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1.  Yeast Sec1p Functions before and after Vesicle Docking 
Molecular Biology of the Cell  2009;20(22):4673-4685.
Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.
PMCID: PMC2777098  PMID: 19776355
2.  Single Particle Fluorescence Burst Analysis of Epsin Induced Membrane Fission 
PLoS ONE  2015;10(3):e0119563.
Vital cellular processes, from cell growth to synaptic transmission, rely on membrane-bounded carriers and vesicles to transport molecular cargo to and from specific intracellular compartments throughout the cell. Compartment-specific proteins are required for the final step, membrane fission, which releases the transport carrier from the intracellular compartment. The role of fission proteins, especially at intracellular locations and in non-neuronal cells, while informed by the dynamin-1 paradigm, remains to be resolved. In this study, we introduce a highly sensitive approach for the identification and analysis of membrane fission machinery, called burst analysis spectroscopy (BAS). BAS is a single particle, free-solution approach, well suited for quantitative measurements of membrane dynamics. Here, we use BAS to analyze membrane fission induced by the potent, fission-active ENTH domain of epsin. Using this method, we obtained temperature-dependent, time-resolved measurements of liposome size and concentration changes, even at sub-micromolar concentration of the epsin ENTH domain. We also uncovered, at 37°C, fission activity for the full-length epsin protein, supporting the argument that the membrane-fission activity observed with the ENTH domain represents a native function of the full-length epsin protein.
PMCID: PMC4370887  PMID: 25799353
3.  Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1 
Molecular Biology of the Cell  2012;23(2):337-346.
The Sec6 subunit of the multisubunit exocyst tethering complex interacts with the Sec1/Munc18 protein Sec1 and with the t-SNARE Sec9. Assembly of the exocyst upon vesicle arrival at sites of secretion is proposed to release Sec9 for SNARE complex assembly and to recruit Sec1 for interaction with SNARE complexes to facilitate fusion.
Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.
PMCID: PMC3258177  PMID: 22114349
4.  At the Junction of SNARE and SM Protein Function 
Current opinion in cell biology  2010;22(4):488-495.
Sec1/Munc18 (SM) proteins bind to and function with soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) at each vesicle fusion site in the cell. The purpose for these interactions is becoming clearer, as what had been interpreted as functional divergence between SM proteins acting at different vesicle trafficking steps, or in specialized cells, is giving way to more recent evidence for common functions among all SM proteins. What is emerging is a picture of SM proteins acting not merely as SNARE regulators, but also as central components of the membrane fusion apparatus. The available data suggest sequential models that describe how the soluble SM protein might first regulate SNARE complex assembly and then cooperate with SNAREs to stimulate membrane fusion.
PMCID: PMC2923694  PMID: 20471239
5.  AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd in endocytic recycling 
Nature cell biology  2009;11(12):1399-1410.
RME-1/EHD1 family proteins are key residents of the recycling endosome required for endosome to plasma membrane transport in C. elegans and mammals. Recent studies suggest parallels of the RME-1/EHD proteins to the Dynamin GTPase superfamily of mechanochemical pinchases that promote membrane fission. Here we show that that endogenous C. elegans AMPH-1, the only C. elegans member of Amphiphysin/BIN1 family of BAR-domain proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1 deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 NPF (D/E) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BIN1/Amphyphysin 2 in EHD1-regulated endocytic recycling. In vitro we find that the purified recombinant AMPH-1/RME-1 complexes produce short, coated, membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, likely through functions required for the production of cargo carriers exiting the recycling endosome for the cell surface.
PMCID: PMC2788952  PMID: 19915558
Endocytosis; endocytic recycling; RME-1; EHD1; Amphiphysin; Bin1; Dynamin; mechanoenzyme
6.  Ordering the Final Events in Yeast Exocytosis 
The Journal of Cell Biology  2000;151(2):439-452.
In yeast, assembly of exocytic soluble N-ethylmaleimide–sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.
PMCID: PMC2192655  PMID: 11038189
NSF; membrane fusion; SNAREs; exocyst; Sec1
7.  Sec1p Binds to Snare Complexes and Concentrates at Sites of Secretion 
The Journal of Cell Biology  1999;146(2):333-344.
Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.
PMCID: PMC3206579  PMID: 10427089
Sec1 proteins; syntaxin proteins; SNARE complex; secretion; yeast

Results 1-7 (7)