Yeast homologues of lethal giant larvae and type V myosin cooperate in the regulation of Rab-dependent vesicle clustering and polarized exocytosis

Lgl family members play an important role in the regulation of cell polarity in eukaryotic cells. The yeast homologues Sro7 and Sro77 are thought to act downstream of the Rab GTPase Sec4 to promote soluble N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) function in post-Golgi transport. In this article, we characterize the interaction between Sro7 and the type V myosin Myo2 and show that this interaction is important for two distinct aspects of Sro7 function. First, we show that this interaction plays a positive role in promoting the polarized localization of Sro7 to sites of active growth. Second, we find evidence that Myo2 negatively regulates Sro7 function in vesicle clustering. Mutants in either Myo2 or Sro7 that are defective for this interaction show hypersensitivity to Sro7 overexpression, which results in Sec4-dependent accumulation of large groups of vesicles in the cytoplasm. This suggests that Myo2 serves a dual function, to both recruit Sro7 to secretory vesicles and inhibit its Rab-dependent tethering activity until vesicles reach the plasma membrane. Thus Sro7 appears to coordinate the spatial and temporal nature of both Rab-dependent tethering and SNARE-dependent membrane fusion of exocytic vesicles with the plasma membrane.     

The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p

Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.     

Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.     

A Highlights from MBoC Selection: Structural basis for recognition of the Sec4 Rab GTPase by its effector,the Lgl/tomosyn homologue,Sro7

Members of the tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in tomosyn vertebrate homologues.     

Reciprocal Intramolecular Interactions of Tomosyn Control Its Inhibitory Activity on SNARE Complex Formation

Neurotransmitter release from presynaptic nerve terminals is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn, a negative regulator of neurotransmitter release, which is composed of N-terminal WD40 repeats, a tail domain, and a C-terminal VAMP-like domain, is known to inhibit SNARE complex formation by sequestering target SNAREs (t-SNAREs) upon interaction of its C-terminal VAMP-like domain with t-SNAREs. However, it remains unclear how the inhibitory activity of tomosyn is regulated. Here we show that the tail domain functions as a regulator of the inhibitory activity of tomosyn through intramolecular interactions. The binding of the tail domain to the C-terminal VAMP-like domain interfered with the interaction of the C-terminal VAMP-like domain with t-SNAREs, and thereby repressed the inhibitory activity of tomosyn on the SNARE complex formation. The repressed inhibitory activity of tomosyn was restored by the binding of the tail domain to the N-terminal WD40 repeats. These results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain controls its inhibitory activity on the SNARE complex formation, leading to a regulated inhibition of neurotransmitter release.Synaptic vesicles are transported to the presynaptic plasma membrane where Ca²⁺ channels are located. Depolarization induces Ca²⁺ influx into the cytosol of nerve terminals through the Ca²⁺ channels, and this Ca²⁺ influx initiates the fusion of the vesicles with the plasma membrane, finally leading to exocytosis of neurotransmitters (1). Soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP)² receptors (SNAREs) are essential for synaptic vesicle exocytosis (2-5). Synaptic vesicles are endowed with vesicle-associated membrane protein 2 (VAMP-2) as a vesicular SNARE, whereas the presynaptic plasma membrane is endowed with syntaxin-1 and SNAP-25 as target SNAREs. VAMP-2 interacts with SNAP-25 and syntaxin-1 to form a stable SNARE complex (6-9). The formation of the SNARE complex then brings synaptic vesicles and the plasma membrane into close apposition, and provides the energy that drives the mixing of the two lipid bilayers (3-5, 9).Tomosyn is a syntaxin-1-binding protein that we originally identified (10). Tomosyn contains N-terminal WD40 repeats, a tail domain, and a C-terminal domain homologous to VAMP-2. The C-terminal VAMP-like domain (VLD) of tomosyn acts as a SNARE domain that competes with VAMP-2. Indeed, a structural study of the VLD revealed that the VLD, syntaxin-1, and SNAP-25 assemble into a SNARE complex-like structure (referred to as tomosyn complex hereafter) (11). Tomosyn inhibits SNARE complex formation by sequestering t-SNAREs through the tomosyn complex formation, and thereby inhibits SNARE-dependent neurotransmitter release. The large N-terminal region of tomosyn shares similarity to the Drosophila tumor suppressor lethal giant larvae (Lgl), the mammalian homologues M-Lgl1 and M-Lgl2, and yeast proteins Sro7p and Sro77p (12, 13). Consistent with the function of tomosyn, Lgl family members play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane in yeast and epithelial cells (12, 13). However, only tomosyn, Sro7, and Sro77 have the tail domains and the VLDs, suggesting that their structural regulation is evolutionally conserved. Recently, the crystal structure of Sro7 was solved and revealed that the tail domain of Sro7 binds its WD40 repeats (14). Sec9, a yeast counterpart of SNAP-25, also binds the WD40 repeats of Sro7. This binding inhibits the SNARE complex formation and exocytosis by sequestering Sec9. In addition, binding of the tail domain to the WD40 repeats causes a conformational change of Sro7 and prevents the interaction of the WD40 repeats with Sec9, leading to regulation of the inhibitory activity of Sro7 on the SNARE complex formation (14). However, the solved structure of Sro7 lacks its VLD. Therefore, involvement of the activity of the VLD in the conformational change of Sro7 remains elusive.Genetic studies in Caenorhabditis elegans showed that TOM-1, an ortholog of vertebrate tomosyn, inhibits the priming of synaptic vesicles, and that this priming is modulated by the balance between TOM-1 and UNC-13 (15, 16). Tomosyn was also shown to be involved in inhibition of the exocytosis of dense core granules in adrenal chromaffin cells and PC12 cells (17, 18). Thus, evidence is accumulating that tomosyn acts as a negative regulator for formation of the SNARE complex, thereby inhibiting various vesicle fusion events. However, the precise molecular mechanism regulating the inhibitory action of tomosyn has yet to be elucidated.In the present study, we show that the tail domain of tomosyn binds both the WD40 repeats and the VLD and functions as a regulator for the inhibitory activity of tomosyn on the SNARE complex formation. Our results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain serves for controlling the inhibitory activity of the VLD.     

Sec2p Mediates Nucleotide Exchange on Sec4p and Is Involved in Polarized Delivery of Post-Golgi Vesicles

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.     

HOPS Interacts with Apl5 at the Vacuole Membrane and Is Required for Consumption of AP-3 Transport Vesicles

Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae, AP-3 mediates vesicle traffic from the late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 δ subunit, Apl5, binds Vps41 directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi (Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles, interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or fusion.     

A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast

SEC4, one of the 10 genes involved in the final stage of the yeast secretory pathway, encodes a ras-like, GTP-binding protein. In wild-type cells, Sec4 protein is located on the cytoplasmic face of both the plasma membrane and the secretory vesicles in transit to the cell surface. In all post-Golgi blocked sec mutants, Sec4p is predominantly associated with the secretory vesicles that accumulate as a result of the secretory block. Sec4p is synthesized as a soluble protein that rapidly (t1/2 less than or equal to 1 min) and tightly associates with secretory vesicles and the plasma membrane by virtue of a conformational change of a covalent modification. These data suggest that Sec4p may function as a "G" protein on the vesicle surface to transduce an intracellular signal needed to regulate transport between the Golgi apparatus and the plasma membrane.     

Quantitative proteomics of yeast post-Golgi vesicles reveals a discriminating role for Sro7p in protein secretion

We here report the first comparative proteomics of purified yeast post-Golgi vesicles (PGVs). Vesicle samples isolated from PGV-accumulating sec6-4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle-associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.     

Modeling Curvature-Dependent Subcellular Localization of the Small Sporulation Protein SpoVM in Bacillus subtilis

Recent in vivo experiments suggest that in the bacterium, Bacillus subtilis, the cue for the localization of the small sporulation protein, SpoVM, an essential factor in spore coat formation, is curvature of the bacterial plasma membrane. In vitro measurements of SpoVM adsorption to vesicles of varying sizes also find high sensitivity of adsorption to vesicle radius. This curvature-dependent adsorption is puzzling given the orders of magnitude difference in length scale between an individual protein and the radius of curvature of the cell or vesicle, suggesting protein clustering on the membrane. Here we develop a minimal model to study the relationship between curvature-dependent membrane adsorption and clustering of SpoVM. Based on our analysis, we hypothesize that the radius dependence of SpoVM adsorption observed in vitro is governed primarily by membrane tension, while for in-vivo localization of SpoVM, we propose a highly sensitive mechanism for curvature sensing based on the formation of macroscopic protein clusters on the membrane.     

The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the “K loop,” which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.     

The role of Sec3p in secretory vesicle targeting and exocyst complex assembly

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.     

The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions.     

Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex

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.     

The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP‐25

The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNARE‐mediated membrane fusion. Recently, a new protein–protein interaction site in Sec1p, designated the groove, was proposed. Here, we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature‐sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18‐1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP‐25b, respectively. Incubation of SNAP‐25b with the Munc18‐1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro when compared with wild‐type Munc18‐1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p–SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP‐25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly.      

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.     

Attachment of 99mTc to lipid vesicles containing the lipophilic chelate dipalmitoylphosphatidylethanolamine-DTTA

The binding of ^99mTc to negatively-charged and neutral unilamellar lipid vesicles was investigated in the absence and presence of the ligand diethylenetriaminepentaacetic acid (DTPA) covalently attached to the headgroup of phosphatidylethanolamine at the surface of the membrane. Even in the absence of DTPA on the membrane surface, ^99mTc reduced by Sn bound to the membrane surface but rapidly dissociated from the vesicles in the presence of plasma in vitro. When DTPA was present on the membrane surface, dissociation of ^99mTc from the vesicle surface in plasma was much reduced. The dissociation of ^99mTc from the surface of negatively-charged vesicles was less than for neutral vesicles in the absence of ligand but was markedly greater than for vesicles containing the ligand DTPA, suggesting that the binding of ^99mTc to vesicles with surface-attached DTPA could not be explained solely on the basis of the negative charge provided by the DTPA. In vitro experiments using ¹⁴C-labeled lipids as well as in vivo imaging studies indicated that dissociation of ^99mTc from the surface of the vesicle did not arise predominantly because of lipid exchange with plasma components or due to cleavage of Tc-DTPA from the vesicle surface. For vesicles with surface-attached DTPA, ^99mTc dissociation from the vesicle surface in plasma was further reduced by addition of the antioxidant ascorbate.     

The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface

The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.     

Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation in Saccharomyces cerevisiae

During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.     

The Candida albicans Exocyst Subunit Sec6 Contributes to Cell Wall Integrity and Is a Determinant of Hyphal Branching

The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans.