Calcium-dependent Regulation of SNARE-mediated Membrane Fusion by Calmodulin

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca²⁺ sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca²⁺-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca²⁺/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K_D = 500 nm) and syntaxin 1 (K_D = 2 μm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca²⁺ sensors act antagonistically in SNARE-mediated fusion.     

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.     

Secretory Vesicle Pools and Rate and Kinetics of Single Vesicle Exocytosis in Neurosecretory Cells

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca²⁺ and the cortical F-actin network. Cortical F-actin disassembly, a Ca²⁺-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.     

Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca²⁺ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr²⁺ and Ba²⁺ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca²⁺, Sr²⁺, and Ba²⁺-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>_Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>_Max and the Ca²⁺ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca²⁺-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca²⁺, Sr²⁺, and Ba²⁺, the complex remains Ca²⁺- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca²⁺ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca²⁺-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca²⁺ sensitivity of fusion.     

A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.     

Phosphatidylinositol-4,5-Bisphosphate and Phospholipase D-Generated Phosphatidic Acid Specify SNARE-Mediated Vesicle Fusion for Prospore Membrane Formation

The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of proteins is required for eukaryotic intracellular membrane fusions. Vesicle fusion for formation of the prospore membrane (PSM), a membrane compartment that forms de novo during yeast sporulation, requires SNARE function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂], and the activity of the phospholipase D (PLD) Spo14p, which generates phosphatidic acid (PA). The SNARE syntaxin Sso1p is essential for PSM production while the functionally redundant homolog in vegetative growth, Sso2p, is not. We demonstrate that Sso1p and Sso2p bind similarly in vitro to PA or phosphoinositide-containing liposomes and that the conserved SNARE (H3) domain largely mediates PA-binding. Both green fluorescent protein-Sso fusion proteins localize to the developing PSM in wild-type cells and to the spindle pole body in spo14Δ cells induced to sporulate. However, the autoregulatory region of Sso1p binds PI(4,5)P₂-containing liposomes in vitro with a greater ability than the equivalent region of Sso2p. Overexpression of the phosphatidylinositol-4-phosphate 5-kinase MSS4 in sso1Δ cells induced to sporulate stimulates PSM production; PLD activity is not increased under these conditions, indicating that PI(4,5)P₂ has roles in addition to stimulating PLD in PSM formation. These data suggest that PLD-generated PA and PI(4,5)P₂ collaborate at multiple levels to promote SNARE-mediated fusion for PSM formation.The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins is required for the fusion of vesicles to target membranes in eukaryotic cells (53). The process of SNARE-mediated fusion is both structurally and mechanistically similar in different intracellular transport pathways and is evolutionarily conserved from yeast to human (18, 31, 34). In vitro experiments demonstrated that SNAREs have the ability to effect fusions of liposomes in the absence of other components, indicating that these proteins directly mediate the fusion event (56). SNAREs can be broadly categorized as either vesicle SNAREs (v-SNAREs) or target membrane SNAREs (t-SNAREs), respectively. The interaction of SNAREs on apposed membranes can overcome the energy barrier generated by charged headgroups of lipids comprising the bilayers. As an incoming vesicle approaches its target membrane, the v-SNAREs and t-SNAREs assemble via their SNARE domains into a four-helix bundle termed a SNAREpin, bringing the two bilayers into closer proximity (3, 55, 56). The outer membrane layers of both the vesicle and target membrane mix, forming a hemifusion intermediate before full fusion of the membranes occurs (23, 24, 29, 58).The helices comprising the SNAREpin are supplied by three different SNARE subfamilies. Two of these subfamily members, syntaxin and SNAP-25, are t-SNAREs; the former contributes one helix while the latter contributes two helices (16). The syntaxin and SNAP-25 homologs heterodimerize to form the t-SNARE complex before the trans-interaction with the helix of vesicle-associated membrane protein/synaptobrevin v-SNARE (42). Discrete intracellular fusion events are mediated by SNAREpins comprising different constituent syntaxin, SNAP-25, and vesicle-associated membrane protein homologs (18, 53).In addition to SNAREs, lipids facilitate membrane fusion events for both membrane curvature induction required for procession through intermediate states of fusion and direct regulation of SNARE molecules (32, 33). Cone-shaped lipids such as diacylglycerol and phosphatidic acid (PA) induce negative (concave) curvature while inverted cone shapes, such as lysophosphatidic acid (LPA), have the opposite effect (26, 27). The assembly of SNARE complexes requires correct lipid composition at the fusion site; addition of inverted cone-shaped lipids antagonized in vitro SNARE complex assembly (35). Recent studies have shown that phosphatidylinositides also play roles in SNARE-mediated fusions. Phosphatidylinositol-3-phosphate [PI(3)P] interacts with the Saccharomyces cerevisiae SNARE Vam7p via its phox homology domain and appears to facilitate targeting to the vacuole (15). Additionally, phosphoinositides increased the rates of in vitro fusion of proteoliposomes that approximated physiological protein and lipids in vivo (36). Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] was shown to bind to the juxtamembrane region of syntaxin-1 in PC12 cells and has both stimulatory and inhibitory effects on in vitro fusion rates (20).The activity of the lipid-modifying enzyme phospholipase D (PLD) also appears to be important for vesicle fusions. PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to PA in a PI(4,5)P₂-dependent manner (22, 49). In S. cerevisiae, PLD activity is required for the de novo formation of a novel compartment, the prospore membrane (PSM), during sporulation (48). Vesicles trafficked from the Golgi and endosomal compartments dock at the spindle pole body (SPB) and participate in SNARE-mediated fusions for PSM formation (38, 40, 41). Cells induced to sporulate that lack the yeast PLD Spo14p show docked but unfused vesicles at the SPB (40, 44). Interestingly, cells lacking Sso1p, a syntaxin functionally redundant with Sso2p at the plasma membrane (PM), display a similar phenotype while sso2Δ cells display no sporulation defect (2, 21, 40). The specific requirement for Sso1p in sporulation is not fully understood although the Sso1p autoregulatory Habc motif is important (43).In this study, we demonstrate that Sso1p acts downstream of Spo14p (PLD)-generated PA during PSM formation. Sso1p and Sso2p bind PA and additional phosphoinositide species; PA binding is mediated by the conserved H3 motif. Additionally, the Sso1p Habc domain shows a greater ability to interact with PIP₂-containing liposomes in vitro than the equivalent region of Sso2p. Overexpression of the PI(4)P 5-kinase Mss4p results in PSM formation in sso1Δ cells induced to sporulate. Together, these data indicate that both PA and PI(4,5)P₂ are required for efficient fusion and furthermore suggest a novel role for PI(4,5)P₂ in the regulation of specialized SNARE fusion events.     

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca²⁺-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.     

In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein

The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.Dengue virus (DV) is a flavivirus that is spread by mosquitoes and causes millions of cases of disease each year worldwide (2, 9, 17). DV infection can result in dengue hemorrhagic fever, a more lethal disease that leads to ∼500,000 hospitalizations and ∼12,500 deaths per year (10, 39). DV is currently endemic in more than 100 countries, including the United States (17), and the World Health Organization estimates that about 40% of the world''s population lives in areas where dengue fever is endemic (39). As yet, there is no licensed DV vaccine or antiviral therapy. Studies of the molecular mechanisms of the virus life cycle are important to the development of new antiviral strategies.Flaviviruses such as DV are small, highly organized enveloped viruses with plus-sense single-stranded RNA genomes (reviewed in references 21 and 25). The flavivirus particle contains 3 structural proteins: a capsid protein, which associates with the genomic RNA to form the viral core, and two membrane proteins, the M protein and the membrane fusion protein E. Like many enveloped viruses, flaviviruses infect cells via endocytic uptake and a membrane fusion reaction triggered by the low pH within endosomes (38). Low-pH-triggered membrane fusion is mediated by conformational changes in the viral E protein, which converts from a prefusion E homodimer to a target membrane-inserted homotrimer. The structure of the DV E ectodomain in the prefusion form shows an elongated finger-like molecule with three domains (DI, DII, and DIII) composed primarily of β-sheets (22, 24, 42) (Fig. ​(Fig.1A;1A; see also Fig. ​Fig.7).7). The central DI is connected to DII. The distal tip of DII contains the hydrophobic fusion loop, the region of E that inserts into the target membrane during fusion. On the other side, DI connects via a short linker to DIII, an immunoglobulin-like domain. In the full-length viral E protein, DIII is followed by the stem, which contains 2 helical regions (H1 and H2) connected by a conserved sequence (CS). The stem connects to the C-terminal transmembrane (TM) anchor. The E-protein homodimer is arranged in a head-to-tail fashion, with the fusion loop on DII of each E protein hidden in a pocket formed by DI and DIII of its dimeric E partner.Open in a separate windowFIG. 1.Production and characterization of truncated DV2 E proteins. (A) Constructs used to express truncated forms of the DV2 E protein. At the top is a linear diagram of the full-length DV2 E protein, with DI indicated in red, DII in yellow, the fusion loop in green, the DI-DIII linker in cyan, DIII in dark blue, and the stem and TM regions in gray. L indicates the linker, and H1, CS, and H2 indicate the stem regions helix1, conserved sequence, and helix2, respectively. The residue numbers of the domain boundaries are listed below the diagram. The four S2 expression constructs primarily used in this work are shown in the middle rows. The E′-ST protein is truncated at residue 395 (DV2-NGC E-protein numbering), DI/II is truncated at residue 291, DI/II-L is truncated at residue 301, and the sequences are joined to the Strep or His tag (underlined) used for protein purification. The four DIII constructs are shown in the bottom rows, where LDIII comprises E residues 289 to 395, DIIIH1 residues 296 to 415, LDIIIH1 residues 289 to 415, and LDIIIH1CS residues 289 to 430. (B) Purified truncated E proteins were electrophoresed on SDS gels (left, 4 to 20% acrylamide; right, 10% acrylamide) under nonreducing conditions unless indicated and stained with Coomassie blue. The calculated mass of each protein (without modifications) is shown in kDa below each lane. DTT, dithiothreitol. (C) Sedimentation analysis of E proteins. Samples of purified E proteins were separated on sucrose sedimentation gradients in TAN buffer, pH 8.0, without detergent. Fractions were analyzed by SDS-PAGE, Western blotting, and Licor quantitation, all as described in Materials and Methods. Fraction 1 is the top of the gradient. (D) Inhibition of DV2 fusion by DIII proteins. Serial dilutions of DV2 were bound to BHK cells on ice and treated at pH 5.7 in the presence of the indicated DIII proteins at a final concentration of 50 μM or in buffer alone (control). Cells infected by virus fusion with the plasma membrane were quantitated by immunofluorescence. The data shown are the averages and standard deviations of three independent experiments.Open in a separate windowFIG. 7.Model for the steps in rearrangement of the dengue virus E protein during membrane fusion. DI, DII, and DIII are colored red, yellow, and blue, respectively. The hydrophobic fusion loop at the tip of DII is shown as a green star. The stem region is shown in gray and the TM domains in black. The virus membrane is shown in pink and the target membrane in blue. (I) At the top is shown the prefusion E-protein dimer, with the orientation looking down on the virus membrane. During the initial step of the fusion protein conformational change, the dimer dissociates upon exposure to low pH (bottom). (II to V) Side views of the trimerization reaction with the target membrane at the top. (II) The E fusion loops insert into the target membrane, and initial trimerization occurs between the DII tips. (III) Trimerization continues with contacts between DI and the β-strand exchange reaction. (IV) The DI-DIII linker inserts into the groove formed by strand exchange. DIII folds back against the core trimer, locking the linker into place. The trimer is now irreversible and stable in detergent. (V) In the final postfusion trimer, the stem has packed against the core trimer. The exact disposition of the fusion loops versus the stem and TM domains is not known, except that they are at the same end of the trimer, as shown in the model.Upon exposure to low pH, the homodimer dissociates and the E proteins insert their fusion loops into the target membrane and form very stable homotrimers (reviewed in reference 12). The structure of the DV E ectodomain trimer reveals that trimerization is mediated by dramatic domain movements (23, 26). The central region of the trimer is composed of DI and DII. DIII rotates by about 70°, folds back toward the target membrane, and packs against the grooves formed by DI and DII in the central trimer. During this refolding, part of the DI-DIII linker region inserts into a β-sheet of DI. These linker-DI rearrangements produce significant intersubunit contacts at the membrane-distal region of the trimer. The DV E protein stem region is not present in the trimer structure, but its length is sufficient to extend along the central trimer and connect with the TM domain. The final postfusion trimer thus has a hairpin-like conformation with the fusion loops and TM domains at the same end of the molecule. The pre- and postfusion structures of the alphavirus E1 protein (8, 18, 29) are very similar to those of the flavivirus E proteins, suggesting common features of membrane fusion between the two virus groups.Biogenesis of flavivirus particles occurs by budding into the endoplasmic reticulum (ER) and transit through the secretory pathway. The M protein is synthesized in the ER as a precursor protein termed prM, which forms a heterodimer with the E protein in the ER and on the nascent immature virus particle (19, 40, 41). Exposure to low pH in the trans-Golgi network mediates rearrangement of the viral envelope proteins and allows furin processing of prM to produce pr peptide and the mature M protein (32). The pr peptide remains associated with E throughout the low-pH environment of the secretory pathway, thereby protecting the virus from premature fusion until it is released from the cell (19, 40, 41). In the mature virus particle, the prefusion E homodimers are oriented tangentially to the virus membrane and form a herringbone-like pattern on the virus surface, essentially covering the virus membrane (16, 25).Thus, extensive structural information is available for both the DV E protein homodimer and the low-pH-induced E homotrimer. In contrast, the intermediates and mechanisms involved in the dramatic conformational transition from prefusion to postfusion E are relatively undefined. Recent studies of the flavivirus West Nile virus (WNV) suggest that an early fusion intermediate involves an extension of the stem region prior to dimer dissociation (15). Studies of the flavivirus tick-borne encephalitis (TBE) virus at pH 10 suggest that initial membrane insertion occurs via an E monomer (36). DV fusion and infection are inhibited by the addition of exogenous DIII during the E conformational change (20), implying that the central trimer region is formed before complete foldback of DIII. The presence of stem peptides can inhibit infection by DV and WNV, indicating the importance of stem interactions during hairpin formation (13). The dissociation of the TBE virus E dimer at low pH is dependent on a key histidine residue on DIII (H323; TBE virus numbering), which also promotes formation of the stable E trimer (5). However, studies of WNV indicate that viral E triggering is not controlled by protonation of a critical histidine residue (27). A better understanding of E-protein conformational changes during trimerization is important to define such intermediate steps and to evaluate their usefulness as targets for fusion inhibitors.Toward this end, in this study we expressed truncated forms of the DV E protein and used them to reconstitute steps in the trimerization reaction. This in vitro system allowed us to characterize the features of E protein involved in the formation of a stable central trimer and in DIII foldback. Our results suggest that monomeric DI/II proteins insert their fusion loops into target membranes and form a core trimer at either neutral or low pH. This core trimer is relatively unstable and can be stabilized by the binding of DIII, thus reconstituting hairpin formation.     

Fusion Proteins and Select Lipids Cooperate as Membrane Receptors for the Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Vam7p

Vam7p, the vacuolar soluble Q_c-SNARE, is essential for yeast vacuole fusion. The large tethering complex, homotypic fusion and vacuole protein sorting complex (HOPS), and phosphoinositides, which interact with the Vam7p PX domain, have each been proposed to serve as its membrane receptors. Studies with the isolated organelle cannot determine whether these receptor elements suffice and whether ligands or mutations act directly or indirectly on Vam7p binding to the membrane. Using pure components that are active in reconstituted vacuolar fusion, we now find that Vam7p binds to membranes through its combined affinities for several vacuolar membrane constituents: HOPS, phosphatidylinositol 3-phosphate, SNAREs, and acidic phospholipids. Acidic lipids allow low concentrations of Vam7p to suffice for fusion; without acidic lipids, the block to fusion is partially bypassed by high concentrations of Vam7p.     

CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.     

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.     

The Yeast ATP-binding Cassette (ABC) Transporter Ycf1p Enhances the Recruitment of the Soluble SNARE Vam7p to Vacuoles for Efficient Membrane Fusion

The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd²⁺ transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p^K669M) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.     

Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, “slow and fast,” emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a “hair trigger.” Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion.     

Multicolour Fluorescence-Detection Size-Exclusion Chromatography for Structural Genomics of Membrane Multiprotein Complexes

Many interesting and important membrane proteins are hetero-oligomeric. However, besides naturally abundant examples, the structures of relatively few such complexes are known. Partly, this is due to difficulties in expression, stoichiometric assembly, and in the evaluation of their stability prior to crystallization trials. Here we describe a new approach, which allows rapid assessment of protein complex quality, assembly and stoichiometry, simplifying the search for conditions conducive to long-term stability and crystallization. Multicolour fluorescence size-exclusion chromatography (MC-FSEC) is used to enable tracking of individual subunits through expression, solubilization and purification steps. We show how the method has been applied to the heterodimeric transporter associated with antigen processing (TAP) and demonstrate how it may be extended in order to analyse membrane multisubunit assemblies.     

Kinetics of complexin binding to the SNARE complex: correcting single molecule FRET measurements for hidden events

Virtually all measurements of biochemical kinetics have been derived from macroscopic measurements. Single-molecule methods can reveal the kinetic behavior of individual molecular complexes and thus have the potential to determine heterogeneous behaviors. Here we have used single-molecule fluorescence resonance energy transfer to determine the kinetics of binding of SNARE (soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor) complexes to complexin and to a peptide derived from the central SNARE binding region of complexin. A Markov model was developed to account for the presence of unlabeled competitor in such measurements. We find that complexin associates rapidly with SNARE complexes anchored in lipid bilayers with a rate constant of 7.0 × 10⁶ M⁻¹ s⁻¹ and dissociates slowly with a rate constant of 0.3 s⁻¹. The complexin peptide associates with SNARE complexes at a rate slower than that of full-length complexin (1.2 × 10⁶ M⁻¹ s⁻¹), and dissociates much more rapidly (rate constant >67 s⁻¹). Comparison of single-molecule fluorescence resonance energy transfer measurements made using several dye attachment sites illustrates that dye labeling of complexin can modify its rate of unbinding from SNAREs. These rate constants provide a quantitative framework for modeling of the cascade of reactions underlying exocytosis. In addition, our theoretical correction establishes a general approach for improving single-molecule measurements of intermolecular binding kinetics.     

Kinetics of Reduction of Fe(III) Complexes by Outer Membrane Cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

Because of their cell surface locations, the outer membrane c-type cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 have been suggested to be the terminal reductases for a range of redox-reactive metals that form poorly soluble solids or that do not readily cross the outer membrane. In this work, we determined the kinetics of reduction of a series of Fe(III) complexes with citrate, nitrilotriacetic acid (NTA), and EDTA by MtrC and OmcA using a stopped-flow technique in combination with theoretical computation methods. Stopped-flow kinetic data showed that the reaction proceeded in two stages, a fast stage that was completed in less than 1 s, followed by a second, relatively slower stage. For a given complex, electron transfer by MtrC was faster than that by OmcA. For a given cytochrome, the reaction was completed in the order Fe-EDTA > Fe-NTA > Fe-citrate. The kinetic data could be modeled by two parallel second-order bimolecular redox reactions with second-order rate constants ranging from 0.872 μM⁻¹ s⁻¹ for the reaction between MtrC and the Fe-EDTA complex to 0.012 μM⁻¹ s⁻¹ for the reaction between OmcA and Fe-citrate. The biphasic reaction kinetics was attributed to redox potential differences among the heme groups or redox site heterogeneity within the cytochromes. The results of redox potential and reorganization energy calculations showed that the reaction rate was influenced mostly by the relatively large reorganization energy. The results demonstrate that ligand complexation plays an important role in microbial dissimilatory reduction and mineral transformation of iron, as well as other redox-sensitive metal species in nature.     

Optimal Testing for Serial Correlation in a Large Number of Small Samples

In recent years, there has been an increased awareness of the potential one-sided nature of many testing problems in applied sciences. Usually, these testing problems can be reduced, either by conditioning on sufficient statistics or by invariant techniques. COX and SOLOMON (1988) considered testing the serial correlation coefficient of a stationary first order autoregressive process and concentrated on four independent samples, with each of size three. We outline a general method for testing the serial correlation coefficient, using locally best invariant, point optimal invariant and locally most mean powerful invariant test procedures. The first procedure optimizes power near the null hypothesis, the second optimizes it at a pre-determined point away from the null while the third optimizes the average curvature of the power hypersurface in the neighbourhood of the null hypothesis.     

Optimal Number of Embryos for Transplantation in Obtaining Genetic-Modified Mice and Goats

Russian Journal of Developmental Biology - The technology of creating genetically modified animals (placental mammals) by microinjection into the pronucleus of a fertilized egg suggests, as one of...