Characterization of vesicular membrane-bound α-SNAP and NSF in adrenal chromaffin cells

被引：7

作者：

Banaschewski, C ^{[1
]}

Höhne-Zell, B ^{[1
]}

Ovtscharoff, W ^{[1
]}

Gratzl, M ^{[1
]}

机构：

[1] Tech Univ Munich, Inst Anat, D-80802 Munich, Germany

来源：

BIOCHEMISTRY | 1998年 / 37卷 / 47期

关键词：

D O I：

10.1021/bi981339p

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

alpha-SNAP and NSF are thought to act as soluble factors, which transiently bind to a complex formed between syntaxin and SNAP-25 located at the plasma membrane and synaptobrevin at the secretory vesicle membrane, at the moment of exocytosis. Here we present data which permit the novel conclusion that alpha-SNAP and NSF are not soluble in adrenal chromaffin cells but are rather membrane-bound in particular to undocked chromaffin vesicles. Evidence for this new paradigm is derived from several experimental approaches. First, alpha-SNAP and NSF were found predominantly at cellular membranes and not in the cytosol of cracked chromaffin cells. Second, alpha-SNAP and NSF were not released from membranes by Mg(2+)ATP, which causes priming of vesicles. Third, immune electron microscopy and immunoblotting of chromaffin vesicles purified by immunoisolation or density gradient centrifugation revealed the presence of alpha-SNAP and NSF together with typical vesicular proteins such as synaptobrevin and synaptotagmin. In the sucrose gradient 30% alpha-SNAP and 27% NSF were recovered with chromaffin vesicles. Bound alpha-SNAP was quantified (14 molecules/vesicle), and binding was characterized with recombinant hiss-tagged alpha-SNAP. Overlay blots revealed that alpha-SNAP is bound to vesicular SNAP-25 and endogenous NSF. Ow data show that mature chromaffin vesicles already contain specifically bound alpha-SNAP and NSF before docking at the plasmalemma.

引用

页码：16719 / 16727

页数：9

共 56 条

[11]

FUTTERER A, 1995, MOL BIOL CELL, V6, P161

[12] Botulinum neurotoxin light chains inhibit both Ca2+-induced and CTP analogue-induced catecholamine release from permeabilised adrenal chromaffin cells [J].

Glenn, DE ;

Burgoyne, RD .

FEBS LETTERS, 1996, 386 (2-3) :137-140

[13] DISTRIBUTION OF CHROMAFFIN SECRETORY VESICLES, ACETYLCHOLINESTERASE, AND LYSOSOMAL-ENZYMES IN SUCROSE AND PERCOLL GRADIENTS [J].

GRATZL, M .

ANALYTICAL BIOCHEMISTRY, 1984, 142 (01) :148-154

[14] LATENT ACETYLCHOLINESTERASE IN SECRETORY VESICLES ISOLATED FROM ADRENAL-MEDULLA [J].

GRATZL, M ;

KRIEGERBRAUER, H ;

EKERDT, R .

BIOCHIMICA ET BIOPHYSICA ACTA, 1981, 649 (02) :355-366

[15] Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) [J].

Haas, A ;

Wickner, W .

EMBO JOURNAL, 1996, 15 (13) :3296-3305

[16] THE N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN AND ALPHA-SNAP INDUCE A CONFORMATIONAL CHANGE IN SYNTAXIN [J].

HANSON, PI ;

OTTO, H ;

BARTON, N ;

JAHN, R .

JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (28) :16955-16961

[17] DISASSEMBLY OF THE RECONSTITUTED SYNAPTIC VESICLE MEMBRANE-FUSION COMPLEX IN-VITRO [J].

HAYASHI, T ;

YAMASAKI, S ;

NAUENBURG, S ;

BINZ, T ;

NIEMANN, H .

EMBO JOURNAL, 1995, 14 (10) :2317-2325

[18] Functional importance of synaptobrevin and SNAP-25 during exocytosis of histamine by rat gastric enterochromaffin-like cells [J].

HohneZell, B ;

Galler, A ;

Schepp, W ;

Gratzl, M ;

Prinz, C .

ENDOCRINOLOGY, 1997, 138 (12) :5518-5526

[19] Adrenal chromaffin cells contain functionally different SNAP-25 monomers and SNAP-25/syntaxin heterodimers [J].

HohneZell, B ;

Gratzl, M .

FEBS LETTERS, 1996, 394 (02) :109-116

[20] SYNAPTOBREVIN CLEAVAGE BY THE TETANUS TOXIN LIGHT-CHAIN IS LINKED TO THE INHIBITION OF EXOCYTOSIS IN CHROMAFFIN CELLS [J].

HOHNEZELL, B ;

ECKER, A ;

WELLER, U ;

GRATZL, M .

FEBS LETTERS, 1994, 355 (02) :131-134

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