TgMORN1 Is a Key Organizer for the Basal Complex of Toxoplasma gondii

Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (ΔTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in ΔTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, ΔTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis.     

The SNARE Machinery Is Involved in Apical Plasma Membrane Trafficking in MDCK Cells

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide–sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and α-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.     

Stimulation of NSF ATPase Activity by α-SNAP Is Required for SNARE Complex Disassembly and Exocytosis

N-ethylmaleimide–sensitive fusion protein (NSF) and α-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH₂- and COOH-terminal truncation mutants of α-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH₂-terminal amino acids had little effect on the ability of α-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH₂-terminal (1–160) and COOH-terminal (160–295) fragments of α-SNAP were able to bind to NSF, suggesting that α-SNAP contains distinct NH₂- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of α-SNAP. Mutation of leucine 294 to alanine (α-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. α-SNAP (1–285) and α-SNAP (L294A) were unable to stimulate Ca²⁺-dependent exocytosis in permeabilized chromaffin cells. In addition, α-SNAP (1–285), and α-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous α-SNAP. α-SNAP, α-SNAP (1–285), and α-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, α-SNAP (1–285) and α-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that α-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the α-SNAP stimulation of exocytosis.     

A Coiled Coil Trigger Site Is Essential for Rapid Binding of Synaptobrevin to the SNARE Acceptor Complex

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight α-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1·SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil “trigger site.” This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1·SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.     

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

In neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca²⁺ sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc18 proteins in exocytosis. However, when full-length Syt1 is present, no enhancement of SNARE zippering and no acceleration of Ca²⁺-triggered content mixing by Munc18-1 are observed. Thus, our results show that Syt1 acts as an antagonist for Munc18-1 in SNARE zippering and fusion pore opening. Although the Sec1/Munc18 family may serve as part of the fusion machinery in other exocytotic pathways, Munc18-1 may have evolved to play a different role, such as regulating syntaxin-1a in neuroexocytosis.     

Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment

Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5′UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5′UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5′UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site.     

Protein N-terminal Acetylation by the NatA Complex Is Critical for Selective Mitochondrial Degradation

Mitophagy is an evolutionarily conserved autophagy pathway that selectively degrades mitochondria. Although it is well established that this degradation system contributes to mitochondrial quality and quantity control, mechanisms underlying mitophagy remain largely unknown. Here, we report that protein N-terminal acetyltransferase A (NatA), an enzymatic complex composed of the catalytic subunit Ard1 and the adaptor subunit Nat1, is crucial for mitophagy in yeast. NatA is associated with the ribosome via Nat1 and acetylates the second amino acid residues of nascent polypeptides. Mitophagy, but not bulk autophagy, is strongly suppressed in cells lacking Ard1, Nat1, or both proteins. In addition, loss of NatA enzymatic activity causes impairment of mitochondrial degradation, suggesting that protein N-terminal acetylation by NatA is important for mitophagy. Ard1 and Nat1 mutants exhibited defects in induction of Atg32, a protein essential for mitophagy, and formation of mitochondria-specific autophagosomes. Notably, overexpression of Atg32 partially recovered mitophagy in NatA-null cells, implying that this acetyltransferase participates in mitophagy at least in part via Atg32 induction. Together, our data implicate NatA-mediated protein modification as an early regulatory step crucial for efficient mitophagy.     

SNARE Complex Zipping as a Driving Force in the Dilation of Proteinaceous Fusion Pores

The assembly of SNARE proteins into a tight complex has been hypothesized to drive membrane fusion. A model of the initial fusion pore as a proteinaceous channel formed by SNARE proteins places their membrane anchors in separate membranes. This leaves the possibility of a final assembly step that brings the membrane anchors together and drives fusion pore expansion. The present study develops a model for expansion in which the final SNARE complex zipping step drives a transition from a proteinaceous fusion pore to a lipidic fusion pore. An estimate of the energy released upon merger of the helical segments of the SNARE motifs with the helical segments of the membrane anchors indicates that completing the assembly of a few SNARE complexes can overcome the elastic energy that opposes lipid bilayer deformation into a narrow fusion pore. The angle between the helical axes of the membrane anchor and SNARE motif serves as a useful reaction coordinate for this transition. Energy was calculated as a function of this angle, incorporating contributions from membrane bending, SNARE complex assembly, membrane anchor flexing and hydrophobic interactions. The rate of this transition was evaluated as a process of diffusion over the barrier imposed by these combined energies, and the rates estimated were consistent with experimental measurements.     

AtVPS45 Is a Positive Regulator of the SYP41/SYP61/VTI12 SNARE Complex Involved in Trafficking of Vacuolar Cargo

We report a functional characterization of AtVPS45 (for vacuolar protein sorting 45), a protein from the Sec1/Munc18 family in Arabidopsis (Arabidopsis thaliana) that interacts at the trans-Golgi network (TGN) with the SYP41/SYP61/VTI12 SNARE complex. A null allele of AtVPS45 was male gametophytic lethal, whereas stable RNA interference lines with reduced AtVPS45 protein levels had stunted growth but were viable and fertile. In the silenced lines, we observed defects in vacuole formation that correlated with a reduction in cell expansion and with autophagy-related defects in nutrient turnover. Moreover, transport of vacuolar cargo with carboxy-terminal vacuolar sorting determinants was blocked in the silenced lines, suggesting that AtVPS45 functions in vesicle trafficking to the vacuole. These trafficking defects are similar to those observed in vti12 mutants, supporting a functional relationship between AtVPS45 and VTI12. Consistent with this, we found a decrease in SYP41 protein levels coupled to the silencing of AtVPS45, pointing to instability and malfunction of the SYP41/SYP61/VTI12 SNARE complex in the absence of its cognate Sec1/Munc18 regulator. Based on its localization on the TGN, we hypothesized that AtVPS45 could be involved in membrane fusion of retrograde vesicles recycling vacuolar trafficking machinery. Indeed, in the AtVPS45-silenced plants, we found a striking alteration in the subcellular fractionation pattern of vacuolar sorting receptors, which are required for sorting of carboxy-terminal vacuolar sorting determinant-containing cargo. We propose that AtVPS45 is essential for recycling of the vacuolar sorting receptors back to the TGN and that blocking this step underlies the defects in vacuolar cargo trafficking observed in the silenced lines.     

Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.     

Munc18-1 Controls SNARE Protein Complex Assembly during Human Sperm Acrosomal Exocytosis

The spermatozoon is a very specialized cell capable of carrying out a limited set of functions with high efficiency. Sperm are then excellent model cells to dissect fundamental processes such as regulated exocytosis. The secretion of the single dense-core granule of mammalian spermatozoa relies on the same highly conserved molecules and goes through the same stages as exocytosis in other types of cells. In this study, we describe the presence of Munc18-1 in human sperm and show that this protein has an essential role in acrosomal exocytosis. We observed that inactivation of endogenous Munc18-1 with a specific antibody precluded the stabilization of trans-SNARE complexes and inhibited acrosomal exocytosis. Addition of recombinant Munc18-1 blocked secretion by sequestering monomeric syntaxin, an effect that was rescued by α-soluble NSF attachment protein. By electron microscopy, we observed that both the anti-Munc18-1 antibody and recombinant Munc18-1 inhibited the docking of the acrosome to the plasma membrane. In conclusion, our results indicate that Munc18-1 plays a key role in the dynamics of trans-SNARE complex assembly and/or stabilization, a process that is necessary for the docking of the outer acrosomal membrane to the plasma membrane and subsequent fusion pore opening.     

Subtle Interplay between Synaptotagmin and Complexin Binding to the SNARE Complex

Ca^2 +-triggered neurotransmitter release depends on the formation of SNARE complexes that bring the synaptic vesicle and plasma membranes together, on the Ca^2 + sensor synaptotagmin-1 and on complexins, which play active and inhibitory roles. Release of the complexin inhibitory activity by binding of synaptotagmin-1 to the SNARE complex, causing complexin displacement, was proposed to trigger exocytosis. However, the validity of this model was questioned based on the observation of simultaneous binding of complexin-I and a fragment containing the synaptotagmin-1 C₂ domains (C₂AB) to membrane-anchored SNARE complex. Using diverse biophysical techniques, here we show that C₂AB and complexin-I do not bind to each other but can indeed bind simultaneously to the SNARE complex in solution. Hence, the SNARE complex contains separate binding sites for both proteins. However, total internal reflection fluorescence microscopy experiments show that C₂AB can displace a complexin-I fragment containing its central SNARE-binding helix and an inhibitory helix (Cpx26-83) from membrane-anchored SNARE complex under equilibrium conditions. Interestingly, full-length complexin-I binds more tightly to membrane-anchored SNARE complex than Cpx26-83, and it is not displaced by C₂AB. These results show that interactions of N- and/or C-terminal sequences of complexin-I with the SNARE complex and/or phospholipids increase the affinity of complexin-I for the SNARE complex, hindering dissociation induced by C₂AB. We propose a model whereby binding of synaptotagmin-1 to the SNARE complex directly or indirectly causes a rearrangement of the complexin-I inhibitory helix without inducing complexin-I dissociation, thus relieving the inhibitory activity and enabling cooperation between synaptotagmin-1 and complexin-I in triggering release.     

Dual Roles of the Mammalian GARP Complex in Tethering and SNARE Complex Assembly at the trans-Golgi Network

Tethering factors and SNAREs control the last two steps of vesicular trafficking: the initial interaction and the fusion, respectively, of transport vesicles with target membranes. The Golgi-associated retrograde protein (GARP) complex regulates retrograde transport from endosomes to the trans-Golgi network (TGN). Although GARP has been proposed to function as a tethering factor at the TGN, direct evidence for such a role is still lacking. Herein we report novel and specific interactions of the mammalian GARP complex with SNAREs that participate in endosome-to-TGN transport, namely, syntaxin 6, syntaxin 16, and Vamp4. These interactions depend on the N-terminal regions of Vps53 and Vps54 and the SNARE motif of the SNAREs. We show that GARP functions upstream of the SNAREs, regulating their localization and assembly into SNARE complexes. However, interactions of GARP with SNAREs are insufficient to promote retrograde transport, because deletion of the C-terminal region of Vps53 precludes GARP function without affecting GARP-SNARE interactions. Finally, we present in vitro data consistent with a tethering role for GARP, which is disrupted by deletion of the Vps53 C-terminal region. These findings indicate that GARP orchestrates retrograde transport from endosomes to the TGN by promoting vesicle tethering and assembly of SNARE complexes in consecutive, independent steps.Conveyance of cargo among organelles of the secretory and endosomal-lysosomal pathways is mediated by transport vesicles that bud from a donor compartment and fuse with an acceptor compartment in a specific and regulated manner (2, 25, 42). The accuracy and efficiency of vesicle fusion with the target compartment are provided by the concomitant actions of at least three protein families: tethers, small GTPases, and SNAREs. The general view is that a transport vesicle first finds its target organelle through interaction with tethering factors and then fuses with it through assembly of SNARE proteins while small GTPases of the Rab and Arl subfamilies orchestrate multiple steps of the overall process (1, 38, 44). The mechanistic details, however, are far from being completely understood and might vary depending on the transport pathway considered.Tethering represents the first step in the interaction between a transport vesicle and its target membrane and results in the formation of physical links between two membranes that are bound to fuse. Two types of tethering factor, long coiled-coil proteins (e.g., p115, GCC185, and GM-130) and multisubunit complexes (e.g., HOPS/Vps-C, exocyst, COG, and GARP/VFT) have been implicated in nearly all vesicular transport routes (19, 38), although their direct role in connecting two opposing membranes has been documented for only a few (7, 40). Fusion is triggered by the assembly of SNAREs on the transport vesicle (v-SNAREs) with their cognate SNAREs on the target membranes (t-SNAREs) to form a SNARE pin or SNARE complex (12, 35). SNARE complex assembly involves the formation of a four-helix bundle that drives fusion of the two lipid bilayers (10, 14). Small GTPases participate in the initial recruitment of tethering factors and other peripherally associated effectors to specific locations on membranes, as well as in the subsequent fusion events (21). For example, the long coiled-coil protein GCC185 binds different GTPases, Rab9 on transport vesicles through the middle part and Rab6 and Arl1 at the trans-Golgi network (TGN) through the C-terminal part, thereby facilitating the recognition and connection of both membrane-bound compartments (11, 33). Other coiled-coil tethers have the ability to bind several different Rabs through domains that are not required for Golgi apparatus targeting. This supports a general model for a tentacular Golgi complex in which coiled-coil proteins capture and retain Rab-containing vesicles (33).In addition to bringing together transport vesicles with target organelles, tethers may also regulate SNARE complex assembly, thus coordinating these two steps of vesicular transport. Several examples of tether-SNARE interactions have been reported, but no consensus for a mechanism of interaction or functional significance has yet emerged. For example, the HOPS complex associates with v- and t-SNARE complexes on Saccharomyces cerevisiae vacuoles both before and after fusion (37). Sec6p, a member of the exocyst complex, binds to the plasma membrane t-SNARE Sec9p, preventing its interaction with the cognate t-SNARE Sso1p (34). The COG complex binds the Golgi t-SNARE syntaxin 5 and enhances intra-Golgi SNARE complex stability (29). The long coiled-coil protein p115 also stimulates SNARE complex assembly (30).The Golgi-associated retrograde protein (GARP) complex, also named the Vps fifty-three (VFT) complex, together with COG and the exocyst, belongs to the quatrefoil family of multisubunit tethering complexes (43), a structurally diverse group of peripheral membrane protein assemblies. Defects in the GARP, COG, or exocyst complexes cause accumulation of untethered vesicles that are scattered throughout the cytoplasm and contain different cargo proteins (18, 20, 45, 47). Direct proof of a tethering function for the GARP complex is still lacking, although its inactivation leads to defects consistent with a prominent role in the fusion of endosome-derived transport intermediates with the TGN (4-6, 20, 31). The yeast GARP complex is composed of four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Mutations in any of these subunits impair the retrieval of the secretory vesicle v-SNARE Snc1p and the carboxypeptidase Y receptor, Vps10p, from endosomes (5, 23, 32). The mammalian GARP complex also comprises Vps52, Vps53, and Vps54 subunits, but no Vps51 subunit has been identified to date (13). Depletion of the mammalian GARP complex prevents the delivery of Shiga toxin B subunit and the retrieval of TGN-localized proteins, such as TGN46, from endosomes to the TGN (20). Moreover, GARP depletion blocks the recycling of the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the TGN, leading to missorting of the CI-MPR cargo, lysosomal hydrolases, into the extracellular space (20). The essential nature of mammalian GARP function in endosome-to-TGN transport is highlighted by the embryonic lethality of mice with ablation of the Vps54 subunit gene (27) and the motor neuron degeneration of Wobbler mice bearing a Vps54 hypomorphic mutation (27).In yeast, the GARP subunit Vps51p specifically binds to the conserved N-terminal regulatory domain of the t-SNARE Tlg1p (5, 32). This finding led to the proposal that GARP tethers endosome-derived vesicles through its interaction with Tlg1p. However, deletions or point mutations that eliminate the binding of Vps51p to Tlg1p do not show any functional phenotype in vivo (8). Binding of Tlg1p to Vps51p is thus not essential for GARP-mediated vesicle tethering. In this work, we set out to study the possible link between the mammalian GARP complex and SNAREs. We found that GARP specifically and directly interacts with SNAREs that participate in the endosome-to-TGN retrograde route (i.e., syntaxin 6 [Stx6], Stx16, and Vamp4). These interactions depend on the fusion-inducing SNARE “motif” of the SNAREs and the N-terminal regions of Vps53 and Vps54. Functional analyses place the GARP complex upstream of the SNAREs, regulating their localization and assembly into SNARE complexes. In addition, we demonstrate that the GARP complex has a vesicle tethering function independent of its interaction with the SNAREs.     

Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase.