Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Background

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.

Methodology/Principal Findings

Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.

Conclusion/Significance

Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.     

Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation

Background

Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited.

Methodology/Principal Findings

We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins.

Conclusion/Significance

These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.     

Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

Background

The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.

Methodology

To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.

Principal Findings

We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.

Conclusions/Signficance

Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.     

The syntaxins

Summary  

The SNARE hypothesis predicts that a family of SNAP receptors are localized to and function in diverse intracellular membrane compartments where membrane fusion processes take place. Syntaxins, the prototype family of SNARE proteins, have a carboxy-terminal tail-anchor and multiple coiled-coil domains. There are 15 members of the syntaxin family in the human genome and 7 syntaxin-like genes in the yeast Saccharomyces cerevisiae. In conjunction with other SNAREs and with the cytoplasmic NSF and SNAP proteins, syntaxins mediate vesicle fusion in diverse vesicular transport processes along the exocytic and the endocytic pathway. They are crucial components that both drive and provide specificity to the myriad vesicular fusion processes that characterize the eukaryotic cell.     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

Probing Subunit-Subunit Interactions in the Yeast Vacuolar ATPase by Peptide Arrays

Background

Vacuolar (H⁺)-ATPase (V-ATPase; V₁V_o-ATPase) is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where its proton pumping action serves to acidify subcellular organelles. In the plasma membrane of certain specialized tissues, V-ATPase functions to pump protons from the cytoplasm into the extracellular space. The activity of the V-ATPase is regulated by a reversible dissociation mechanism that involves breaking and re-forming of protein-protein interactions in the V₁-ATPase - V_o-proton channel interface. The mechanism responsible for regulated V-ATPase dissociation is poorly understood, largely due to a lack of detailed knowledge of the molecular interactions that are responsible for the structural and functional link between the soluble ATPase and membrane bound proton channel domains.

Methodology/Principal Findings

To gain insight into where some of the stator subunits of the V-ATPase associate with each other, we have developed peptide arrays from the primary sequences of V-ATPase subunits. By probing the peptide arrays with individually expressed V-ATPase subunits, we have identified several key interactions involving stator subunits E, G, C, H and the N-terminal domain of the membrane bound a subunit.

Conclusions

The subunit-peptide interactions identified from the peptide arrays complement low resolution structural models of the eukaryotic vacuolar ATPase obtained from transmission electron microscopy. The subunit-subunit interaction data are discussed in context of our current model of reversible enzyme dissociation.     

Fusogenic pairings of vesicle-associated membrane proteins (VAMPs) and plasma membrane t-SNAREs--VAMP5 as the exception

Background

Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities.

Methodology/Principal Findings

In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30–50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction.

Conclusions/Significance

These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.     

Crystal structure of the Habc domain of neuronal syntaxin from the squid <Emphasis Type="Italic">Loligo pealei</Emphasis> reveals conformational plasticity at its C-terminus

Background

Intracellular membrane fusion processes are mediated by the spatial and temporal control of SNARE complex assembly that results in the formation of a four-helical bundle, composed of one vesicle SNARE and three target membrane SNARE polypeptide chains. Syntaxins are essential t-SNAREs and are characterized by an N-terminal Habc domain, a flexible linker region, a coiled-coil or SNARE motif and a membrane anchor. The N-terminal Habc domain fulfills important regulatory functions while the coiled-coil motif, present in all SNAREs, is sufficient for SNARE complex formation, which is thought to drive membrane fusion.

Results

Here we report the crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei, s-syntaxin. Squid Habc crystallizes as a dimer and the monomer structure consists of a three-helical bundle. One molecule is strikingly similar to mammalian syntaxin 1A while the second one shows a structural deviation from the common fold in that the C-terminal part of helix C unwinds and adopts an extended conformation.

Conclusion

Conservation of surface residues indicates that the cytosolic part of s-syntaxin can adopt an auto-inhibitory closed conformation that may bind squid neuronal Sec1, s-Sec1, in the same manner as observed in structure of the rat nSec1/syntaxin 1A complex. Furthermore, despite the overall structural similarity, the observed changes at the C-terminus of one molecule indicate structural plasticity in neuronal syntaxin. Implications of the structural conservation and the changes are discussed with respect to potential Habc domain binding partners such as Munc13, which facilitates the transition from the closed to the open conformation.

     

Binding interactions control SNARE specificity in vivo

Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.     

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.     

Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

Mso1 is a novel component of the yeast exocytic SNARE complex

The yeast exocytic SNARE complex consists of one molecule each of the Sso1/2 target SNAREs, Snc1/2 vesicular SNAREs, and the Sec9 target SNARE, which form a fusion complex that is conserved in evolution. Another protein, Sec1, binds to the SNARE complex to facilitate assembly. We show that Mso1, a Sec1-interacting protein, also binds to the SNARE complex and plays a role in mediating Sec1 functions. Like Sec1, Mso1 bound to SNAREs in cells containing SNARE complexes (i.e. wild-type, sec1-1, and sec18-1 cells), but not in cells in which complex formation is inhibited (i.e. sec4-8 cells). Nevertheless, Mso1 remained associated with Sec1 even in sec4-8 cells, indicating that they act as a pair. Mso1 localized primarily to the plasma membrane of the bud when SNARE complex formation was not impaired but was mostly in the cytoplasm when assembly was prevented. Genetic studies suggest that Mso1 enhances Sec1 function while attenuating Sec4 GTPase function. This dual action may impart temporal regulation between Sec4 turnoff and Sec1-mediated SNARE assembly. Notably, a small region at the C terminus of Mso1 is conserved in the mammalian Munc13/Mint proteins and is necessary for proper membrane localization. Overexpression of Mso1 lacking this domain (Mso1-(1-193)) inhibited the growth of cells bearing an attenuated Sec4 GTPase. These results suggest that Mso1 is a component of the exocytic SNARE complex and a possible ortholog of the Munc13/Mint proteins.     

Plasma membrane targeting of exocytic SNARE proteins

SNARE proteins play a central role in the process of intracellular membrane fusion. Indeed, the interaction of SNAREs present on two opposing membranes is generally believed to provide the driving force to initiate membrane fusion. Eukaryotic cells express a large number of SNARE isoforms, and the function of individual SNAREs is required for specific intracellular fusion events. Exocytosis, the fusion of secretory vesicles with the plasma membrane, employs the proteins syntaxin and SNAP-25 as plasma membrane SNAREs. As a result, exocytosis is dependent upon the targeting of these proteins to the plasma membrane; however, the mechanisms that underlie trafficking of exocytic syntaxin and SNAP-25 proteins to the cell surface are poorly understood. The intracellular trafficking itinerary of these proteins is particularly intriguing as syntaxins are tail-anchored (or Type IV) membrane proteins, whereas SNAP-25 is anchored to membranes via a central palmitoylated domain-there is no common consensus for the trafficking of such proteins within the cell. In this review, we discuss the plasma membrane targeting of these essential exocytic SNARE proteins.     

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells

Background

Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear.

Methodology/Principal Finding

In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion.

Conclusion/Significance

Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.     

Topological arrangement of the intracellular membrane fusion machinery

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form a four-helix coiled-coil bundle that juxtaposes two bilayers and drives a basal level of membrane fusion. The Sec1/Munc18 (SM) protein binds to its cognate SNARE bundle and accelerates the basal fusion reaction. The question of how the topological arrangement of the SNARE helices affects the reactivity of the fusion proteins remains unanswered. Here we address the problem for the first time in a reconstituted system containing both SNAREs and SM proteins. We find that to be fusogenic a SNARE topology must support both basal fusion and SM stimulation. Certain topological combinations of exocytic SNAREs result in basal fusion but cannot support SM stimulation, whereas other topologies support SM stimulation without inducing basal fusion. It is striking that of all the possible topological combinations of exocytic SNARE helices, only one induces efficient fusion. Our results suggest that the intracellular membrane fusion complex is designed to fuse bilayers according to one genetically programmed topology.     

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.     

A search for synthetic peptides that inhibit soluble N-ethylmaleimide sensitive-factor attachment receptor-mediated membrane fusion

Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.     

Ordering the final events in yeast exocytosis

In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.     

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims

Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.

Methods

Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.

Key Results

VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.

Conclusions

These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.     

Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background

Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.

Methodology/Principal Findings

We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.

Conclusions/Significance

Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.