS

guanosine 5’-3’- O-(thio)triphosphate

MVB

multivesicular body

OD

optical density

SNARE

soluble N-ethylmaleimide-sensitive factor adaptor protein receptor

TEV

tobacco etch virus

TRITC

tetramethylrhodamine isothiocyanate

YPD

yeast peptone dextrose

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E23-08-0311) on January 10, 2024.

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Supplementary Materials

mbc-35-br8-s001.pdf (1.7MB, pdf)

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