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From the
Syntaxin 1 (HPC-1), a component of the receptor for SNAPs
(soluble N-ethylmaleimide-sensitive factor attachment
proteins), has been implicated in the docking and fusion of synaptic
vesicles with the plasma membrane. It was reported that syntaxin 1 in
rat brain and chromaffin cells (PC12) is exclusively located on the
plasma membrane (Bennett, M. K., Calakos, N., and Scheller, R. H.(1992) Science 257, 255-259; Söllner, T., Bennett, M. K.,
[Medline]
Whiteheart, S. W., Scheller, R. H., and Rothman, J. E.(1993) Cell 75, 409-418). By means of biochemical and morphological
analyses, we now show that syntaxin 1 is associated with chromaffin
granules in the adrenal medulla. This finding raises the possibility
that syntaxin 1 in chromaffin cells is a component of vesicle-SNAP
receptor as well as one of target-SNAP receptor on the plasma membrane.
Syntaxin was discovered as a protein that interacts with the
synaptic vesicle membrane protein, synaptotagmin(1, 2) .
This protein was also identified as a surface protein of various
neurons recognized by clone HPC-1(3, 4) . Bennett et
al.(5) identified a family of syntaxin-related proteins in
rat that share significant sequence similarity. Microinjection of
syntaxin 1A fragments and anti-syntaxin 1A antibodies into
neuroendocrine PC12 cells inhibited calcium-regulated
secretion(5) , indicating the involvement of this protein in
neurotransmitter secretion.
Syntaxin 1 forms a complex with
SNAP-25,
In this study
we show, by means of biochemical and morphological analyses, that
syntaxin 1 is associated with chromaffin granules, which mediate the
secretion of catecholamine from adrenal chromaffin cells. The present
finding raises the possibility that syntaxin is a component of v-SNARE
as well as one of t-SNARE on the plasma membrane.
For equilibrium density
gradient centrifugation, a postnuclear supernatant obtained from
adrenal medullae was layered on a discontinuous gradient formed from
0.9 ml each of 0.6, 0.9, 1.2, 1.5, and 1.8 M sucrose in 4
mM EDTA, 10 mM MOPS, pH 8.0, and then centrifuged at
100,000
Since chromaffin granules are highly dense organelles, they
can be isolated to near homogeneity (more than 90% pure) by
centrifugation (15, 18). Fig. 1A shows the purity of
chromaffin granules isolated as described under ``Experimental
Procedures.'' The granule fraction contained dopamine
Subcellular fractionation revealed that syntaxin 1B is
associated with chromaffin granules. The possibility that syntaxin 1B
detected in isolated granule membranes was derived from contaminating
plasma membranes was unequivocally excluded by the fact that three
plasma membrane marker proteins,
Na
The present finding that
syntaxin 1 is associated with chromaffin granules is consistent with
recently accumulated results suggesting that syntaxin is a component of
transport vesicles as well as one of the target membranes. Dascher et al.(24) showed that syntaxin 5 is mainly localized
in pre-Golgi vesicular-tubular intermediates in mammalian cells. In
yeast, the early effect of depletion of Sed5p, which is likely to be
the yeast homologue of syntaxin 5, leads to elaboration of the
endoplasmic reticulum rather than a change in the Golgi
apparatus(25) . This effect can be explained by the idea that
Sed5p interacts with the endoplasmic reticulum, in which transport
vesicles are formed. On electron microscopic analysis, Koh et al.(17) detected immunoreactivity for syntaxin 1 not only on
the plasma membrane but also on synaptic vesicles. Schulze et al.(26) demonstrated, by means of subcellular fractionation,
that syntaxin 1A is associated with Drosophila synaptic
vesicles. The present finding combined with these results may raise the
possibility that syntaxin plays a role as v-SNARE in addition to the
previously suggested role as t-SNARE.
Gold
particles on the cross-sectional profiles of chromaffin granule
membrane, plasma membrane, and mitochondrial outer membrane within 20
nm from the center of the membranes were counted. About 50 µm of
each membrane was analyzed.
We thank Drs. M. Kawakita, M. Sakaguchi, and J. E.
Rothman for the generous gifts of the
anti-Na
Volume 270,
Number 27,
Issue of July 07, pp. 15930-15933, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
()VAMP/synaptobrevin-2, and Rab
3A(6, 7, 8) , as well as
synaptotagmin(1, 2) . Recently, Söllner et al.(9) demonstrated that syntaxin 1, VAMP/synaptobrevin-2, and
SNAP-25 are membrane-embedded components of a 20 S NSF
SNAP
complex. NSF and SNAPs were initially identified as factors essential
for vesicle-mediated intra-Golgi protein transport (for a review, see
Ref. 10). Based on the localization of
SNAREs(1, 5, 11, 12) ,
VAMP/synaptobrevin-2 was classified as vesicle (v)-SNARE, and syntaxin
1 and SNAP-25 as target (t)-SNARE(9) . The SNARE hypothesis
predicts that all eukaryotic cells should have families of v- and
t-SNAREs that mediate different secretory pathways(9) . Indeed,
homologues of SNAREs were identified in yeast as components involved in
distinct steps of the secretory pathways(13) .
Materials
Monoclonal antibodies that recognize
both brain syntaxins 1A and 1B (mAb 10H5), and brain syntaxin 1B (mAb
6H1) were produced as described previously(2) . A polyclonal
antibody against a fusion protein comprising 
-galactosidase and
brain syntaxin 1 was produced as described previously(14) . An
anti-chromogranin A antibody was produced by immunizing rabbits with
the antigen. Anti-VAMP/synaptobrevin-2 and anti-SNAP-25 antibodies were
raised in rabbits using synthetic peptides corresponding to residues
2-18 (SATAATVPPAAPAGEGP) of VAMP/synaptobrevin-2 and
carboxyl-terminal residues 195-206 (ANQRATKMLGSG) of SNAP-25,
respectively. Anti-Na,H
exchanger and
anti-adrenodoxin antibodies were generous gifts from Dr. M. Kawakita
(Tokyo Metropolitan Institute of Medical Science) and Dr. M. Sakaguchi
(Kyushyu University), respectively. A polyclonal anti-SNAP antibody
that recognizes
- and -isoforms was kindly donated by Dr. J.
E. Rothman (Memorial Sloan-Kettering Cancer Center). An antibody
against dopamine -hydroxylase was obtained from Eugene Tech
International, Inc.
Subcellular Fractionation
Chromaffin granules from
bovine adrenal medulla were isolated according to the method of Nelson et al.(15) . All steps were carried out at 0-4
°C. Ten bovine adrenal medullae were homogenized in 130 ml of
homogenation buffer (0.3 M sucrose, 4 mM EDTA, 10
mM MOPS, pH 8.0) containing 1 mM phenylmethylsulfonyl
fluoride with a Polytron homogenizer. The postnuclear supernatant was
centrifuged at 13,000 g for 20 min. The pellet was
suspended in 130 ml of homogenation buffer and then centrifuged once
again. The resulting pellet was suspended in 20 ml of homogenization
buffer, and 10-ml portions of the suspension were layered on
discontinuous gradients formed from 16 ml of 1.2 M sucrose and
11 ml of 1.8 M sucrose in 4 mM EDTA, 10 mM MOPS, pH 8.0. After centrifugation at 53,000
g for 14 h in a Beckman SW28 rotor, chromaffin granules were
recovered as a pellet. After hypoosmotic treatment of the isolated
granules, chromaffin granule membranes were recovered by centrifugation
at 200,000
g for 40 min.
g for 17 h in a Beckman SW 50.1 rotor.
Fractions of 0.5 ml each were recovered from the top, and the
precipitate at the bottom was dissolved in 0.5 ml of homogenation
buffer. The proteins were precipitated with 6% trichloroacetic acid in
the presence of 0.02% deoxycholic acid and then subjected to SDS-PAGE
as described by Laemmli(16) .
Electron Microscopy
Immunogold labeling of frozen
sections of rat adrenal medullae and cerebellum was performed as
described previously (17) using gold conjugates of goat
anti-rabbit IgGs (5 nm in diameter, A = 0.08).
Immunoprecipitation
Chromaffin granule membranes
were solubilized with 4 mM EDTA, 1 mM dithiothreitol,
0.5 mM ATP, 250 mM KCl, 1% Triton X-100, 25 mM PIPES, pH 7.2. After centrifugation at 200,000 g for 30 min, 500 µl of the supernatant was incubated with 5
µg of a control antibody (mAb MAC-L1, which reacts with chicken
Ca
channel but not Ca
channel from
other sources) or 5 µg of mAb 10H5 at 0 °C for 1 h. To this
solution was added 30 µl of protein A/G-agarose (Santa Cruz
Biotechnology). After gentle shaking overnight, the resin was washed
three times with 2 mM EDTA, 1 mM dithiothreitol, 0.5
mM ATP, 50 mM KCl, 10% (v/v) glycerol, 0.1% Triton
X-100, 25 mM PIPES, pH 7.2. The bound proteins were eluted
with SDS sample buffer and then analyzed by SDS-PAGE, followed by
immunoblotting with anti-syntaxin 1 and anti-VAMP/synaptobrevin-2
antibodies.
-hydroxylase and chromogranin A, membrane-bound and soluble marker
proteins of chromaffin granules, respectively, but did not contain
significant amounts of plasma membrane marker proteins
(Na,K
exchanger and
Na
,K
-ATPase) or a mitochondrial
marker protein, adrenodoxin. Fig. 1B shows the results
of immunoblotting with antibodies against components of the SNARE
complex. Consistent with the results of Baumert et
al.(19) , and Hodel et al.(20) ,
VAMP/synaptobrevin-2 was present in the isolated granule fraction.
SNAP-25 was not detected in either the total membrane fraction or the
granule membrane fraction. This probably reflects the fact that the
level of SNAP-25 in the adrenal medulla is about 20-fold lower than
that in the brain(21) . As will be described below, SNAP-25 was
detected when larger amounts of proteins were subjected to
immunoblotting. Although Hodel et al.(20) showed that
syntaxin 1 is exclusively localized in the plasma membrane fraction, we
did detect a protein that is recognized by a monoclonal anti-brain
syntaxin 1 antibody (mAb 10H5) in the chromaffin granule membrane.
Immunoblotting with a brain syntaxin 1B-specific monoclonal antibody
(mAb 6H1) revealed that this protein is syntaxin 1B (Fig. 1C). Similar results were obtained when chromaffin
granules were purified by centrifugation on a discontinuous Percoll
gradient (18) (data not shown).
Figure 1:
Syntaxin 1B is co-purified with
chromaffin granules. A, appropriate amounts of total membranes
of adrenal medulla (TOTALMEM.) or chromaffin granule
membranes (C.G.M.) were subjected to SDS-PAGE, and then
immunoblotted with antibodies against dopamine -hydroxylase (DBH), Na,K
-ATPase (NaK), and Na
,H
exchanger (NHE). A postnuclear supernatant (PNS) or isolated
chromaffin granules (C.G.) were used for blotting with
antibodies against chromogranin A (CgA) and
adrenodoxin (Ad). Dopamine
-hydroxylase and adrenodoxin
are known to exist as multiple forms. B, 50 µg of total
membranes (TOTAL MEM.) or granule membrane proteins (C.G.M.) was subjected to SDS-PAGE, and then immunoblotted
with antibodies against SNAREs. C, a rat brain homogenate (BRAIN), chromaffin granule membranes (C.G.M.), and
total membranes of adrenal medulla (TOTALMEM.) were
subjected to SDS-PAGE, and then immunoblotted with antibodies against
brain syntaxins 1A and 1B (10H5), brain syntaxin 1B (6H1), and a fusion protein comprising -galactosidase and
brain syntaxin 1 (POLY). A minor 17-kDa band was not obvious
in this figure because it was very faint. Arrows indicate the
positions of brain syntaxins 1A (1A) and 1B (1B).
We next examined whether or not
syntaxin 1 is cosedimented with chromaffin granules on equilibrium
centrifugation. As shown in Fig. 2, minor amounts of chromogranin
A were detected in fractions 1 and 2, but the majority was recovered in
higher density fractions, especially in the precipitate. This implies
that the homogenation process did not markedly disrupt chromaffin
granules and that the fractionation was satisfactory. Fractions
4-6 contained SNAP-25, VAMP/synaptobrevin-2, and syntaxin 1,
suggesting that these fractions comprise the plasma membrane and small
light vesicles. Consistent with our previous finding that NSF is
tightly associated with rat brain synaptic vesicles(22) , NSF
and SNAPs were detected in fractions 4-6, which contain small
light vesicles. Morgan and Burgoyne (23) also showed that significant
amounts of NSF and SNAPs are associated with membranes in chromaffin
cells, although Rothman (10) insisted that NSF and SNAPs, which
are involved in the formation of the 20 S NSFSNAPSNARE
complex, are cytosolic proteins. Fractions 9 and 10 and the precipitate
contained VAMP/synaptobrevin-2 and syntaxin 1, with small amounts of
NSF and SNAPs. These results again suggest that syntaxin 1 is
associated with chromaffin granules.
Figure 2:
Subcellular fractionation of adrenal
medullae. A postnuclear supernatant obtained from adrenal medullae was
fractionated by equilibrium centrifugation as described under
``Experimental Procedures.'' Appropriate amounts of proteins
were subjected to SDS-PAGE, and then immunodetected with a mixture of
monoclonal antibodies against NSF (firstrow) and
syntaxin 1 (thirdrow), a mixture of polyclonal
antibodies against SNAP (secondrow), SNAP-25 (fourthrow), and VAMP/synaptobrevin-2 (fifthrow), and a polyclonal antibody against chromogranin A (bottomrow). Arrows indicate the positions
of individual protein bands. The lower molecular band at the top of the
gradient in the second row was not SNAP. - and -SNAPs were
not resolved with this electrophoresis system. For the detection of
syntaxin 1, mAb 10H5, which recognizes both brain syntaxins 1A and 1B,
was used. The higher molecular band for fraction 3 in the third row was
not syntaxin 1.
To confirm the association of
syntaxin 1 with chromaffin granules, we investigated the localization
of syntaxin 1 in frozen ultrathin sections of adrenal medulla by the
immunogold labeling method. For this purpose, a polyclonal antibody
against a fusion protein comprising -galactosidase and syntaxin 1 (14) was used because monoclonal antibodies against syntaxin 1
(mAbs 10H5 and 6H1) are not applicable to the immunogold labeling
method. The polyclonal antibody mainly recognized two bands of about 35
kDa, and a minor fuzzy band of about 17 kDa, of an adrenal medulla
membrane preparation (the minor band is not obvious in Fig. 1C because it is very faint). The minor band was
most likely of a proteolytic fragment of syntaxin 1, because it was
also recognized by monoclonal antibodies against syntaxins 1A and 1B
(mAb 10H5), syntaxin 1B (mAb 6H1), and HPC-1. As shown in Fig. 3(A and B), a significant number of gold
particles was observed on the cytoplasmic side of chromaffin granules.
Quantitative analysis confirmed the presence of syntaxin 1 on
chromaffin granules (). This labeling pattern was quite
different from that of cerebellum. In the cerebellum, syntaxin 1 was
mainly detected on the plasma membrane and less on synaptic vesicles (Fig. 3C and Ref. 17).
Figure 3:
Comparison of the distributions of
syntaxin 1 in adrenal medulla and brain. A and B,
immunogold localization of syntaxin 1 in a frozen ultrathin section of
adrenal medulla. Chromaffin granules are large and dense vesicles. Gold
particles were detected on the cytoplasmic side of chromaffin granules (CG) and fewer on the plasma membrane (PM), but
essentially not on mitochondria (MT). C, immunogold
localization of syntaxin 1 in a frozen ultrathin section of cerebellum.
Gold particles were mainly detected on the plasma membrane (PM). Bar, 0.2 µm.
Syntaxin 1 on the plasma
membrane can form a complex with VAMP/synaptobrevin-2, SNAP-25,
synaptotagmin, and Rab
3A(1, 2, 6, 7, 8) . We wondered
whether or not syntaxin 1 associated with chromaffin granules is
functionally equivalent to that on the plasma membrane. To answer this
question, we examined whether syntaxin 1 and VAMP/synaptobrevin-2 in
chromaffin granules are co-immunoprecipitated or not. As shown in Fig. 4, a significant amount of VAMP/synaptobrevin-2 was
co-immunoprecipitated with mAb 10H5. This suggests that syntaxin 1 in
chromaffin granules is functionally equivalent to that on the plasma
membrane.
Figure 4:
Co-immunoprecipitation of syntaxin 1 with
VAMP/synaptobrevin-2. Triton X-100 extracts of isolated chromaffin
granule membranes (470 µg of protein) were incubated with a control
antibody (control) or mAb 10H5 (anti-syntaxin) and
then analyzed by SDS-PAGE and immunoblotting with anti-syntaxin 1 (mAb
10H5) and anti-VAMP/synaptobrevin-2
antibodies.
,K
-ATPase,
Na
,H
exchanger (Fig. 1A), and SNAP-25 (Fig. 2), were not
significantly present in the chromaffin granule fraction. Hodel et
al.(20) reported that syntaxin 1A or 1B is not present in
chromaffin granules. Since their anti-syntaxin antibody reacted well
with syntaxin 1A but poorly with syntaxin 1B, it is likely that the
amount of syntaxin 1B in their chromaffin granule preparation subjected
to immunoblotting was lower than the limit of detection with the poorly
reactive antibody. The association of syntaxin 1 with chromaffin
granules was confirmed by means of the immunogold labeling method (Fig. 3), although we could not determine which isoform of
syntaxin 1 is associated with chromaffin granules by this method
because the polyclonal antibody used for labeling recognizes isoforms
of syntaxin 1 (Fig. 1C).
Table: Density of gold particles on membranes
,H
exchanger,
anti-adrenodoxin, and anti-
-SNAP antibodies, respectively. We also
thank A. Furuno for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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