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

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.

Allosteric regulation of exocyst: Discrete activation of tethering by two spatial signals.

The exocyst imparts spatial control during exocytic vesicle tethering through its interactions with proteins and lipids on the vesicle and the plasma membrane. One such interaction is with the vesicle tether Sro7, although the outcome of this interaction is poorly understood. Here, we describe how Sro7 binding to the Exo84 subunit results in activation of the exocyst complex which leads to an increase in avidity for the Rab GTPase Sec4 and an increase in exocyst-mediated vesicle tethering. Gain-of-function (GOF) mutations in Exo84 that mimic Sro7 activation replicate these biochemical changes and result in allosteric changes within the complex. Direct comparison of GOF mutants which mimic Sro7- and Rho/Cdc42-activation of the exocyst reveals distinct mechanisms and outcomes. We propose a model by which these two activation pathways reside within the same tethering complex but remain insulated from one another. Structural modeling suggests a related mechanism for Sro7 activation of the exocyst in yeast and Ral GTPase activation of the exocyst in animal cells.

Schizophrenia-Linked Protein tSNARE1 Regulates Endosomal Trafficking in Cortical Neurons.

TSNARE1, which encodes the protein tSNARE1, is a high-confidence gene candidate for schizophrenia risk, but nothing is known about its cellular or physiological function. We identified the major gene products of TSNARE1 and their cytoplasmic localization and function in endosomal trafficking in cortical neurons. We validated three primary isoforms of TSNARE1 expressed in human brain, all of which encode a syntaxin-like Qa SNARE domain. RNA-sequencing data from adult and fetal human brain suggested that the majority of tSNARE1 lacks a transmembrane domain that is thought to be necessary for membrane fusion. Biochemical data demonstrate that tSNARE1 can compete with Stx12 for incorporation into an endosomal SNARE complex, supporting its possible role as an inhibitory SNARE. Live-cell imaging in cortical neurons from mice of both sexes demonstrated that brain tSNARE1 isoforms localized to the endosomal network. The most abundant brain isoform, tSNARE1c, localized most frequently to Rab7+ late endosomes, and endogenous tSNARE1 displayed a similar localization in human neural progenitor cells and neuroblastoma cells. In mature rat neurons from both sexes, tSNARE1 localized to the dendritic shaft and dendritic spines, supporting a role for tSNARE1 at the postsynapse. Expression of either tSNARE1b or tSNARE1c, which differ only in their inclusion or exclusion of an Myb-like domain, delayed the trafficking of the dendritic endosomal cargo Nsg1 into late endosomal and lysosomal compartments. These data suggest that tSNARE1 regulates endosomal trafficking in cortical neurons, likely by negatively regulating early endosomal to late endosomal trafficking.SIGNIFICANCE STATEMENT Schizophrenia is a severe and polygenic neuropsychiatric disorder. Understanding the functions of high-confidence candidate genes is critical toward understanding how their dysfunction contributes to schizophrenia pathogenesis. TSNARE1 is one of the high-confidence candidate genes for schizophrenia risk, yet nothing was known about its cellular or physiological function. Here we describe the major isoforms of TSNARE1 and their cytoplasmic localization and function in the endosomal network in cortical neurons. Our results are consistent with the hypothesis that the majority of brain tSNARE1 acts as a negative regulator to endolysosomal trafficking.

Exocyst structural changes associated with activation of tethering downstream of Rho/Cdc42 GTPases.

The exocyst complex plays a critical role in determining both temporal and spatial dynamics of exocytic vesicle tethering and fusion with the plasma membrane. However, the mechanism by which the exocyst functions and how it is regulated remain poorly understood. Here we describe a novel biochemical assay for the examination of exocyst function in vesicle tethering. Importantly, the assay is stimulated by gain-of-function mutations in the Exo70 component of the exocyst, selected for their ability to bypass Rho/Cdc42 activation in vivo. Single-particle electron microscopy and 3D reconstructions of negatively stained exocyst complexes reveal a structural change in the mutant exocyst that exposes a binding site for the v-SNARE. We demonstrate a v-SNARE requirement in our tethering assay and increased v-SNARE binding to exocyst gain-of-function complexes. Together, these data suggest an allosteric mechanism for activation involving a conformational change in one subunit of the complex, which is relayed through the complex to regulate its biochemical activity in vitro, as well as overall function in vivo.

Comprehensive Risk Management in Arrhythmogenic Cardiomyopathy Associated With Autosomal Dominant Carvajal Syndrome: Protecting Families.

In a 37-year-old cardiac arrest survivor with autosomal dominant Carvajal syndrome and arrhythmogenic cardiomyopathy, a desmoplakin mutation was identified. Cascade screening identified 2 affected family members and 2 healthy children carrying the mutation. Strategies for primary and secondary risk prevention emphasize the role of genetic testing in rare cardiomyopathies. (Level of Difficulty: Advanced.).

Eighty-two per cent of male professional football (soccer) players return to play at the previous level two seasons after Achilles tendon rupture treated with surgical repair.

OBJECTIVE: To evaluate the time to return to playing following acute Achilles tendon rupture (ATR) and surgical repair in professional male football (soccer) players. METHODS: Professional male football (soccer) players who sustained an ATR and underwent surgical repair were identified through internet-based injury reports from January 2008 to August 2018. Only League 1 and 2 players with injuries who had at least 1 year of follow-up from the search date were included. Injury history and time to return to play were retrieved from the public platform transfermarkt.com. For athletes who competed for at least two seasons after returning to play, re-ruptures and number of matches played were reported. RESULTS: 118 athletes (mean age 27.2±7.2 years) were included. 113 (96%) returned to unrestricted practice after a mean of 199±53 days, with faster recovery in players involved in national teams. Return to competition was after a mean of 274±114 days. In the 76 athletes with at least two seasons of follow-up, 14 (18%) did not compete at the pre-injury level during the two seasons following the index injury. Six players (8%) sustained a re-rupture within the first two seasons after return to play; four re-ruptures were in footballers who returned to play <180 days after injury. Age >30 years and re-ruptures had higher odds ratios of not returning to the same level of play. CONCLUSIONS: 96% of professional male football players who underwent surgery to repair an ATR returned to unrestricted practice and then competition after an average time of 7 and 9 months, respectively. However, 18% did not return to the same level of play within the two seasons following their return, with a higher risk in those experiencing a re-rupture.

The tomosyn homologue, Sro7, is a direct effector of the Rab GTPase, Sec4, in post-Golgi vesicle tethering.

The tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7's ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7's functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.

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 ß-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.

In vitro reconstitution of Rab GTPase-dependent vesicle clustering by the yeast lethal giant larvae/tomosyn homolog, Sro7.

Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers that tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here, we describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 that results in a protein that is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this, we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9, which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.

Quantitative analysis of membrane trafficking in regulation of Cdc42 polarity.

Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post-Golgi secretory vesicles in intact yeast cells, we: (1) determined that endocytosis plays an important role in Cdc42's association with secretory vesicles (2) found that a GFP-tag placed on the N-terminus of Cdc42 negatively impacts this vesicle association and (3) quantified the surface densities of Cdc42 on post-Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.

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.

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.

Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1.

At steady state, most Rho GTPases are bound in the cytosol to Rho guanine nucleotide dissociation inhibitors (RhoGDIs). RhoGDIs have generally been considered to hold Rho proteins passively in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while activating the remaining membrane-bound fraction. Because RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression itself but from additional effects on the levels and activity of other Rho GTPases as a result of competition for binding to RhoGDI1; this may require a re-evaluation of previously published studies that rely exclusively on these techniques.

The ghost in the machine: small GTPases as spatial regulators of exocytosis.

Temporal and spatial regulation of membrane-trafficking events is crucial to both membrane identity and overall cell polarity. Small GTPases of the Rab, Ral and Rho protein families have been implicated as important regulators of vesicle docking and fusion events. This review focuses on how these GTPases interact with the exocyst complex, which is a multisubunit tethering complex involved in the regulation of cell-surface transport and cell polarity. The Rab and Ral GTPases are thought to function in exocyst assembly and vesicle-tethering processes, whereas the Rho family GTPases seem to function in the local activation of the exocyst complex to facilitate downstream vesicle-fusion events. The localized activation of the exocyst by Rho GTPases is likely to have an important role in spatial regulation of exocytosis.

Spatial regulation of exocytosis and cell polarity: yeast as a model for animal cells.

Exocytosis is the major mechanism by which new membrane components are delivered to the cell surface. In most, if not all, eukaryotic cells this is also a highly spatially regulated process that is tightly coordinated with the overall polarity of a cell. The Rho/Cdc42 family of GTPases and the lethal giant larvae/Sro7 family are two highly conserved families of proteins which appear to have dual functions both in cell polarity and exocytosis. Analysis of their functions has begun to unravel the coordination between these processes and propose a model for polarized vesicle docking and fusion at the site of asymmetric cell growth.

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.

Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex.

Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3-Exo70 and Rho1-Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.

Structurally conserved interaction of Lgl family with SNAREs is critical to their cellular function.

The Lethal giant larvae (Lgl) tumor suppressor family is conserved from yeast to mammals and plays a critical yet controversial role in cell polarity. Studies on Drosophila Lgl suggest that its function in polarity is through regulation of the acto-myosin cytoskeleton. In contrast, studies on the yeast Lgl homologs, Sro7/Sro77, suggest a function in exocytosis through interaction with the t-SNARE Sec9. Using yeast/mammalian Lgl chimeras, we demonstrate that the overall architecture of Lgl proteins is highly conserved and that the C-terminal domain is the major site of SNARE interaction within both yeast and mammalian homologs. Importantly, we find that the ability of Lgl chimeras to function as the only source of Lgl in yeast correlates precisely with the ability to interact with the yeast t-SNARE. We report a novel interaction between Sro7 and the yeast myosin V, Myo2. However, we find that interactions with either Myo2 or Myo1 (myosin II) cannot account for the dramatic functional differences observed for these chimeras in yeast. These results provide the first demonstration that the interaction of an Lgl family member with a specific effector is critical to its function in vivo. These data support the model that the Lgl family functions in cell polarity, at least in part, by regulating SNARE-mediated membrane delivery events at the cell surface.

The yeast par-1 homologs kin1 and kin2 show genetic and physical interactions with components of the exocytic machinery.

Kin1 and Kin2 are Saccharomyces cerevisiae counterparts of Par-1, the Caenorhabditis elegans kinase essential for the establishment of polarity in the one cell embryo. Here, we present evidence for a novel link between Kin1, Kin2, and the secretory machinery of the budding yeast. We isolated KIN1 and KIN2 as suppressors of a mutant form of Rho3, a Rho-GTPase acting in polarized trafficking. Genetic analysis suggests that KIN1 and KIN2 act downstream of the Rab-GTPase Sec4, its exchange factor Sec2, and several components of the vesicle tethering complex, the Exocyst. We show that Kin1 and Kin2 physically interact with the t-SNARE Sec9 and the Lgl homologue Sro7, proteins acting at the final stage of exocytosis. Structural analysis of Kin2 reveals that its catalytic activity is essential for its function in the secretory pathway and implicates the conserved 42-amino acid tail at the carboxy terminal of the kinase in autoinhibition. Finally, we find that Kin1 and Kin2 induce phosphorylation of t-SNARE Sec9 in vivo and stimulate its release from the plasma membrane. In summary, we report the finding that yeast Par-1 counterparts are associated with and regulate the function of the exocytic apparatus via phosphorylation of Sec9.