Adrenal chromaffin cells contain functionally different SNAP‐25 monomers and SNAP‐25/syntaxin heterodimers

Höhne-Zell, Barbara; Gratzl, Manfred

doi:10.1016/0014-5793(96)00931-3

Cited by 63 publications

(43 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In our highly purified vesicle fraction, virtually all syntaxin and SNAP-25 was resistant to toxin, documenting that the majority is bound in ternary complexes. Similarly, SDS-resistant complexes recently have been observed in purified fractions of chromaffin granules (19). The proteins readily associate with each other as soon as NSF is removed (Fig.…”

Section: Discussionsupporting

confidence: 66%

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles

Otto

Hanson

Jahn

1997

Proc. Natl. Acad. Sci. U.S.A.

258

207

View full text Add to dashboard Cite

The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimidesensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.Synaptobrevin (also referred to as VAMP), SNAP-25, and syntaxin are crucial components of the exocytotic apparatus in neurons (1-4). Synaptobrevin is exclusively localized to synaptic vesicles whereas syntaxin and SNAP-25 are mainly localized to the neuronal plasma membrane. Any interference with the function of these proteins, e.g., proteolysis by clostridial neurotoxins (5, 6) or genetic deletion (7), inhibits exocytotic neurotransmitter release. Relatives of these proteins participate in many intracellular membrane traffic steps in all eukaryotic cells (3), suggesting that membrane fusion is mediated by a common and conserved mechanism.Despite compelling evidence linking syntaxin, SNAP-25, and synaptobrevin to exocytosis, it is not understood how these proteins operate in the sequence of events that leads to vesicle docking and membrane fusion. In detergent extracts, the three proteins form a stable complex that binds the soluble proteins ␣͞␤͞␥-SNAP and N-ethylmaleimide-sensitive factor (NSF), leading to their designation as v-SNAREs (synaptobrevin, vesicular SNAp-REceptors) and t-SNAREs (syntaxin and SNAP-25, target membrane SNAp REceptor) (8, 9). ATPhydrolysis by NSF causes disassembly of the complex. A complex with very similar properties also can be formed from recombinant proteins lacking their transmembrane domains (10-12). Based on these, observations it has been suggested that v-SNARE-t-SNARE interactions are responsible for docking of vesicles at their target membrane and that the conformational change caused by NSF contributes to membrane fusion (1, 9). The specificity of v-SNARE-t-SNARE pairing would ensure that trafficking vesicles only dock at an appropriate target membrane. These proposals are referred to as the SNARE hypothesis (1) and have gained wide acceptance in the field (1-3).Recently, we have observed that, unlike other residents of the presynaptic plasma membrane, significant amounts of syntaxin and SNAP-25 are also localized to synaptic vesicles (13). Using two independent and complementary approaches, we now report that a ternary complex containing syntaxin, synaptobrevin, and SNAP-25 can form in the membrane of synaptic vesicles in the absence of plasm...

show abstract

Section: Discussionsupporting

confidence: 66%

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles

Otto

Hanson

Jahn

1997

Proc. Natl. Acad. Sci. U.S.A.

258

207

View full text Add to dashboard Cite

show abstract

“…An earlier study on chromaffin cells revealed the presence of multiple SDSresistant SNARE complexes only in a lysosomal fraction (Hohne-Zell and Gratzl, 1996). It might therefore be that, variations in the purification protocols used by us and by Lawrence and Dolly (Lawrence and Dolly, 2002) lead to different contamination by sources other than the plasmalemma, which in turn might be the basis for the diverging results.…”

Section: Discussionmentioning

confidence: 89%

Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

Kubista

Edelbauer

Boehm

2004

Journal of Cell Science

View full text Add to dashboard Cite

The core complex, formed by the SNARE proteins synaptobrevin 2, syntaxin 1 and SNAP-25, is an important component of the synaptic fusion machinery and shows remarkable in vitro stability, as exemplified by its SDS-resistance. In western blots, antibodies against one of these SNARE proteins reveal the existence of not only an SDS-resistant ternary complex but also as many as five bands between 60 and >200 kDa. Structural conformation as well as possible functions of these various complexes remained elusive. In western blots of protein extracts from PC12 cell membranes, an antibody against SNAP-25 detected two heat-sensitive SDS-resistant bands with apparent molecular weights of 100 and 230 kDa. A syntaxin antibody recognized only the 230 kDa band and required heat-treatment of the blotting membrane to detect the 100 kDa band. Various antibodies against synaptobrevin failed to detect SNARE complexes in conventional western blots and detected either the 100 kDa band or the 230 kDa band on heat-treated blotting membranes. When PC12 cells were exposed to various extracellular K+-concentrations (to evoke depolarization-induced Ca2+ influx) or permeabilized in the presence of basal or elevated free Ca2+, levels of these SNARE complexes were altered differentially: moderate Ca2+ rises (≤1 μM) caused an increase, whereas Ca2+ elevations of more than 1 μM led to a decrease in the 230 kDa band. Under both conditions the 100 kDa band was either increased or remained unchanged. Our data show that various SDS-resistant complexes occur in living cells and indicate that they represent SNARE complexes with different structures and diverging functions. The distinct behavior of these complexes under release-promoting conditions indicates that these SNARE structures have different roles in exocytosis.

show abstract

“…Functional studies have revealed that these toxins can also inhibit exocytosis of SGs from chromaffin cells (Hohne-Zell et al, 1994, Hohne-Zell andGratzl, 1996) as well as rat parotid cells (Fujita-Yoshigaki et al, 1996). Thus, toxins not only support a role for a SNARE complex in membrane fusion in non-neuronal cells, but will also help define their specific interactions and contributions to exocytosis.…”

Section: Introductionmentioning

confidence: 97%

GTP-Binding Proteins and Regulated Exocytosis

Watson

1999

Critical Reviews in Oral Biology & Medicine

View full text Add to dashboard Cite

Regulated exocytosis, which occurs in response to stimuli, is a two-step process involving the docking of secretory granules (SGs) at specific sites on the plasma membrane (PM), with subsequent fusion and release of granule contents. This process plays a crucial role in a number of tissues, including exocrine glands, chromaffin cells, platelets, and mast cells. Over the years, our understanding of the proteins involved in vesicular trafficking has increased dramatically. Evidence from genetic, biochemical, immunological, and functional assays supports a role for ras-like monomeric GTP-binding proteins (smgs) as well as heterotrimeric GTP-binding protein (G-protein) subunits in various steps of the vesicular trafficking pathway, including the transport of secretory vesicles to the PM. Data suggest that the function of GTP-binding proteins is likely related to their localization to specific cellular compartments. The presence of both G-proteins and smgs on secretory vesicles/granules implicates a role for these proteins in the final stages of exocytosis. Molecular mechanisms of exocytosis have been postulated, with the identification of a number of proteins that modify, regulate, and interact with GTP-binding proteins, and with the advent of approaches that assess the functional importance of GTP-binding proteins in downstream, exocytotic events. Further, insight into vesicle targeting and fusion has come from the characterization of a SNAP receptor (SNARE) complex composed of vesicle, PM, and soluble membrane trafficking components, and identification of a functional linkage between GTP-binding and SNARES.

show abstract

Adrenal chromaffin cells contain functionally different SNAP‐25 monomers and SNAP‐25/syntaxin heterodimers

Cited by 63 publications

References 56 publications

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles

Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

GTP-Binding Proteins and Regulated Exocytosis

Contact Info

Product

Resources

About