Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7342-7346, August 1995 Neurobiology

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules (small dense core vesicles/sympathetic neurons) RUDOLF BAUERFEIND, RuTH JELINEK, ANDREA HELLWIG, AND WIELAND B. HUrTNER* Institute for Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Communicated by Philip Siekevitz, The Rockefeller University, New York, NY, March 27, 1995

ABSTRACT We have investigated the relationship of the This implies that only the regulated, but not the constitutive, so-called small dense core vesicle (SDCV), the major cate- pathway of protein secretion contributes to the biogenesis of cholamine-containing neurosecretory vesicle of sympathetic SDCVs. This concept has been extended to the biogenesis of neurons, to synaptic vesicles containing classic neurotrans- synaptic vesicles in general (4, 9, 10) but has not provided a mitters and secretory granules containing neuropeptides. satisfactory explanation for the very low abundance, in secre- SDCVs contain membrane proteins characteristic of synaptic tory granules, of certain membrane proteins that are major vesicles such as synaptophysin and synaptoporin. However, components of classic synaptic vesicles. The other concept, SDCVs also contain membrane proteins characteristic of which originated from the demonstration that the classic certain secretory granules like the vesicular monoamine synaptic vesicle membrane is distinct from that ofthe secretory transporter and the membrane-bound form of dopamine granule, has viewed the SDCVs, which lack secretory proteins f3-hydroxylase. In neurites of sympathetic neurons, synapto- and morphologically resemble classic synaptic vesicles, as physin and dopamine 8-hydroxylase are found in distinct synaptic vesicles that simply contain vesicular amine transport- vesicles, consistent with their transport from the trans-Golgi ers instead of uptake systems for classic neurotransmitters (1, network to the site of SDCV formation in constitutive secre- 8). Since only the constitutive, but not the regulated, pathway tory vesicles and secretory granules, respectively. Hence, of protein secretion is thought to contribute to the biogenesis SDCVs constitute a distinct type ofneurosecretory vesicle that of classic synaptic vesicles from early endosomes (2), this is a hybrid of the synaptic vesicle and the secretory granule concept has not provided a satisfactory explanation for the membranes and that originates from the contribution of both presence, in SDCVs, of certain secretory granule membrane the constitutive and the regulated pathway of protein secre- proteins such as cytochrome b561 (11). Herein we show that tion. SDCVs are hybrids of the classic synaptic vesicle and the secretory granule membranes. Two distinct types of neurosecretory vesicles are thought to mediate the regulated release of neurotransmitters and neu- MATERIALS AND METHODS ropeptides (1). One type is the synaptic vesicles that store and release classic neurotransmitters such as glutamate, acetylcho- Electron Microscopy. Rat vas deferens was fixed with (i) 1% line, glycine, and y-aminobutyric acid but do not contain glutaraldehyde in 100 mM sodium cacodylate (pH 7.2) (caco- secretory proteins. We shall refer to these as classic synaptic dylate buffer) overnight (Fig. 1A); (ii) 2% (wt/vol) paraform- vesicles because of the data described below and the concept aldehyde in 200 mM Hepes-NaOH (pH 7.4) for 5 hr, followed derived therefrom. Classic synaptic vesicles are thought to by a postfixation with 8% paraformaldehyde in 200 mM originate from early endosomes after delivery of their mem- Hepes-NaOH (pH 7.4) for 16 hr (Fig. 1B); (iii) dichromate brane proteins via the constitutive pathway of protein secre- fixative [3% (wt/vol) potassium dichromate/4.8% paraform- tion (for review, see ref. 2). The other type is the secretory aldehyde/0.1 M sodium acetate, pH 5.8] for 1 hr (Fig. 1 C and granules (in neurons also called large dense core vesicles) that D); or (iv) 3% (wt/vol) potassium permanganate in 150 mM store and release neuropeptides, originate from the trans- sodium phosphate (pH 7.4) for 1 hr (Fig. 1E). For Epon Golgi network, and constitute the regulated pathway ofprotein embedding, fixed samples were washed in cacodylate buffer secretion (3). In certain neurons and endocrine cells, secretory and postfixed in 1% OS04 plus 1.5% (wt/vol) magnesium granules also store and release biogenic amines such as ferricyanide for 1 hr. After washes in cacodylate buffer and catecholamines (4). water, samples were incubated for 30 min in 1.5% (wt/vol) An enigma has been the nature of the so-called small dense magnesium uranyl acetate in water. Samples were then dehy- core vesicles (SDCVs) that are found in certain nerve cells, drated in ethanol and embedded in Epon. Ultrathin sections such as catecholaminergic sympathetic neurons, and store and were contrasted with lead citrate and uranyl acetate. Ultrathin release biogenic amines (e.g., catecholamines) (for review, see cryosections were prepared from samples fixed with paraform- ref. 4). SDCVs do not contain secretory proteins (5) and show aldehyde or dichromate and immunogold-labeled as described an electron dense core only after certain chemical fixations (12, 13), by using the mouse monoclonal antibody against (for example, permanganate or dichromate) that prevent the synaptophysin (14) (Boehringer Mannheim) (1 jig/ml), a loss of catecholamines (6, 7). secondary rabbit anti-mouse IgG antibody (Cappel) (50 ,ug/ The relation of SDCVs to classic synaptic vesicles and ml), and protein A-colloidal gold (9 nm), prepared as de- secretory granules has been controversial (1, 4, 8, 9). One scribed by Slot and Geuze (15), at an OD520 of 0.098. concept has been that SDCVs, because of their ability to store Subcellular Fractionation. A post-10,000 x g high-speed and take up catecholamines like certain secretory granules membrane pellet from vas deferens, prepared from 12-16 rats such as chromaffin granules, are simply formed from the as described (5, 16), was resuspended in 1 ml of in vitro buffer membrane of secretory granules after their exocytosis (4, 9). (250 mM sucrose/10 mM KCl/10 mM MgCl2/10 mM Hepes-KOH, pH 7.2) and centrifuged for 5 min at 14,000 x g,V The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: SDCV, small dense core vesicle. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

7342 Downloaded by guest on September 26, 2021 Neurobiology: Bauerfeind et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7343

FIG. 1. Presence of synaptophysin on SDCVs by immunoelectron microscopy. Rat vas deferens was fixed with aldehyde (A and B), dichromate (C and D), or permanganate (E), followed by the prepara- i tion of Epon sections (A, C, and E) or cryosections with (B) or without (D) immunogold labeling for synaptophysin. The sections show axonal varicosities innervating smooth muscle. Note the presence of dense cores in SDCVs (arrowheads) after fixation with dichromate and permanganate, but not alde- hyde, whereas the dense cores in secretory granules (arrows) are observed irrespective of the type of fixation. In cryosections prepared after aldehyde fixation, gold particles indicative of synaptophysin immunoreactivity are associatedwith SDCVs but not secretory granules (B). (Inset) Immunogold-labeled vesicular profiles (50-60 nm in diameter) at higher magnifications. No synaptophysin immunoreactivity could be detected in cryosections prepared after dichromate fixation (data not shown). (Bars: A-E, 200 nm.) The pellet was resuspended in 1 ml of in vitro buffer and lected from the bottom of the gradient. Aliquots of each centrifuged for 5 min at 14,000 X gay, and the supernatants fraction were (i) analyzed for [3H]noradrenaline by liquid from the two 14,000 x g centrifugations were pooled. The scintillation counting or (ii) subjected to SDS/PAGE and pooled supernatant (1.0-1.6 ml) was supplemented with (final immunoblot analysis using the following antibodies: the rabbit concentrations) 5 mM creatine phosphate (from a 0.8 M anti-hSgIIi_2o peptide antiserum against secretogranin II (18) stock), creatine phosphokinase at 40 units/ml (from a 3200 (1:200 dilution); a rabbit antiserum (gift of T. Flatmark, units/ml stock), and 5 mM ATP (from a 100 mM stock). In Bergen, Norway) against a synthetic 21-mer peptide, corre- some experiments, the supplemented supernatant was split sponding to the cytoplasmically located portion [Gln2-Ala22 into two aliquots and 3 ,uM reserpine (from a 200 ,uM stock in (19)] of the uncleaved signal peptide (20) of rat dopamine ethanol) or the corresponding amount of ethanol only was 3-hydroxylase, specific for the membrane-bound, but not the added. The supplemented supernatant was then preincubated soluble, form of dopamine f3-hydroxylase (1:500 dilution); the for 5 min at 32°C, followed by the addition of 1-[7,8- mouse monoclonal antibody against synaptophysin (14) (0.2 3H]noradrenaline at 7.2 ,uCi/ml (180 nM) (40 Ci/mmol; 1 Ci ,g/ml); a rabbit antiserum against synaptoporin (21) (1:500 = 37 GBq; Amersham TRK.584) and a 30-min incubation. At dilution); and mouse monoclonal antibody 41.1 against syn- the end of the incubation, as shown in Fig. 2, samples were aptotagmin (22) (1 ,ug/ml). Rabbit primary antibodies were either subjected to immunoadsorption or directly applied to a detected by incubation with 125I-labeled protein A (0.12 ,uCil linear 0.6-1.6 M sucrose gradient (Beckman SW40 rotor) and ml; NEN). Monoclonal primary antibodies were detected by centrifuged at 25,000 rpm for 5 hr, with the brake off. incubation with rabbit anti-mouse IgG (Cappel) at 1 ,ug/ml Immunoadsorption was performed as described (17) by using and 1251-labeled protein A (0.12 ACi/ml; NEN). Immunore- immunobeads (Bio-Rad) precoated without or with the mouse activity was quantitated by using a Fuji BioImaging analyzer. monoclonal antibody against synaptophysin at 10 ,ug/ml (14). Immunofluorescence. Primary culture of sympathetic neu- At the end of the immunoadsorption, the unbound material rons from the superior cervical ganglion of newborn rats was was loaded onto the linear sucrose gradient. After equilibrium performed as described (23). After 11 days in culture, cells sucrose gradient centrifugation, fractions (0.5 ml) were col- were fixed and processed for double immunofluorescence as Downloaded by guest on September 26, 2021 7344 Neurobiology: Bauerfeind et al. Proc. Natl. Acad. Sci. USA 92 (1995)

secretory granules SDCVs described (24), by using the mouse monoclonal antibody against synaptophysin (14) (0.5 ,.g/ml) followed by a fluores- cein isothiocyanate-conjugated secondary antibody and the rabbit antisera against secretogranin II (18) (1:50 dilution) or qMc the membrane-bound form of dopamine j3-hydroxylase (1:100 .s- dilution) followed by tetramethylrhodamine P isothiocyanate- e conjugated secondary antibodies. The confocal laser scanning s- microscope used was a Leica TCS4D. Scanning of optical 1000 Z E sections in the x and y axes was performed with a pinhole setting of 80, and a single pixel corresponded to 110 nm. 0 i RESULTS SDCVs Contain Synaptophysin, an Integral Membrane :5 Protein Characteristic of Classic Synaptic Vesicles. We first tco examined whether SDCVs contain the integral membrane 500- C protein synaptophysin, a major component of classic synaptic E vesicles but not secretory granules (8, 25). SDCVs were E nus1 identified in sympathetic neurons of the rat vas deferens by using permanganate (Fig. 1E) or dichromate (Fig. 1 C and D) fixation (6, 7). After such fixation, both the numerous SDCVs :> (arrowheads) and the few secretory granules (arrows) ob- Cd served in axonal varicosities are characterized by electron- 2 dense cores that result from the fixation of catecholamines and o condensed secretory proteins. In contrast, after aldehyde E0 fixation (Fig. 1 A and B), secretory granules (arrows) but not 0 SDCVs (arrowheads) appear with electron-dense cores be- cause of the differential effect ofthis fixation on the condensed

Ca3000 secretory proteins and the catecholamines. Immunogold la- beling with an anti-synaptophysin antibody of cryosections > 2000 prepared after aldehyde fixation (Fig. 1B) revealed immuno- CaCu 2 reactivity over membrane profiles corresponding to SDCVs ° 1 000. (arrowheads, compare with Fig. 1D) but not over those corresponding to secretory granules (arrows). Hence, SDCVs E o04 contain the classic synaptic vesicle marker protein synapto- , 2000- physin. Cd Coexistence ofClassic Synaptic Vesicle and Secretory Gran- 2 1500- ule Membrane Proteins in SDCVs. Rat vas deferens SDCVs :> and secretory granules were separated from each other by u 1000- 12 equilibrium sucrose gradient centrifugation and the gradient

500- fractions were analyzed for the presence of various marker E proteins (Fig. 2, solid circles). Two populations of vesicles E A-1 capable of [3H]noradrenaline uptake in vitro were observed (Fig. 2A). The sensitivity of this uptake to reserpine (Fig. 24, 600 crosses), a specific inhibitor of vesicular but not plasma Cd membrane catecholamine uptake (26), indicates that both of .g 400 these catecholamine-containing vesicles possess the vesicular amine transporter. The vesicles in the denser fractions of the Es 200 gradient were identified as secretory granules because of the presence of secretogranin II (Fig. 2B), a secretory protein

0 present in the matrix of secretory granules in a wide variety of 0 5 10 15 20 endocrine cells and neurons (27). The catecholamine- Fraction containing vesicles in the lighter fractions of the gradient were identified as SDCVs because of the presence of synaptophysin FIG. 2. Comparison of SDCVs and secretory granules after their (Fig. 2D), which is specifically associated with SDCVs as shown separation by equilibrium sucrose gradient centrifugation. A mem- in Fig. 1B. brane fraction containing secretory granules (solid arrow) and SDCVs Both secretory granules and SDCVs contained synaptotag- (open arrow) was prepared from rat vas deferens and subjected to catecholamine uptake followed by immunoadsorption of vesicles with min (Fig. 2F), a protein implicated in calcium-dependent control immunobeads (solid circles) or immunobeads coated with exocytosis (28). Synaptoporin, which like synaptophysin is an synaptophysin antibody (open circles). The nonimmunoadsorbed integral membrane protein characteristic of classic synaptic membranes were then analyzed by equilibrium sucrose gradient cen- vesicles but not secretory granules (21), was associated with the trifugation (fraction 1 = bottom of gradient), and fractions were SDCVs of the rat vas deferens but not the secretory granules subjected to catacholamine determination (A) and immunoblot anal- (Fig. 2E). In contrast, the membrane-bound form of dopamine ysis for secretogranin II (B), the membrane-bound form of dopamine f-hydroxylase, a protein characteristic of certain secretory ,B-hydroxylase (C), synaptophysin (D), synaptoporin (E), and synap- granules but not classic synaptic vesicles (29), was found not totagmin (F). A also shows catecholamine uptake in the presence of only in secretory granules of the rat vas deferens but also in reserpine (crosses) into vesicles subjected directly to equilibrium 2C). sucrose gradient centrifugation; in this experiment, catecholamine SDCVs (Fig. recovery in the absence of reserpine (data not shown) was 20-25% To determine whether the various proteins detected in the higher than in the nonimmunodepleted sample (solid circles) shown in SDCV-containing fractions of the gradient were indeed local- A. a.u., Arbitrary units. ized in the same membrane vesicle as the classic synaptic Downloaded by guest on September 26, 2021 Neurobiology: Bauerfeind et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7345 vesicle marker synaptophysin, we subjected a membrane frac- cretory vesicles (although other types of vesicles cannot be tion from vas deferens to immunoadsorption by using immu- excluded). Our results are consistent with the hypothesis that nobeads coated with an anti-synaptophysin antibody, followed both the constitutive (synaptophysin) and the regulated (do- by equilibrium sucrose gradient centrifugation of the nonad- pamine 83-hydroxylase) pathway of protein secretion contrib- sorbed membrane vesicles (Fig. 2, open circles). This immu- ute to the biogenesis of SDCVs (for details, see Fig. 4). noadsorption resulted in the removal of only a small portion of the proteins studied from the sucrose gradient fractions con- taining secretory granules. [This is consistent with previous DISCUSSION observations that the abundance of synaptophysin on secretory Our study resolves a long-standing controversy about the granules is very low (8, 25) but may be sufficient for their biogenesis of synaptic vesicles and calls for a modification of partial immunoadsorption (10).] In contrast, the immunoad- the classification of neurosecretory vesicles. Because SDCVs sorption led to the almost complete depletion of each of the have been regarded as model synaptic vesicles by some inves- proteins analyzed from the sucrose gradient fractions contain- tigators (4, 9), the fact that they contain secretory granule ing SDCVs, indicating that each of these proteins resided in membrane components (5, 11) has led to the general hypoth- synaptophysin-containing vesicles. We conclude that SDCVs esis that synaptic vesicles are formed from the membrane of contain membrane proteins of both classic synaptic vesicles secretory granules after their exocytosis. On the other hand, (synaptophysin and synaptoporin) and secretory granules (ve- the characterization of highly purified brain synaptic vesicles, sicular monoamine transporter and membrane-bound form of which for the vast majority are classic synaptic vesicles as they dopamine P-hydroxylase) and, thus, are hybrids of these two do not contain biogenic amines but classic neurotransmitters, types of neurosecretory vesicles (for synaptotagmin, see Dis- has established that their membrane protein composition is cussion). clearly distinct from that of secretory granules (1, 25). This has Delivery of Classic Synaptic Vesicle and Secretory Granule Membrane Proteins to SDCVs via Distinct Vesicles. How are the membrane proteins characteristic of either classic synaptic vesicles or secretory granules delivered to SDCVs? To obtain TRANS GOLGI NETWORK a first clue toward answering this question, we studied the localization of synaptophysin and dopamine f3-hydroxylase in the neurites of primary sympathetic neurons in culture, by using double immunofluorescence and confocal laser scanning microscopy (Fig. 3). Consistent with the results of subcellular $aaTrov;uvU'SECRETORY GRANULE fractionation and immunoadsorption, synaptophysin and do- $E')It,"Ar,CgtYO v MD LE, Ssecretory proteins pamine 3-hydroxylase were colocalized in varicosities and syn-a,lDphy-sirl VMAT, DBH cytochrome b561 terminals (data not shown), where SDCVs are known to (synnptu~rromir3 ?3 synaptotagmin accumulate. However, in the thin segments of the neurites, analysis of the fine immunoreactive puncta, which are likely to correspond to vesicles en route to varicosities and terminals, EARLY ENDOSOME revealed that synaptophysin and dopamine ,3-hydroxylase were localized in distinct vesicles (Fig. 3A). As SDCVs of cat- echolaminergic neurons characteristically contain both synap- tophysin and dopamine /3-hydroxylase (see Fig. 2), neither of these vesicles was SDCVs. The vesicles immunoreactive for 83jLAA\Lq_DENSE dopamine ,B-hydroxylase contained secretogranin II (data not shown) and, thus, were likely to be secretory granules. In ...... ; contrast, the vesicles immunoreactive for synaptophysin lacked ...... - .. secretogranin II (Fig. 3B) and, thus, were not secretory syaptojApor1ri granules. Given the previous observation that in the neuroen- sy npift4agmin docrine cell line PC12, newly synthesized synaptophysin is VMAT, DBH delivered from the trans-Golgi network to the cell periphery in cytochromeb56l constitutive secretory vesicles (2), at least some of these synaptophysin-positive but secretogranin II- and dopamine PLASMA MEMBRANE f3-hydroxylase-negative vesicles may well be constitutive se- FIG. 4. Proposed biogenesis of the SDCV, a hybrid between the classic synaptic vesicle and the secretory granule membranes. Char- acteristics of classic synaptic vesicles and secretory granules are indicated by the use of open and solid type, respectively. In the perikaryon, the membrane proteins characteristic of either classic synaptic vesicles (e.g., syn4ptophysin) or secretory granules [e.g., dopamine f3-hydroxylase (DBH) or vesicular monoamine transporter -El. (VMAT)] are segregated, in the trans-Golgi network, into distinct secretory vesicles-i.e., the constitutive secretory vesicle and the secretory granule, respectively. After their exocytosis at the cell FIG. 3. Localization of synaptophysin, the membrane-bound form periphery, both the membrane proteins characteristic of classic syn- of dopamine f3-hydroxylase, and secretogranin II in the neurites of rat aptic vesicles and secretory granules are intemalized into early endo- sympathetic neurons in primary culture studied by double immuno- somes. In neurons that form secretory granules and classic synaptic fluorescence and confocal laser scanning microscopy. (A) Synapto- vesicles (e.g., glutamatergic neurons), the membrane proteins char- physin (green) and dopamine 3-hydroxylase (red). (B) Synaptophysin acteristic of secretory granules and classic synaptic vesicles are segre- (green) and secretogranin II (red). The two micrographs are single gated, in early endosomes, into vesicles recycling back to the trans- optical sections. Note that in the thin segments ofthe neurites, the fine Golgi network and into classic synaptic vesicles, respectively (not puncta of immunoreactivity for synaptophysin (A and B) (arrows) are illustrated). In contrast, in neurons that form secretory granules and distinct from those for the membrane-bound form of dopamine SDCVs (e.g., catecholaminergic sympathetic neurons), the membrane ,3-hydroxylase (A) (arrowheads) and secretogranin II (B) (arrow- proteins characteristic of secretory granules and classic synaptic heads). (Bar in B = 5 ,um.) vesicles are assembled, in early endosomes, to generate SDCVs. Downloaded by guest on September 26, 2021 7346 Neurobiology: Bauerfeind et al. Proc. Natl. Acad Sci. USA 92 (1995) led to the realization that the biogenesis of classic synaptic microscopy and comments on the manuscript; and Alan Summerfield vesicles should be independent from that of secretory granules for excellent photography and artwork. W.B.H. was supported by a (1, 25), a concept supported by experimental evidence (2) and grant from the Deutsche Forschungsgemeinschaft (SFB 317). also consistent with the fact that in many neurons the abun- dance of secretory granules simply seems too low for this 1. De Camilli, P. & Jahn, R. (1990)Annu. Rev. Physiol. 52,625-645. 2. Regnier-Vigouroux, A. & Huttner, W. B. (1993) Neurochem. Res. organelle to provide the membrane for classic synaptic vesicles. 18, 59-64. Our observations that the SDCV is neither derived only from 3. Burgess, T. L. & Kelly, R. B. (1987) Annu. Rev. Cell Biol. 3, the secretory granule membrane nor a classic synaptic vesicle 243-293. lacking secretory granule membrane components but, rather, 4. Winkler, H., Sietzen, M. & Schober, M. (1987)Ann. N.Y Acad. is a vesicle composed of membrane constituents of both classic Sci. 493, 3-19. synaptic vesicles (synaptophysin and synaptoporin) and secre- 5. Neuman, B., Wiedermann, C. J., Fischer-Colbrie, R., Schober, tory granules (vesicular amine transporter and membrane- M., Sperk, G. & Winkler, H. (1984) Neuroscience 13, 921-931. bound form of dopamine f-hydroxylase) demonstrate that 6. Smith, A. D. (1972) Pharmacol. Rev. 24, 435-457. each of the two previous concepts as to the nature of SDCVs 7. Klein, R. L., Lagercrantz, H. & Zimmermann, H. (1982) Neu- was partially correct, but incomplete. The present data and rotransmitter Vesicles (Academic, London). 8. De Camilli, P. & Navone, F. (1987) Ann. N.Y Acad. Sci. 493, previous observations (5, 11, 29, 30) require that the concept 461-479. of the existence of two types of neurosecretory vesicles, each 9. Winkler, H. & Fischer-Colbrie, R. (1990) Neurochem. Int. 17, originating independently from the other via either the con- 245-262. stitutive (classic synaptic vesicle) or the regulated (secretory 10. Lowe, A. W., Maddedu, L. & Kelly, R. B. (1988) J. CellBio. 106, granule) pathway of protein secretion, needs to be modified 51-59. (see Fig. 4) to include the SDCV as a hybrid type of neuro- 11. Schwarzenbrunner, U., Schmidle, T., Obendorf, D., Scherman, secretory vesicle. D., Hook, V., Fischer-Colbrie, R. & Winkler, H. (1990) Neuro- Synaptophysin and the membrane-bound form of dopamine science 37, 819-827. f3-hydroxylase, though colocalized in SDCVs, were found in 12. Griffiths, G., Simons, K., Warren, G. & Tokuyasu, K T. (1983) distinct vesicles in the neurites of sympathetic neurons. We Methods Enzymol. 96, 466-485. 13. Griffiths, G., McDowall, A., Back, R. & Dubochet, J. (1984) J. interpret this as an indication that both constitutive secretory Ultrastruct. Res. 89, 65-78. vesicles (containing synaptophysin but lacking dopamine (-hy- 14. Wiedenmann, B. & Franke, W. W. (1985) Cell 41, 1017-1028. droxylase and secretogranin II) and secretory granules (con- 15. Slot, J. W. & Geuze, H. J. (1985) Eur. J. Cell Biol. 38, 87-93. taining dopamine f-hydroxylase and secretogranin II but 16. Fried, G., Lagercrantz, H. & H6kfelt, T. (1978) Neuroscience 3, lacking synaptophysin) deliver membrane proteins to the site 1271-1291. of SDCV biogenesis. Our concept, though speculative at 17. Gruenberg, J. & Howell, K. E. (1985) Eur. J. Cell Biol. 38, present, is illustrated in Fig. 4. Based on our observations, we 312-321. propose that the membrane proteins of SDCVs (i) are segre- 18. Rosa, P., Bassetti, M., Weiss, U. & Huttner, W. B. (1992) J. gated from each other into constitutive secretory vesicles and Histochem. Cytochem. 40, 523-533. upon exit from the trans-Golgi network 19. McMahon, A., Geertman, R. & Sabban, E. L. (1990) J. Neurosci. secretory granules Res. 25, 395-404. and, after exocytosis of these vesicles, (ii) meet again in early 20. Taljanidisz, J., Stewart, L., Smith, A. J. & Klinman, J. P. (1989) endosomes [into which both classic synaptic vesicle (31) and Biochemistry 28, 10054-10061. secretory granule (32) membrane proteins are internalized 21. Knaus, P., Marqueze-Pouey, B., Scherer, H. & Betz, H. (1990) after exocytosis] to assemble into the SDCV membrane. This Neuron 5, 453-462. concept not only is consistent with our data but also provides 22. Brose, N., Petrenko, A. G., Sudhof, T. C. & Jahn, R. (1992) a satisfactory explanation for the differential distribution of Science 256, 1021-1025. synaptophysin and various secretory granule membrane pro- 23. Higgins, D., Lein, P. J., Osterhout, D. J. & Johnson, M. I. (1991) teins in sympathetic axons observed by other investigators (11, in CulturingNerve Cells, eds. Banker, G. & Goslin, K (MIT Press, 30, 33). Cambridge, MA), pp. 177-205. It remains to be 24. Rosa, P., Weiss, U., Pepperkok, R., Ansorge, W., Niehrs, C., determined whether synaptotagmin, which Stelzer, E. H. K. & Huttner, W. B. (1989) J. CellBiol. 109, 17-34. is found in secretory granules, classic synaptic vesicles, and 25. Jahn, R. & De Camilli, P. (1991) in Markers for Neural and SDCVs, exits from the trans-Golgi network only in secretory Endocrine Cells, eds. Gratzl, M. & Langley, K. (VCH, New York), granules or also in constitutive secretory vesicles (Fig. 4). It is pp. 25-92. worth noting that the relative proportion of synaptotagmin 26. Johnson, R. G. (1988) Physiol. Rev. 68, 232-307. immunoreactivity in SDCVs vs. secretory granules (Fig. 2F) 27. Huttner, W. B., Gerdes, H.-H. & Rosa, P. (1991) in Markers for was the same as that of dopamine ,B-hydroxylase immunore- Neural and Endocrine Cells: Molecular and Cell Biology, Diagnos- activity (Fig. 2C). This would be consistent with the possibility ticApplications, eds. Gratzl, M. & Langley, K (VCH, New York), that in SDCV-containing neurons, synaptotagmin is delivered pp. 93-131. to the site of SDCV formation predominantly via secretory 28. Chapman, E. R. & Jahn, R. (1994) Semin. Neurosci. 6, 159-165. 29. Thureson-Klein, A. K. & Klein, R. L. (1990) Int. Rev. Cytol. 121, granules. However, in many neurons the abundance of secre- 67-126. tory granules in comparison to classic synaptic vesicles may be 30. Annaert, W. G., Quatacker, J., Llona, I. & De Potter, W. P. so low as to necessitate delivery of synaptotagmin to the site (1994) J. Neurochem. 62, 265-274. of classic synaptic vesicle formation via the same traffic route 31. McPherson, P. S. & De Camilli, P. (1994) Semin. Neurosci. 6, as synaptophysin (2). 137-147.- 32. Patzak, A. & Winkler, H. (1986) J. Cell Biol. 102, 510-515. We thank Drs. H. Betz, T. Flatmark, H.-H. Gerdes, and R. Jahn for 33. Schmidle, T., Weiler, R., Desnos, C., Scherman, D., Fischer- antibodies; Dr. Hermann Rohrer for advice on the culturing of Colbrie, R., Floor, E. & Winkler, H. (1991) Biochim. Biophys. sympathetic neurons; Dr. Matthew Hannah for help with confocal Acta 1060, 251-256. 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