©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Syntaxin 1 (HPC-1) Is Associated with Chromaffin Granules (*)

Mitsuo Tagaya (§) , Shuichi Toyonaga , Masami Takahashi (1), Akitsugu Yamamoto (2), Tomonori Fujiwara (3), Kimio Akagawa (3), Yoshinori Moriyama (4), Shoji Mizushima

From the (1)School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan, the Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan, the (2)Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570, Japan, the (3)Department of Physiology, School of Medicine, Kyorin University, Mitaka, Tokyo 181, Japan, and the (4)Marine Biological Laboratory, Graduate School of Gene Sciences, Faculty of Sciences, Hiroshima University, Mukaishima, Hiroshima 722, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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,()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 NSFSNAP 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) .

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.


EXPERIMENTAL PROCEDURES

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.

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 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.


RESULTS

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 -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.




DISCUSSION

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,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).

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.

  
Table: Density of gold particles on membranes

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.



FOOTNOTES

*
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and the Ciba-Geigy Foundation (Japan) for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. Fax: 81-426-76-8866.

The abbreviations used are: SNAP-25, synaptosome-associated protein of 25 kDa; NSF, N-ethylmaleimide-sensitive factor; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; v-SNARE, vesicle-SNAP receptor; t-SNARE, target-SNAP receptor; mAb, monoclonal antibody; MOPS, 4-morpholinopropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; VAMP, vesicle-associated membrane protein.


ACKNOWLEDGEMENTS

We thank Drs. M. Kawakita, M. Sakaguchi, and J. E. Rothman for the generous gifts of the anti-Na,H exchanger, anti-adrenodoxin, and anti--SNAP antibodies, respectively. We also thank A. Furuno for technical assistance.


REFERENCES
  1. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science257, 255-259
  2. Yoshida, A., Oho, C., Omori, A., Kuwahara, R., Ito, T., and Takahashi, M. (1992) J. Biol. Chem.267, 24925-24928 [Abstract/Free Full Text]
  3. Akagawa, K., and Barnstable, C. J. (1986) Brain Res.383, 110-120 [Medline] [Order article via Infotrieve]
  4. Inoue, A., Obata, K., and Akagawa, K. (1992) J. Biol. Chem267, 10613-10619 [Abstract/Free Full Text]
  5. Bennett, M. K., Garcia-Arrarás, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell74, 863-873 [Medline] [Order article via Infotrieve]
  6. Horikawa, H. P. M., Saisu, H., Ishizuka, T., Sekine, Y., Tsugita, A., Odani, S., and Abe, T. (1993) FEBS Lett.330, 236-240 [CrossRef][Medline] [Order article via Infotrieve]
  7. Calakos, N., Bennett, M. R., Peterson, K. E., and Scheller, R. H. (1994) Science263, 1146-1149 [Medline] [Order article via Infotrieve]
  8. Pevsner, J., Hsu, S.-C., Braun, J. E. A., Calakos, N., Ting, A. E., Bennett, M. K., and Scheller, R. H. (1994) Neuron13, 353-361 [Medline] [Order article via Infotrieve]
  9. Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993) Nature362, 318-324 [CrossRef][Medline] [Order article via Infotrieve]
  10. Rothman, J. E. (1994) Nature372, 55-63 [CrossRef][Medline] [Order article via Infotrieve]
  11. Elferink, L. A., Trimble, W. S., and Scheller, R. H. (1989) J. Biol. Chem.264, 11061-11064 [Abstract/Free Full Text]
  12. Oyer, G. A., Higgins, G. A., Hart, R. A., Battenberg, E., Billingsley, M., Bloom, F. E., and Wilson, M. C. (1989) J. Cell Biol.109, 3039-3052 [Abstract]
  13. Bennett, M. K., and Scheller, R. H. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 2559-2563 [Abstract]
  14. Inoue, A., and Akagawa, K. (1992) Biochem. Biophys. Res Commun.187, 1144-1150 [Medline] [Order article via Infotrieve]
  15. Nelson, N., Cidon, S., and Moriyama, Y. (1988) Methods Enzymol.157, 619-633 [Medline] [Order article via Infotrieve]
  16. Laemmli, U. K. (1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  17. Koh, S., Yamamoto, A., Inoue, A., Inoue, Y., Akagawa, K., Kawamura, Y., Kawamoto, K., and Tashiro, Y. (1993) J. Neurocytol.22, 995-1005 [Medline] [Order article via Infotrieve]
  18. Meyer, D. I., and Burger, M. M. (1979) J. Biol. Chem.254, 9854-9859 [Abstract]
  19. Baumert, M., Maycox, P. R., Navone, F., De Camilli, P., and Jahn, R. (1989) EMBO J.8, 379-384 [Abstract]
  20. Hodel, A., Schäfer, T., Gerosa, D., and Burger, M. M. (1994) J. Biol. Chem.269, 8623-8626 [Abstract/Free Full Text]
  21. Roth, D., and Burgoyne, R. D. (1994) FEBS Lett.351, 207-210 [CrossRef][Medline] [Order article via Infotrieve]
  22. Hong, R.-M., Mori, H., Fukui, T., Moriyama, Y., Futai, M.,Yamamoto, A., Tashiro, Y., and Tagaya, M. (1994) FEBS Lett350, 253-257 [CrossRef][Medline] [Order article via Infotrieve]
  23. Morgan, A., and Burgoyne, R. D. (1995) EMBO J.14, 232-239 [Abstract]
  24. Dascher, C., Matteson, J., and Balch, W. E. (1994) J. Biol. Chem.269, 29363-29366 [Abstract/Free Full Text]
  25. Hardwick, K. G., and Pelham, H. R. B. (1992) J. Cell Biol119, 513-521 [Abstract]
  26. Schulze, K. L., Broadie, K., Perin, M. S., and Bellen, H. J. (1995) Cell80, 311-320 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.