A t-SNARE of the endocytic pathway must be activated for fusion.

The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast. We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p. In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion. Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell. Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

Molecular mass, stoichiometry, and assembly of 20 S particles.

N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.

The use of pHluorins for optical measurements of presynaptic activity.

Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. pHluorins have a pK approximately 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theoretical analysis of the expected signals using this approach and guidelines for future reporter development are provided.

Compartmental specificity of cellular membrane fusion encoded in SNARE proteins.

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.

Topological restriction of SNARE-dependent membrane fusion.

To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1 (refs 2-6), each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex.

Functional architecture of an intracellular membrane t-SNARE.

Lipid bilayer fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, of which one is supplied by the v-SNARE and the other three by the t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and a SNAP-25 protein contributes the other two. Although there are numerous homologues of syntaxin on intracellular membranes, there are only two SNAP-25-related proteins in yeast, Sec9 and Spo20, both of which are localized to the plasma membrane and function in secretion and sporulation, respectively. What replaces SNAP-25 in t-SNAREs of intracellular membranes? Here we show that an intracellular t-SNARE is built from a 'heavy chain' homologous to syntaxin and two separate non-syntaxin 'light chains'. SNAP-25 may thus be the exception rather than the rule, having been derived from genes that encoded separate light chains that fused during evolution to produce a single gene encoding one protein with two helices.

Exclusion of golgi residents from transport vesicles budding from Golgi cisternae in intact cells.

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells. In both cases, these abundant cisternal constituents were strongly excluded from buds and vesicles. This result suggests that cisternal progression takes place substantially more slowly than most protein transport and therefore is unlikely to be the predominant mechanism of anterograde movement.

Megavesicles implicated in the rapid transport of intracisternal aggregates across the Golgi stack.

Engineered protein aggregates ranging up to 400 nm in diameter were selectively deposited within the cis-most cisternae of the Golgi stack following a 15 degrees C block. These aggregates are much larger than the standard volume of Golgi vesicles, yet they are transported across the stack within 10 min after warming the cells to 20 degrees C. Serial sectioning reveals that during the peak of anterograde transport, about 20% of the aggregates were enclosed in topologically free "megavesicles" which appear to pinch off from the rims of the cisternae. These megavesicles can explain the rapid transport of aggregates without cisternal progression on this time scale.

Anterograde flow of cargo across the golgi stack potentially mediated via bidirectional "percolating" COPI vesicles.

How do secretory proteins and other cargo targeted to post-Golgi locations traverse the Golgi stack? We report immunoelectron microscopy experiments establishing that a Golgi-restricted SNARE, GOS 28, is present in the same population of COPI vesicles as anterograde cargo marked by vesicular stomatitis virus glycoprotein, but is excluded from the COPI vesicles containing retrograde-targeted cargo (marked by KDEL receptor). We also report that GOS 28 and its partnering t-SNARE heavy chain, syntaxin 5, reside together in every cisterna of the stack. Taken together, these data raise the possibility that the anterograde cargo-laden COPI vesicles, retained locally by means of tethers, are inherently capable of fusing with neighboring cisternae on either side. If so, quanta of exported proteins would transit the stack in GOS 28-COPI vesicles via a bidirectional random walk, entering at the cis face and leaving at the trans face and percolating up and down the stack in between. Percolating vesicles carrying both post-Golgi cargo and Golgi residents up and down the stack would reconcile disparate observations on Golgi transport in cells and in cell-free systems.

Close is not enough: SNARE-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors.

Is membrane fusion an essentially passive or an active process? It could be that fusion proteins simply need to pin two bilayers together long enough, and the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning two bilayers, exerting force in the bilayer in one or another way to direct the fusion process. To distinguish these alternatives, we replaced one or both of the peptidic membrane anchors of exocytic vesicle (v)- and target membrane (t)-SNAREs (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor) with covalently attached lipids. Replacing either anchor with a phospholipid prevented fusion of liposomes by the isolated SNAREs, but still allowed assembly of trans-SNARE complexes docking vesicles. This result implies an active mechanism; if fusion occurred passively, simply holding the bilayers together long enough would have been sufficient. Studies using polyisoprenoid anchors ranging from 15-55 carbons and multiple phospholipid-containing anchors reveal distinct requirements for anchors of v- and t-SNAREs to function: v-SNAREs require anchors capable of spanning both leaflets, whereas t-SNAREs do not, so long as the anchor is sufficiently hydrophobic. These data, together with previous results showing fusion is inhibited as the length of the linker connecting the helical bundle-containing rod of the SNARE complex to the anchors is increased (McNew, J.A., T. Weber, D.M. Engelman, T.H. Sollner, and J.E. Rothman, 1999. Mol. Cell. 4:415-421), suggests a model in which one activity of the SNARE complex promoting fusion is to exert force on the anchors by pulling on the linkers. This motion would lead to the simultaneous inward movement of lipids from both bilayers, and in the case of the v-SNARE, from both leaflets.

SNAREpins are functionally resistant to disruption by NSF and alphaSNAP.

SNARE (SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.

Receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways.

Heat shock proteins (HSPs) derived from tumors or virally infected cells can stimulate antigen-specific CD8(+) T cell responses in vitro and in vivo. Although this antigenicity is known to arise from HSP-associated peptides presented to the immune system by major histocompatibility complex (MHC) class I molecules, the cell biology underlying this presentation process remains poorly understood. Here we show that HSP 70 binds to the surface of antigen presenting cells by a mechanism with the characteristics of a saturable receptor system. After this membrane interaction, processing and MHC class I presentation of the HSP-associated antigen can occur via either a cytosolic (transporter associated with antigen processing [TAP] and proteasome-dependent) or an endosomal (TAP and proteasome-independent) route, with the preferred pathway determined by the sequence context of the optimal antigenic peptide within the HSP-associated material. These findings not only characterize two highly efficient, specific pathways leading to the conversion of HSP-associated antigens into ligands for CD8(+) T cells, they also imply the existence of a mechanism for receptor-facilitated transmembrane transport of HSP or HSP-associated ligands from the plasma membrane or lumen of endosomes into the cytosol.

Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors.

It has recently been reported that N-ethylmaleimide-sensitive fusion ATPase (NSF) can fuse protein-free liposomes containing substantial amounts of 1,2-dioleoylphosphatidylserine (DOPS) and 1, 2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999). The authors impart physiological significance to this observation and propose to re-conceptualize the general role of NSF in fusion processes. We can confirm that isolated NSF can fuse liposomes of the specified composition. However, this activity of NSF is resistant to inactivation by N-ethylmaleimide and does not depend on the presence of alpha-SNAP (soluble NSF-attachment protein). Moreover, under the same conditions, either alpha-SNAP, other proteins apparently unrelated to vesicular transport (glyceraldehyde-3-phosphate dehydrogenase or lactic dehydrogenase) or even 3 mM magnesium ions can also cause lipid mixing. In contrast, neither NSF nor the other proteins nor magnesium had any significant fusogenic activity with liposomes composed of a biologically occurring mixture of lipids. A straightforward explanation is that the lipid composition chosen as optimal for NSF favors non-specific fusion because it is physically unstable when formed into liposomes. A variety of minor perturbations could then trigger coalescence.

Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70.

Heat shock proteins 70 (hsp70) derived from tissues and cells can elicit cytotoxic T lymphocyte (CTL) responses against peptides bound to hsp70. However, peptides can markedly differ in their affinity for hsp, and this potentially limits the repertoire of peptides available to induce CTL by the hsp immunization. Hybrid peptides consisting of a high-affinity ligand for the peptide-binding site of hsp70 joined to T cell epitopes by a glycine-serine-glycine linker were constructed. Immunization with hybrid peptides complexed to mouse hsp70 effectively primed specific CTL responses in mice and were more potent than T cell peptide epitopes alone with hsp70. In vivo immunization with hsp70 and hybrid peptides led to rejection of tumors expressing antigen with greater efficacy than immunization with peptide epitope plus hsp70. Induction of CTL responses occurred independently of CD4(+) T cells, suggesting that immunization directly primed antigen-presenting cells to elicit CD8(+) cytotoxic T cell responses without T cell help. Both peptide/hsp70 complexes and mouse hsp70 alone were able to induce cultures of mouse bone marrow-derived dendritic cells (DC) to release cytokines, including DC from endotoxin-resistant C57BL/10Sc mice. Thus, hsp70/hybrid peptide complexes can activate DC for cytokine release, providing a potential adjuvant effect that could bypass T cell help.

Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

The length of the flexible SNAREpin juxtamembrane region is a critical determinant of SNARE-dependent fusion.

The topology of a SNARE complex bridging two docked vesicles could act as a mechanical couple to do work on the lipid bilayer resulting in fusion. To test this, we prepared a series of modified SNARE proteins and determined their effects on SNARE-dependent membrane fusion. When two helix-breaking proline residues are introduced into the juxtamembrane region of VAMP, there is little or no effect on fusion, and the same change in syntaxin 1A only reduced the extent and rate of fusion by half. The insertion of a flexible linker between the transmembrane domain and the conserved coiled-coil domain only moderately affected fusion; however, fusion efficiency systematically decreased with increasing length of the linker. Together, these results rule out a requirement for helical continuity and suggest that distance is a critical factor for membrane fusion.

Rapid and efficient fusion of phospholipid vesicles by the alpha-helical core of a SNARE complex in the absence of an N-terminal regulatory domain.

A protease-resistant core domain of the neuronal SNARE complex consists of an alpha-helical bundle similar to the proposed fusogenic core of viral fusion proteins [Skehel, J. J. & Wiley, D. C. (1998) Cell 95, 871-874]. We find that the isolated core of a SNARE complex efficiently fuses artificial bilayers and does so faster than full length SNAREs. Unexpectedly, a dramatic increase in speed results from removal of the N-terminal domain of the t-SNARE syntaxin, which does not affect the rate of assembly of v-t SNARES. In the absence of this negative regulatory domain, the half-time for fusion of an entire population of lipid vesicles by isolated SNARE cores ( approximately 10 min) is compatible with the kinetics of fusion in many cell types.

Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs.

Membrane bilayer fusion has been shown to be mediated by v- and t-SNAREs initially present in separate populations of liposomes and to occur with high efficiency at a physiologically meaningful rate. Lipid mixing was demonstrated to involve both the inner and the outer leaflets of the membrane bilayer. Here, we use a fusion assay that relies on duplex formation of oligonucleotides introduced in separate liposome populations and report that SNARE proteins suffice to mediate complete membrane fusion accompanied by mixing of luminal content. We also find that SNARE-mediated membrane fusion does not compromise the integrity of liposomes.

Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors.

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.