Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes.

Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.

Dynamic Light Scattering Analysis to Dissect Intermediates of SNARE-Mediated Membrane Fusion.

Dynamic light scattering (DLS) spectroscopy provides rapid information on the size distribution of a large number of particles in a mixture. Vesicle sizes change during the merger of lipid bilayers, and DLS analysis can provide rapid, accurate, and non-perturbative quantification of the size distribution of proteoliposomes in SNARE-dependent membrane fusion. In this chapter, we describe the methodologies and reagents used for DLS spectroscopy in a biochemical and biophysical study of SNARE-mediated membrane fusion.

SNAREpin Assembly: Kinetic and Thermodynamic Approaches.

Proteins constantly interact and often form molecular complexes. The dynamics of most biological processes strongly rely on the kinetics and thermodynamics of assembly and disassembly of these complexes. Consequently an accurate characterization of these kinetics and thermodynamics that underlie them provides key information to better understand these processes. Here, we present two efficient techniques to quantify the assembly and disassembly of protein complexes: isothermal titration calorimetry and fluorescence anisotropy. As an example we focus on the formation of SNAREpins and also present how to prepare SNARE proteins to use in these experimental setups. We then show how to use these techniques to determine the critical factors that activate assembly kinetics.

Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly.

Intracellular membrane fusion mediates material and information exchange among different cells or cellular compartments with high accuracy and spatiotemporal resolution. Fusion is driven by ordered folding and assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) and regulated by many other proteins. Understanding regulated SNARE assembly is key to dissecting mechanisms and physiologies of various fusion processes and their associated diseases. Yet, it remains challenging to study regulated SNARE assembly using traditional ensemble-based experimental approaches. Here, we describe our new method to measure the energy and kinetics of neuronal SNARE assembly in the presence of α-SNAP, using a single-molecule manipulation approach based on high-resolution optical tweezers. Detailed experimental protocols and methods of data analysis are shown. This approach can be widely applied to elucidate the effects of regulatory proteins on SNARE assembly and membrane fusion.

Functional Reconstitution of Intracellular Vesicle Fusion Using Purified SNAREs and Sec1/Munc18 (SM) Proteins.

The fusion of intracellular vesicles with target membranes is mediated by two classes of conserved molecules-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAP receptors or SNAREs) and Sec1/Munc18 (SM) proteins. A conserved function of SM proteins is to recognize their cognate trans-SNARE complexes and accelerate fusion kinetics. Here, we describe a physiologically relevant reconstitution system in which macromolecular crowding agents are included to recapitulate the crowded intracellular environment. Through this system, we elucidate the molecular mechanisms by which SNAREs and SM proteins drive vesicle fusion.

Reconstituted Proteoliposome Fusion Mediated by Yeast SNARE-Family Proteins.

Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular membrane trafficking in all eukaryotic cells, which delivers proteins and lipids to their appropriate subcellular membrane compartments such as organelles and plasma membrane. The molecular basis of SNARE-mediated membrane fusion has been revealed by studying fusion of reconstituted proteoliposomes bearing purified SNARE-family proteins and chemically defined lipid species. This chapter describes the detailed experimental protocols for (1) purification of recombinant SNARE-family and SM (Sec1/Munc18-family) proteins in the yeast Saccharomyces cerevisiae; (2) preparation of reconstituted proteoliposomes bearing purified yeast SNARE proteins; and (3) developing an assay to monitor lipid mixing between reconstituted SNARE-bearing proteoliposomes. Lipid mixing assays for reconstituted SNARE-bearing proteoliposomes are useful for evaluating the intrinsic capacity of SNARE-family proteins to directly catalyze membrane fusion and to determine the specificity of membrane fusion.

In Vitro Reconstitution of Autophagosome-Lysosome Fusion.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins are a highly regulated class of membrane proteins lying in the center of membrane fusion. In conjunction with accessory proteins, SNAREs drive efficient merger of two distinct lipid bilayers into one interconnected structure. This chapter describes our fluorescence resonance energy transfer (FRET)-based proteoliposome fusion assays for the roles of various SNARE proteins, accessory proteins, and effects of different lipid compositions on membrane fusion involved in autophagy.

Detection of SNARE complexes with FRET using the tetracysteine system.

The three proteins synaptosomal-associated protein 25 (SNAP-25), Syntaxin-1a and vesicle-associated membrane protein (VAMP-2) are collectively called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). By assembling into an exocytic complex, the three SNAREs help in catalyzing membrane fusion. Due to lack of probes that adequately reconstitute the intracellular behavior of endogenous SNAREs, the dynamics of SNARE complexes in living cells is poorly understood. Here we describe a new FRET-based probe, called Cerulean-SNAP-25-C4 (CSNAC), that can track the conformational changes undergone by SNAP-25 as exocytic complexes assemble. The fluorescent protein Cerulean was attached to the N terminus and served as a FRET donor. The biarsenical dye FlAsH served as a FRET acceptor and was attached to a short tetracysteine motif (C4) motif inserted into the so-called linker domain of SNAP-25. CSNAC reported successive FRET changes when first Syntaxin-1a and then VAMP-2 were added in vitro. Small tetracysteine insertions used as a FRET acceptor are expected to have less steric hindrance than previously used GFP-based fluorophores. We propose that genetically-encoded tetracysteine tags can be used to study regulated SNARE complex assembly in vivo.

In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release.

Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca(2+) triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca(2+) injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca(2+) injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca(2+) sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca(2+) triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.

Unaltered SNARE complex formation in an in vivo model of prion disease.

The ME7 model of prion disease is a chronic slowly evolving model of neurodegeneration in which cell death is preceded by synaptic dysfunction. Previous studies in cell culture show that accumulation of misfolded prion inhibits the formation of the SNARE complexes involving synaptobrevin, syntaxin and SNAP-25 that play an essential role in neurotransmitter release. Such observations suggest that similar phenomenon may contribute to synaptic dysfunction observed in vivo. We have thus used detergent extraction of hippocampal tissue to investigate the status of SNARE complexes in the ME7 model. In the presence of increasing PrP(Sc) deposition we failed to see a change in the amount of SNARE complexes directly extracted into SDS and resolved by SDS-PAGE. Conversely pre-extraction in Triton X-100, a treatment that promotes SNARE complexes ex vivo, demonstrated a modest reduction in hippocampal SNARE complexes when homogenates were made from tissue at late stage disease. This suggests that accumulated PrP(Sc), or perhaps fibrillar complexes formed of prion only inhibit SNARE complexes that are formed ex vivo following biochemical extraction. Thus the accumulation of PrP(Sc) although deleterious to synaptic function in vivo, does not exert its synaptic effects by disrupting the formation of SNARE complexes that are core to transmitter release.

Mechanism of arachidonic acid action on syntaxin-Munc18.

Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.

Isolation and characterization of a novel v-SNARE family protein that interacts with a calcium-dependent protein kinase from the common ice plant, Mesembryanthemum crystallinum.

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydrationstress responsive. The enzyme also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system, we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2 in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin super-family that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTIl1a, an Arabidopsis v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma membrane along actin microfilaments of the cytoskeleton.

Quantitative proteomics analysis of the secretory pathway.

We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.