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Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins
TLDR
PHluorins are developed pH-sensitive mutants of green fluorescent protein by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles, to monitor vesicle exocytosis and recycling. Expand
SNAP receptors implicated in vesicle targeting and fusion
TLDR
The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicles or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesICLE-to-target specificity. Expand
A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion
TLDR
It is reported that in the absence of SNAP and NSF, these three SNAREs form a stable complex that can also bind synaptotagmin, suggesting that synapttagmin operates as a "clamp" to prevent fusion from proceeding in the absent of a signal. Expand
Membrane Fusion: Grappling with SNARE and SM Proteins
TLDR
The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion and are spectacularly apparent in the exquisite speed and precision of synaptic exocytosis. Expand
SNAREpins: Minimal Machinery for Membrane Fusion
Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes. This leads to spontaneous fusion of the dockedExpand
Mechanisms of intracellular protein transport
TLDR
The general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocyTosis of hormones and neurotransmitters, is uncovered. Expand
Selective Activation of Cognate SNAREpins by Sec1/Munc18 Proteins
TLDR
A fundamental role is demonstrated of the SM protein: to act as a stimulatory subunit of its cognate SNARE fusion machinery, enhancing fusion specificity in a reconstituted system. Expand
The use of pHluorins for optical measurements of presynaptic activity.
TLDR
P pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals are characterized and a theoretical analysis of the expected signals using this approach and guidelines for future reporter development are provided. Expand
Compartmental specificity of cellular membrane fusion encoded in SNARE proteins
TLDR
It is found 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. Expand
Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.
TLDR
This complementation assay has allowed us to purify the NEM-sensitive factor, which is a tetramer of 76-kDa subunits, and appears to act catalytically, one tetramer leading to the metabolism of numerous transport vesicles. Expand
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