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Vesicle transport requires four steps; vesicle formation, movement, tethering and fusion. In yeast, two Rab GTPases, Ypt31/32 are required for post-Golgi vesicle formation. A third Rab GTPase, Sec4, and the exocyst act in tethering and fusion of these vesicles. Vesicle production is coupled to transport via direct interaction between Ypt31/32 and the yeast myosin V, Myo2. Here we show that Myo2 interacts directly with Sec4, and the exocyst subunit Sec15. Disruption of these interactions results in compromised growth and the accumulation of secretory vesicles. We identified the Sec15-binding region on Myo2, and also identified residues on Sec15 required for interaction with Myo2. That Myo2 interacts with Sec15 uncovers additional roles for the exocyst as an adaptor for molecular motors, and implies similar roles for structurally related tethering complexes. Moreover, these studies predict that for many pathways, molecular motors attach to vesicles prior to their formation, and remain attached until fusion.
Vesicle transport is a fundamental process. Vesicles form, move, are tethered at their destination, and ultimately fuse with the acceptor membrane. In secretory vesicle transport, vesicles are produced as post-Golgi vesicles and are moved along the cytoskeleton by motor proteins. The vesicles are then tethered to the plasma membrane by tethering factors, and ultimately the vesicles fuse with the plasma membrane.
The unconventional myosin motor, myosin V moves several caroges including secretory vesicles (Johnston et al., 1991), melanosomes (Wu et al., 1997), the vacuole (Hill et al., 1996), peroxisomes (Hoepfner et al., 2001), synaptic vesicles (Evans et al., 1998; Prekeris and Terrian, 1997) and the endoplasmic reticulum (Estrada et al., 2003; Tabb et al., 1998; Takagishi et al., 1996; Wagner et al., 2011). The globular tail domain (GTD)/cargo-binding domain (CBD) of myosin V, located at its C-terminus, plays a critical role in the attachment of myosin V to cargoes. The cargo-binding domain interacts directly with cargo-specific adaptor proteins that regulate the interaction of myosin V cargoes. Recently we solved the structure of the cargo-binding domain of the yeast myosin V motor, Myo2, and mapped the binding sites for the adaptor/receptor proteins, Vac17, Inp2 and Ypt31/32, which connect myosin V to the vacuole, peroxisome and secretory vesicles, respectively (Fagarasanu et al., 2009; Lipatova et al., 2008; Pashkova et al., 2006).
In the yeast S. cerevisiae, each step in secretory vesicle transport is essential for yeast viability. The Rab GTPases Ypt31/32 (Rab11 homologues) are required for secretory vesicle formation from the trans-Golgi (Jedd et al., 1997). Myo2 is recruited to these vesicles by Ypt31/32 (Lipatova et al., 2008). Ypt31/32, along with phosphatidylinositol 4-phosphate, recruit Sec2, a guanine nucleotide exchange factor (GEF) for the Rab GTPase Sec4 (Mizuno-Yamasaki et al., 2010; Ortiz et al., 2002). Sec2 mediates a Rab GTPase cascade and recruits Sec4 (Rab8/10 homologue) (Ortiz et al., 2002; Walch-Solimena et al., 1997). The nucleotide bound to Sec4 is converted from guanosine diphosphate (GDP) to guanosine triphosphate (GTP) by the GEF function of Sec2. Sec4 also binds to Myo2 (Santiago-Tirado et al., 2011). In addition, Sec2 binds Sec15, a subunit of the vesicle tethering complex, the exocyst (Medkova et al., 2006), and concomitantly Ypt31/32 are released (Mizuno-Yamasaki et al., 2010). Sec15 then binds GTP-bound Sec4 (Guo et al., 1999). Sec4 also functions with the t-SNARE, Sec9 to promote fusion (Brennwald et al., 1994; Grosshans et al., 2006).
Myo2 moves secretory vesicles from the mother cell to the growing tip of the daughter cell (Govindan et al., 1995). The average velocity of movement of the Sec4 positive secretory vesicle is 3 μm/s (Schott et al., 2002). In myo2 mutants, where some or all of the Myo2 IQ motifs are deleted, these Sec4 positive vesicles move at a slower velocity. These observations strongly suggest that movement of Sec4 positive vesicles is mediated by Myo2 (Schott et al., 2002). Ypt31/32 (Lipatova et al., 2008) and Sec4 (Santiago-Tirado et al., 2011) are each candidate molecules that link secretory vesicles to Myo2. In further support of this hypothesis, homologues of Ypt31/32 and Sec4 in other species interact with myosin V motors. Ypt31/32 is a member of the Rab11 family. In Drosophila, Rab11 binds myosin V (Li et al., 2007; Wu et al., 2005). There are three myosin V motors in mammals, Va, Vb and Vc. Mammalian Rab11 binds myosin Vb (Roland et al., 2009). In addition, Sec4 is closely related to the Rab8 and Rab10 families. Rab8 binds myosin Vb and Vc (Ishikura and Klip, 2008; Rodriguez and Cheney, 2002; Roland et al., 2009). Rab10 binds myosin Va, Vb and Vc (Roland et al., 2009).
It is likely that Rab GTPases are not the sole proteins required to tether secretory vesicles to Myo2. Unlike Myo2, neither Ypt31/32 nor Sec4 are present on secretory vesicles from their formation through their fusion. Ypt31/32 are replaced by Sec4 at an as yet undefined point in the transport process. This raises the question of how Myo2 remains attached to these moving vesicles, and leads to the postulate that additional proteins are involved in this attachment.
The cargo-binding domain of myosin V is primarily composed of alpha-helical bundles arranged as extended rods (Pashkova et al., 2006). Remarkably, although there is no sequence similarity, all known structures of the exocyst subunits are similar to each other (Dong et al., 2005; Hamburger et al., 2006; Moore et al., 2007; Munson and Novick, 2006; Sivaram et al., 2006; Wu et al., 2005) and share the same unique fold with the Myo2 cargo-binding domain (Croteau et al., 2009; Moore et al., 2007; Pashkova et al., 2006). These observations raise the intriguing possibility that the cargo-binding domain of myosin V shares a similar molecular function with the exocyst complex, and interacts with secretory vesicles via direct interaction with the exocyst.
Here we show that Ypt31/32 and Sec4 bind to the same site on Myo2. We further show that the exocyst tethering complex is also required to attach Myo2 to secretory vesicles. Myo2 interacts directly with Sec15, at a site distinct from the Rab GTPase site. Moreover, we map the site on Sec15 that binds to Myo2, and identify a mutation on Sec15, that suppresses Myo2 mutants that are defective in binding Sec15. Together these studies show that Myo2 attaches to secretory vesicles through direct binding to multiple proteins required for vesicle fusion, as well as to proteins required for vesicle formation.
Secretory vesicle transport is the only function of Myo2 that is required for yeast viability. An intact motor and cargo-binding (globular tail) domain of Myo2 are each required (Catlett et al., 2000; Reck-Peterson et al., 1999; Schott et al., 1999). We identified surface residues on the Myo2 cargo-binding domain that comprise the secretory vesicle binding site, L1331, L1411, Y1415, K1444 and Q1447 (Fig. 1A)(Pashkova et al., 2006), and subsequently showed that these bind directly to the Rab GTPases Ypt31/32 (Lipatova et al., 2008). Support of the importance of this interaction came from the observation that a lethal point mutation in the center of the secretory vesicle binding site of Myo2 could be rescued by direct fusion of Ypt32 to the C-terminus of Myo2-Y1415R (Lipatova et al., 2008). This finding raised the question of whether the Rab GTPase Sec4, which functions in a Rab cascade after Ypt31/32 and is required for secretory vesicle tethering and fusion, also interacts with Myo2. Indeed, five independent approaches indicate that Sec4 interacts with Myo2.
In a yeast two hybrid test, Sec4 interacts with Myo2, and notably, the Myo2-Y1415R mutation abolishes this interaction (Fig. 1B)(Santiago-Tirado et al., 2011). To test whether Sec4 binds to the same region on Myo2 as Ypt32, we tested each of the secretory vesicle binding site mutants, L1331S, L1411S, Y1415R and Q1447R. Each failed to interact with Sec4 (Fig. 1B). In contrast, a vacuole binding mutant, Myo2-N1304S, which does not interact with Vac17 (Ishikawa et al., 2003), interacts with Sec4 (Fig. 1B). The two hybrid studies strongly suggest that Sec4 and Ypt31/32 bind to the same site (Fig. 1A). We could not determine whether the Myo2-K1444A mutant is defective in binding to Sec4, because the Gal4 DNA binding domain fused to the Myo2-K1444A mutant showed self activation (data not shown).
To further test whether Myo2 interacts with Sec4, we used recombinant proteins and performed pull down experiments from solubilized yeast lysates. Recombinant GSTSec4 pulled down endogenous Myo2 from yeast lysates (Fig. 1C). Note that GST-Sec4 also pulled down endogenous Sec15, a known binding partner of Sec4 (Fig. 1C)(Guo et al., 1999). This result, as well as a previous study (Wagner et al., 2002), suggest that Sec4 is in the same complex with Myo2.
Recombinant Myo2 cargo-binding domain interacts with recombinant Sec4 (Santiago-Tirado et al., 2011). To test whether the interaction between Myo2 and Sec4 is dependent on the nucleotide state of Sec4, we used recombinant purified Sec4-Q79L (GTP locked mutant) (Walworth et al., 1992), Sec4-S34N (a predicted GDP locked mutant) (Collins et al., 1997) and Sec4-N133I (nucleotide-free mutant) (Walworth et al., 1989). We found that His-tagged Myo2 cargo-binding domain bound to GST-Sec4-Q79L but not to GST-Sec4-S34N or GST-Sec4-N133I (Fig. 1D).
To further test the interaction between Myo2 and Sec4, we measured their affinity using surface plasmon resonance (SPR), and observed a dissociation constant (KD) of 4.6 μM, for Myo2 with Sec4(GppNHp), and a KD of 34.8 μM for Myo2 with Sec4 without addition of nucleotide (Table S1 and Fig. S1). The differences in affinity of Myo2 for Sec4 and Sec4(GppNHp) strongly suggest that this interaction requires GTP.
To test whether Myo2 and Sec4 interact in vivo, we used a split GFP assay (Kerppola, 2008). Myo2 was fused to the N-terminal half of the YFP variant Venus (vYFPN), while Sec4 was fused to the C-terminal half of the YFP variant Venus (vYFPC). The YFP signal is only detected when the N-terminal and C-terminal portions of vYFP are brought into close proximity. Co-expression of vYFPN fused to C-terminal end of Myo2, Myo2-vYFPN, and vYFPC fused to N-terminus of Sec4, vYFPC-Sec4, in myo2Δ cells resulted in a YFP signal in the growing bud or in the mother-bud neck, sites where secretory vesicles are delivered (Fig. 1E). These results indicate that Myo2 and Sec4 are in close proximity in vivo. Myo2-vYFPN and vYFPC-Sec4 fusion proteins support viability of the lethal myo2Δ and sec4Δ mutants, respectively (data not shown).
To test whether the lethal point mutation in the center of the Rab binding site, myo2-Y1415R, is inviable due to loss of interaction between Myo2 and Sec4, we expressed Sec4 fused to the C-terminal end of Myo2-Y1415R (Fig. 2A and Fig. S2A). Fusion of the Myo2-Y1415R mutant with Sec4 rescues its lethality (Fig. 2, B and C). In further support that the interactions between Myo2 and Ypt31/32 as well as Myo2 and Sec4 are specific, a fusion of Myo2-Y1415R with the Rab GTPase Ypt1 was generated. Ypt1 is involved in vesicle transport from the ER to the Golgi and does not bind to Myo2 (Jedd et al., 1997; Lipatova et al., 2008; Segev et al., 1988). Fusion with Ypt1 does not rescue the lethality of the myo2-Y1415R mutant (Fig. 2B), which indicates that only specific Rab GTPases are capable of rescuing the lethality. Note that in each experiment, wild-type Ypt31/32 and Sec4 are present. We also tested and found that the affinity of Sec4 (GppNHp) for Myo2, KD = 4.6 μM, is modestly greater than the affinity of Ypt32 (GppNHp) for Myo2, KD = 6.4 μM (Table S1).
Only the prenylation competent form of Sec4 rescues the lethality of myo2-Y1415R mutant (Fig. 2, D and E). The Sec4-SS mutant, mutated in its predicted prenylation site (Calero et al., 2003), cannot attach to membranes, and does not suppress the lethality of the myo2-Y1415R mutant (Fig. 2E). Together these results suggest that the interaction between Myo2 and Sec4 is essential for yeast viability, secretory vesicle transport and Myo2 association with secretory vesicles. All Rab GTPase fusion proteins were expressed at similar levels (Fig. 2, C and F). In addition, over-expression of Sec4 in the myo2-Y1415E temperature sensitive mutant partially rescued its growth defect (Fig. S2B). This finding adds further support to the hypothesis that the myo2-Y1415E/R mutants are defective in binding to Sec4.
The structure of the cargo-binding domain of Myo2 is strikingly similar to the structures of subunits of the exocyst (Dong et al., 2005; Hamburger et al., 2006; Moore et al., 2007; Munson and Novick, 2006; Pashkova et al., 2006; Sivaram et al., 2006; Wu et al., 2005). These proteins consist of α-helical bundles. The elongated sides of the bundles are predicted to form contact sites between the exocyst subunits (Munson and Novick, 2006). The structural similarity between these proteins suggested the intriguing possibility that Myo2 may integrate into the exocyst complex. In addition, one of the exocyst subunits, Sec15, binds directly to Sec4 (Guo et al., 1999). That both Sec15 and Myo2 bind Sec4 potentially places Myo2 near the exocyst.
To test whether the Myo2 cargo-binding domain interacts with the exocyst, we performed GST pull down assays from yeast lysates. Purified recombinant GST-Myo2 cargo-binding domain pulled down the entire exocyst complex (Sec3-vYFP, Sec5-vYFP, Sec6, Sec8-vYFP, Sec10-vYFP, Sec15, Exo70-vYFP and Exo84) from solubilized yeast lysates (Fig. 3A). The low levels of Sec3 that are pulled down by Myo2 are likely due to the degradation of Sec3 (TerBush et al., 1996), and/or due to the absence of Sec3 on secretory vesicles (Boyd et al., 2004). Indeed, GST-Sec15 also pulled down little Sec3 (Fig. 3B). Together, these results show that the Myo2 cargo-binding domain interacts with the exocyst.
Using recombinant proteins, we tested whether Myo2 directly binds to a subunit of the exocyst complex. We tested Sec15, because Sec15 is the most proximal protein in the attachment of the exocyst to secretory vesicles (Guo et al., 1999). Moreover, Myo2 (Fig. 1)(Santiago-Tirado et al., 2011) and Sec15 (Guo et al., 1999) each bind Sec4. We observed that recombinant GST-Sec15, binds to purified His-tagged Myo2 cargo-binding domain, and conversely, recombinant GST-Myo2 cargo-binding domain also binds to purified His-tagged Sec15 in vitro (Fig. 3, C, D and E). We also tested GST-Sec10, which binds to Sec15, and found that GST-Sec10 does not bind Myo2 (Fig. 3E). These results indicate that the cargo-binding domain of Myo2 directly binds with Sec15. To test whether Myo2 and Sec15 interact in vivo, we utilized the split GFP system. Co-expression of Myo2-vYFPN, and Sec15-vYFPC in myo2Δ, sec15Δ double knockout cells displayed a YFP signal in the bud-tip or mother-bud neck, sites of active polarized growth (Fig. 3F). Note that expression of Myo2-vYFPN and Sec15-vYFPC rescues the lethality of the myo2Δ, sec15Δ double mutant (data not shown), which indicates that these fusion proteins are functional. In addition, as a test of the reliability of the split GFP assay, we found that bud-tip localized protein Myo4, tagged with either NYFP or CYFP, gave little to no signal when co-expressed with the appropriate fusions of Myo2, Sec15 or Sec4 (Fig. S3).
Based on the possibility that the surface area on Myo2 that binds Sec15 is not identical to the surface that binds Sec4, we designed a random mutagenesis screen to identify the Sec15 binding site. The screen is based on the postulate that Myo2 utilizes both Sec4 and Sec15 to attach to secretory vesicles. Thus, the lethal Sec4 binding site mutant, myo2-Y1415R, might be suppressed by second site mutations that enhance the ability of Myo2 to bind to Sec15. We postulated that a subset of the suppressors may increase Sec15 binding to Myo2 at the bona fide Sec15 binding region, while other suppressors may act by binding Sec15 through an additional site. Furthermore, some suppressors might function by enhancing the binding of Myo2 to another secretory vesicle protein.
To directly test whether the Sec15 binding region could be narrowed through identification of second site suppressors of myo2-Y1415R, we performed random PCR mutagenesis of the coding region of the Myo2-Y1415R cargo-binding domain, and isolated several viable Myo2-Y1415R containing strains and mapped the locations of the suppressor mutations (Fig. S4A). Notably, 18 out of 23 suppressors mapped to the Myo2 surface, consistent with the postulate that they function by enhancing the ability of Myo2 to interact with another protein. We focused on surface mutations and generated each mutation in the absence of the Y1415R mutation. None of these mutations on their own exhibited a defect in yeast growth. We then focused on the largest cluster of suppressors (Fig. S4A) and made several point mutations in residues adjacent to the cluster (Fig. S4, B and C). In particular, three mutants, R1402E, Q1472E and K1473E exhibited slow growth at all temperatures tested (Fig. 4, A-C). These mutations reveal a patch on the surface of Myo2 that is distinct from the Rab binding region (Fig. 4A). Combining the R1402E mutation with either Q1472E or K1473E resulted in the same growth defect as the R1402E mutation alone (data not shown).
To determine whether these myo2 point mutants have secretion defects, we tested and found that, similar to the myo2-Y1415E mutant (Lipatova et al., 2008), myo2-R1402E, -Q1472E and -K1473E accumulated numerous vesicles as well as other aberrant membranous structures (Fig. 4D). Note that Myo2 has at least five different membranous cargoes. To determine whether some of the small vesicles observed are indeed secretory vesicles, we tested for potential defects in the secretion of the cell wall modification enzyme endo-β1,3-glucanase (Bgl2), a commonly studied secretory cargo (Adamo et al., 1999; Harsay and Bretscher, 1995; He et al., 2007). Invertase secretion was not assayed, because the yeast strains utilized in our studies lack the SUC2 gene. Consistent with the accumulation of vesicles, all three mutants, as well as myo2-Y1415E, retained high levels of Bgl2, when compared with the wild-type control (Fig. 4E). These findings indicate that myo2-R1402E, -Q1472E and -K1473E are each defective in the secretory vesicle transport pathway. Note that these mutants exhibit normal vacuole inheritance (Fig. 4F), suggesting that the overall structure of the cargo-binding domain is retained.
To test whether the mutants were defective in binding to Sec15 in vitro, we chose the two mutations that had the most severe growth defect, myo2-R1402E and –K1473E and performed in vitro binding assays using recombinant proteins. Each mutant was defective in its interaction with GST-Sec15 (Fig. 5A). We also measured their affinity using SPR, and observed a KD of 0.5 μM, for wild-type Myo2 cargo-binding domain with GST-Sec15 and a KD of 2.5 μM for Myo2-R1402E mutant cargo-binding domain (Table S1 and Fig. S5). These results suggest that the growth defects of the myo2-R1402E and –K1473E mutations are due to a defect in binding to Sec15. We tagged myo2-R1402E, -Q1472E and –K1473E with mCherry and found that these mutants localize to sites of polarized growth, which strongly suggests that the motor activity is unaffected by these mutations (Fig. 5B). Importantly, these mutants result in a mislocalization of Sec15, which suggests that Sec15 must bind Myo2 in order to localize to sites of polarized growth (Fig. 5B).
We also tested whether these mutations affect the ability of Myo2 to interact with Sec4. In a yeast two hybrid assay, we found that myo2-R1402E and myo2-K1473E each bind Sec4 (Fig. 5C). In in vitro binding experiments, the mutants displayed a modest defect in binding Sec4, in contrast with myo2-Y1415R, which has a major defect in binding Sec4 (Fig. 5D). These finding support the postulate that the slow growth phenotype of myo2-R1402E and myo2-K1473E is due to a defect in binding to Sec15. Together these studies reveal an additional surface region on the Myo2 cargo-binding domain that is required for yeast growth, and which is distinct from the Rab binding site.
The interaction between Myo2 and Sec15 may be mediated by ionic interactions. Note that two of the three Myo2 mutants (R1402E and K1473E) involve a charge reversal, which suggests that there are negatively charged amino acids on the surface of Sec15 that interact directly with Myo2. While a high resolution structure of yeast Sec15 is not available, we used the high-resolution structure of Drosophila Sec15 (Wu et al., 2005), and the ESyPred3D program (Lambert et al., 2002) to predict a structure for yeast Sec15. From an alignment of Sec15 from four species (human, fly, worm and yeast), we noted a cluster of conserved, surface, negatively charged amino acids – D681, E684, D705 (Fig. 6A and Fig. S6A). We tested several single point mutants, D681K, E684K and D705K as well as E687K, D689K, E694K, D698K, D700K and D765K. Most mutations did not alter the in vivo function of Sec15. However, the sec15-D705K single mutant and sec15-D681K/E684K double mutant were lethal (Fig. 6B). Note that the vYFP-tagged Sec15 mutant proteins were expressed at similar levels (Fig. 6C). Also, the sec15 mutants with or without the vYFP tag are lethal (data not shown). Importantly, the lethal mutants, Sec15-D681K/E684K and –D705K are mis-localized (Fig. 6D). This result parallels the finding that Sec15 is mislocalized in myo2 mutants that have a defect in binding to Sec15 (Fig. 5B). Together, these findings strongly suggest that contact between Myo2 and Sec15 is important for Sec15 localization and yeast viability. Note that the two lethal Sec15 mutants are able to assemble into the exocyst complex. We performed immunoprecipitation experiments using Sec15-TAP, wild-type, D681K/E684K or D705K expressed in a strain containing SEC10-vYFP. These studies showed that wild-type Sec15-TAP as well as mutant Sec15-TAP, D681K/E684K or D705K pulled down the exocyst subunits tested, Sec10-vYFP, Sec6 and Exo84 (Fig. S6B).
To test whether these Sec15 mutants are defective in binding to Myo2, we performed GST pull down experiment using purified proteins from E. coli. Notably, the Sec15-D705K mutant was defective in binding to Myo2, but not in binding to Sec4 (Fig. 6E). To further test the ability of the Sec15 mutants to bind to Myo2, we performed split-GFP experiments using Myo2 fused with vYFPN and tested for interaction with the Sec15 mutants fused with vYFPC. We found that Sec15-D681K/E684K and -D705K are defective in binding to Myo2 in vivo (Fig. S6C). Note that these proteins were expressed at levels similar to wild-type Sec15-vYFPC.
If the interaction between Myo2 and Sec15 is mediated in part by ionic interactions, then charge reversal of one or two amino acids on the surface of Sec15 might potentially suppress the growth defect of myo2-R1402E, -Q1472E or -K1473E. To test this hypothesis, we analyzed each of the Sec15 mutants generated above and found that the sec15-D700K mutant significantly suppressed the growth defect of the myo2-R1402E, -Q1472E or -K1473E mutants (Fig. 6F). That a charge reversal of a single residue on Sec15, compensates each of three mutations caused by introduction of a negatively charged surface residue on Myo2, provides very strong support for the hypothesis that Myo2 and Sec15 interact with each other directly in vivo, and that this interaction is critical for normal yeast growth. Moreover, these results strongly suggest that Myo2 and Sec15 bind in part through ionic interactions, and that amino acids, R1402, Q1472 and K1473 on Myo2, and D681, E684, D700 and D705 on Sec15 are involved in this interaction. Note that we did not pursue approaches to test whether the charge reversal mutation, Sec15-D700K rescues the binding defect of Myo2-R1402E and -K1473E to Sec15 in vitro, because recombinant Sec15-D700K protein is not sufficiently soluble.
Most surface residues on Myo2 that bind Sec15 are conserved from yeast to mammals (Fig. S7A). Moreover most of the predicted surface residues on Sec15 critical for Myo2 interaction with Sec15 are conserved (Fig. 6 and Fig. S6). These observations strongly suggest that the interaction between myosin V and the exocyst is conserved. To test whether mouse myosin Va interacts with the mouse exocyst, we performed GST pull down assays from detergent solubilized lysates prepared from cultured mouse NIH 3T-3 cells. Recombinant GST-myosin Va globular tail domain pulled down the mammalian exocyst, as measured by the presence of the two exocyst subunits tested, Sec6 and Sec8 (Fig. 7A). We also tested and found that recombinant GST-rSec15 binds to purified His-tagged myosin Va globular tail domain in vitro (Fig. 7B). These results indicate that myosin Va directly binds with Sec15 through its globular tail domain, and strongly predict that the interaction between myosin V and the exocyst complex is conserved, and is critical for vesicle transport in mammals.
A striking prediction of these studies is that the myosin V motor, Myo2, is present at each step of secretory vesicle transport (Fig. 8). We previously reported that Myo2 binds directly to Ypt31/32 (Rab11 homologues), the Rab GTPase pair required for secretory vesicle formation. Here we show that Myo2 binds directly to Sec4 (Rab8/10 homologue), the Rab GTPase required for secretory vesicle fusion with the plasma membrane (this study) (Santiago-Tirado et al., 2011). We also show that Myo2 binds directly to Sec15, a subunit of the exocyst tethering complex. Together, the observations that Myo2 contacts Ypt31/32, Sec4, and the exocyst strongly suggest that Myo2 is present prior to the completion of vesicle formation and remains attached during the final steps of vesicle fusion.
Based on previous studies that order the activities of Ypt31/32, Sec4 and the exocyst, we propose a model for how Myo2 is integrated into each step of the secretory vesicle pathway. During production of secretory vesicles via Ypt31/32, Myo2 is recruited to vesicles through interaction of its cargo-binding domain with the Rab GTPases Ypt31/32. The fact that the GTP bound rather than GDP bound Ypt31/32 interacts with the Myo2 cargo-binding domain (Lipatova et al., 2008) provides strong support for the hypothesis that Myo2 is present before post-Golgi vesicles are fully formed.
The cargo-binding domain of Myo2 also binds the Rab GTPase Sec4. That Ypt31/32 and Sec4 bind the same region of Myo2 (this study), and that SEC4 functions downstream of YPT31/32 (Ortiz et al., 2002), suggests that Myo2 may interact first with Ypt31/32 and then with Sec4. We postulate that Ypt32 and Sec4 bind the Myo2 cargo-binding domain at distinct times in the life of post-Golgi vesicles. Myo2 also binds to Sec15 and interacts indirectly with the rest of the exocyst. The attachment of Myo2 to Sec15 may occur simultaneously with Myo2 interaction with Sec4 or may be a separate step.
The Myo2 cargo-binding domain (residues 1131-1574) binds Ypt31/32, Sec4 and Sec15. Notably, other regions of Myo2 also interact with additional proteins required for fusion of secretory vesicles with the plasma membrane. The neck-region of Myo2 (871-1204 a.a.) associates with the small GTPase Rho3 whose effector is the exocyst subunit Exo70 (Robinson et al., 1999). An overlapping region of Myo2 (782-990 a.a.) interacts with Sro7 (Gangar et al., 2005; Rossi and Brennwald, 2011), a conserved Lgl family member, which interacts with Sec4 and the t-SNARE, Sec9 (Grosshans et al., 2006; Lehman et al., 1999), and is required for fusion of secretory vesicles with the plasma membrane. Indeed, a recent study suggests that Myo2 negatively regulates the late fusion pathway via its interaction with Sro7 (Gangar et al., 2005; Rossi and Brennwald, 2011), which in turn binds with Exo84 (Zhang et al., 2005). That Myo2 interacts with so many proteins related to late steps in vesicle fusion, provides further support to the postulate that Myo2 remains attached to vesicles until very late in the fusion pathway. We propose that these proteins anchor Myo2 to secretory vesicles during vesicle transport. Moreover, Myo2 may function as a scaffold that organizes membrane tethering and/or fusion.
We speculate that the Rab cascade in vesicle movement, as well as a role for myosin V in this cascade, is partially conserved. The degree of sequence similarity between yeast Myo2 and mammalian myosin Va, Vb and Vc, enabled us to predict a three-dimensional model for cargo-binding domain of these three mammalian myosin V motors. Alignment of the cargo-binding domains of mammalian myosin Va, Vb and Vc with yeast Myo2, reveal that there is conservation of the predicted surface residues that bind to Ypt31/32 and Sec4, (Fig. S7)(Lipatova et al., 2008), as well as the two predicted surface residues that contact Sec15. Similarly, the predicted Myo2 binding site on Sec15 is conserved (Fig. S6A). Indeed we tested and found that mammalian myosin Va pulls down the mammalian exocyst from cell lysates, and interacts with recombinant mammalian Sec15 (Fig. 7).
Our findings fit with several studies on metazoan myosin V motors. Mammalian myosin Va, Vb and Vc have been shown to interact with multiple Rab GTPases including Rab8, Rab10, Rab11 and Rab27 (Fukuda et al., 2002; Hume et al., 2002; Ishikura and Klip, 2008; Nagashima et al., 2002; Rodriguez and Cheney, 2002; Roland et al., 2009; Strom et al., 2002; Wu et al., 2002). Importantly, myosin Vb associates with Rab11 (Ypt31/32-like) and also Rab8 (Sec4-like) (Roland et al., 2011; Roland et al., 2007). Moreover, Rab11 binds the region of myosin Vb that corresponds precisely with the region on Myo2 that binds to Ypt31/32, while Rab8 binds to a region upstream of the globular tail domain (Roland et al., 2011). Thus, Rab11-related and Rab8-related GTPases may interact sequentially with myosin V motors in several membrane transport pathways in ways that are similar to Rab GTPase interactions with yeast Myo2. In addition, Rab11 also associates with the exocyst both in mammals (Fielding et al., 2005; Zhang et al., 2004), and in Drosophila (Beronja et al., 2005; Jafar-Nejad et al., 2005; Langevin et al., 2005; Wu et al., 2005).
Residues in the Sec15 binding site on mouse myosin Va have also been predicted to function in another capacity. In vitro, mammalian myosin V motors are autoinhibited by the globular tail domain in the absence of cargo-binding. This head-tail interaction inhibits the ATPase cycle of the myosin Va motor (Li et al., 2008; Liu et al., 2006; Olivares et al., 2006; Thirumurugan et al., 2006) Importantly, mutation of the two residues on myosin Va, K1706 and K1779, that correspond with R1402 and K1473 on Myo2, blocks the in vitro interaction of the myosin Va motor and cargo-binding domain (Li et al., 2008). Note that it is not yet known whether the motor and cargo-binding domains of myosin Va interact in vivo. Moreover, the physiological significance of this interaction has not been determined. Importantly, it is not known whether this type of interaction occurs in Myo2. However, collectively these findings raise the possibility that Sec15 and the exocyst compete with the myosin Va motor for access to the myosin Va globular tail domain.
It is not clear whether binding to Rab GTPases and the exocyst is conserved among all myosin V motors. The structure of the cargo-binding domain of Myo4 is similar to Myo2 (Heuck et al., 2010) (Fig. S7E). However, in preliminary studies, Myo4 did not bind to the exocyst (Heuck et al., 2010). Indeed, analysis of the structure of Myo4 does not provide a clear prediction of whether Myo4 binds to either a Rab GTPase or to the exocyst. Two of the five Rab GTPase binding residues are conserved, while only one of the three Sec15 binding residues is conserved in Myo4 (Fig. S7, A and E).
Since there is no sequence similarity, it was initially surprising to discover that the Myo2 cargo-binding domain shares a similar fold with the exocyst subunits. Furthermore, there is very little sequence conservation between the exocyst subunits. Thus, it is currently not possible to predict how many more examples exist of proteins with this same fold. However, judging from recent studies, this fold may be widely shared among diverse tethering factors.
High resolution structures of subunits of the conserved oligomeric Golgi (COG) complex, a vesicle tethering complex for retrograde traffic in the Golgi, have a similar structure with the known exocyst subunits and with Myo2 (Cavanaugh et al., 2007; Richardson et al., 2009). Moreover, the Dsl1 tethering complex, which functions in retrograde traffic from the Golgi to the ER, and Vps53, a member of the Golgi-associated retrograde protein (GARP) tethering complex which functions in retrograde traffic from endosomes to the trans-Golgi, also have a similar structure with exocyst subunits and Myo2 (Perez-Victoria et al., 2010; Ren et al., 2009; Tripathi et al., 2009; Vasan et al., 2010). Thus integration of the myosin V globular tail domain into vesicle tethering complexes may be a common mechanism that couples diverse membrane cargoes to myosin V, and further ensures that the motor remains attached until late in the fusion pathway. Furthermore, other motors may contain cargo-binding domains with similar structures that join with tethering factors to couple vesicle formation, movement, tethering and fusion.
We thank Drs Wei Guo (University of Pennsylvania), Patrick Brennwald (University of North Carolina, Chapel Hill), Ruth Collins (Cornell University), Jeffrey Martens (University of Michigan) and Alan Saltiel (University of Michigan) for plasmids and antibodies. We thank Dr. Tom Kerppola (University of Michigan) for helpful suggestions about the split GFP assay. We thank Drs Nava Segev and Zhanna Lipatova for communicating unpublished results and for helpful discussions. We thank all members of the Weisman lab, especially Dr. Natsuko Jin, P. Taylor Eves and Richard Gar Wai Yau for fruitful discussions and comments on this manuscript. We thank Emily Kauffman for technical assistance. This work was supported by National Institutes of Health grants R37 GM62261 to LSW, R01 GM068803 to MM, Howard Hughes Medical Institute support to RS, and a Science Foundation Ireland Investigator Award (grant 07/IN.1/B975) to ARK.
See Extended Experimental Procedures for further details.
Yeast strains used (Supplementary Table S2). Yeast cultures grown at 24 °C unless stated otherwise. Yeast extract-peptone-dextrose (1% yeast extract, 2% peptone, 2% dextrose; YEPD), synthetic complete (SC) lacking the appropriate supplement(s), and 5-FOA media made as described (Kaiser, 1994). Unless stated otherwise, SC medium contained 2% dextrose.
NIH 3T-3 mouse embryonic fibroblast cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
Plasmids used (Supplementary Table S3).
GST and all GST fusion proteins were expressed in E. coli BL21 DE3 star. Recombinant proteins were purified with glutathione sepharose beads (GE Healthcare).
To obtain a second site mutation that suppressed the lethality of myo2-Y1415R, the myo2-Y1415R tail region from pRS426 myo2-Y1415R was PCR amplified with Taq polymerase.
SDS-PAGE and Western blot analysis were performed using standard procedures.
Biacore X100 system (GE Healthcare).
Bgl2 assays were performed as described (Adamo et al., 1999).
Cells were grown in rich medium to early log phase; 24 °C, fixed and prepared as described (Wuestehube et al., 1996). Thin sections were examined under a CM10 electron microscope (Philips Electron Instruments, Eindhoven, Netherlands).
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