Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor

The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.

Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.

Differential Roles of Syntaxin 7 and Syntaxin 8 in Endosomal Trafficking

To understand molecular mechanisms that regulate the intricate and dynamic organization of the endosomal compartment, it is important to establish the morphology, molecular composition, and functions of the different organelles involved in endosomal trafficking. Syntaxins and vesicle-associated membrane protein (VAMP) families, also known as soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), have been implicated in mediating membrane fusion and may play a role in determining the specificity of vesicular trafficking. Although several SNAREs, including VAMP3/cellubrevin, VAMP8/endobrevin, syntaxin 13, and syntaxin 7, have been localized to the endosomal membranes, their precise localization, biochemical interactions, and function remain unclear. Furthermore, little is known about SNAREs involved in lysosomal trafficking. So far, only one SNARE, VAMP7, has been localized to late endosomes (LEs), where it is proposed to mediate trafficking of epidermal growth factor receptor to LEs and lysosomes. Here we characterize the localization and function of two additional endosomal syntaxins, syntaxins 7 and 8, and propose that they mediate distinct steps of endosomal protein trafficking. Both syntaxins are found in SNARE complexes that are dissociated by α-soluble NSF attachment protein and NSF. Syntaxin 7 is mainly localized to vacuolar early endosomes (EEs) and may be involved in protein trafficking from the plasma membrane to the EE as well as in homotypic fusion of endocytic organelles. In contrast, syntaxin 8 is likely to function in clathrin-independent vesicular transport and membrane fusion events necessary for protein transport from EEs to LEs.

Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex.

We analyzed whether synaptic membrane trafficking proteins are substrates for casein kinase II, calcium/calmodulin-dependent protein kinase II, and cAMP-dependent protein kinase (PKA), three kinases implicated in the modulation of synaptic transmission. Each kinase phosphorylates a specific set of the vesicle proteins syntaxin 1A, N-ethylmaleimide-sensitive factor (NSF), vesicle-associated membrane protein (VAMP), synaptosome-associated 25-kDa protein (SNAP-25), n-sec1, alpha soluble NSF attachment protein (alpha SNAP), and synaptotagmin. VAMP is phosphorylated by calcium/calmodulin-dependent protein kinase II on serine 61. alpha SNAP is phosphorylated by PKA; however, the beta SNAP isoform is phosphorylated only 20% as efficiently. alpha SNAP phosphorylated by PKA binds to the core docking and fusion complex 10 times weaker than the dephosphorylated form. These studies provide a first glimpse at regulatory events that may be important in modulating neurotransmitter release during learning and memory.

Identification of a second homolog of N-ethylmaleimide-sensitive fusion protein that is expressed in the nervous system and secretory tissues of Drosophila.

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. Recently, cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. We now report the identification of a second homolog of NSF, called dNSF-2 within this species and report evidence that this ubiquitous and widely utilized fusion protein belongs to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1), 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases. dNSF-2 is localized to a region between bands 87F12 and 88A3 on chromosome 3, and in situ hybridization techniques revealed expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis.

Purification of the β-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells

The β cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in β cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated β cells. The human cDNA coding for GSF/IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line αTC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed βTC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF/IPF-1, which may be present in limiting amounts in normal as opposed to transformed β cells.

Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex1

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O2-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.

Development of an in vitro reconstitution assay for glucose transporter 4 translocation

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca2+-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5′-[γ-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein–protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca2+ chelator EGTA. Endosome fusion was restored by the addition of Ca2+ with an optimum at a free Ca2+ concentration of 0.3 × 10−6 M. Other divalent cations did not substitute for Ca2+. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca2+. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca2+ from the endosomal interior.

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca2+-dependent activator protein for secretion), a protein required for Ca2+-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca2+ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca2+ dependencies. A threshold of ≈10 μM Ca2+ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca2+ activity < 10 μM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca2+ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Selective Formation of Sed5p-containing SNARE Complexes Is Mediated by Combinatorial Binding Interactions

Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo. We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE–SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts (≈25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

Three SNARE complexes cooperate to mediate membrane fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the syntaxin, SNAP-25, and VAMP families mediate intracellular membrane fusion through the formation of helical bundles that span opposing membranes. Soluble SNARE domains that lack their integral membrane anchors inhibit membrane fusion by forming nonfunctional complexes with endogenous SNARE proteins. In this study we investigate the dependence of membrane fusion on the concentration of a soluble SNARE coil domain derived from VAMP2. The increase in the inhibition of fusion observed with increasing concentration of inhibitor is best fit to a function that suggests three SNARE complexes cooperate to mediate fusion of a single vesicle. These three complexes likely contribute part of a protein and lipidic fusion pore.

Formation and Turnover of NSF- and SNAP-containing “Fusion” Complexes Occur on Undocked, Clathrin-coated Vesicle–derived Membranes

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle–derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules >20 Å and that transcytosis is an N-ethylmaleimide–sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein–lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.