Journal of Cell Science 106, 649-656 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
649
Recycling of a secretory granule membrane protein after stimulated
secretion
Stella M. Hurtley
Department of Biochemistry, University of Edinburgh Medical School, Hugh Robson Building, George Square, Edinburgh,
EH8 9XD, UK
SUMMARY
Recycling of a secretory granule membrane protein,
dopamine- -hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were
stimulated to secrete in the presence of antibodies
directed against the luminal domain of dopamine- hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory
stimulus was assessed using immunofluorescence
microscopy. Stimulation led to the exposure of
dopamine- -hydroxylase at the plasma membrane,
which could be detected by a polyclonal antibody in
living and fixed cells. The plasma membrane dopamine-hydroxylase, either alone or complexed with antibody,
was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained
stable for at least 24 hours of reculture. Twenty four
hours after stimulation the cells with internalized antibody could respond to further stimulation and some of
the antibody was re-exposed at the plasma membrane.
These findings were confirmed using FACS analysis.
This suggests that the antibody-protein complex had
returned to secretory granules that could respond to
further secretagogue stimulation.
INTRODUCTION
by clathrin-coated-pit-mediated endocytosis (Phillips,
1987).
Several studies have demonstrated the delivery of externally applied antibodies recognising secretory granule
membrane proteins to organelles defined by morphological
criteria to be secretory granules (Patzak and Winkler, 1986;
Yamashita and Yasuda, 1992) that have been interpreted as
demonstrating membrane recycling after stimulated secretion. However, morphological criteria alone, in the absence
of additional cytochemical markers, may well be inadequate
in the discrimination of secretory granules from lysosomes
(Patzak et al., 1987) and so it is possible that these studies
only showed endocytosis of secretory granule membrane
proteins, rather than functional recycling. In order to
demonstrate functional recycling it is necessary to show that
the proteins travel to a functional secretory granule, i.e. one
that can respond to a further secretory stimulus.
Here the recycling of a secretory granule membrane protein, dopamine-β-hydroxylase (DBH), has been monitored
by the delivery of antibodies against DBH to an intracellular compartment that can respond to a further secretory
stimulus.
Membrane traffic in mammalian cells occurs via the budding and fusion of transport vesicles. During this process
there is a net transfer of membrane from one organelle to
another. This membrane flow requires recycling pathways
in order for the cell to maintain the size and composition
of organelle membranes.
The secretory pathway is expressed constitutively in all
nucleated mammalian cells (Burgess and Kelly, 1987).
Secretory and membrane proteins enter the secretory pathway by cotranslational translocation at the endoplasmic
reticulum and are then transported via the Golgi complex
to the plasma membrane by vesicular carriers. In addition
to the constitutive secretory pathway, several cell types also
possess a regulated pathway (Burgess and Kelly, 1987). In
this case secretory proteins are stored after exit from the
Golgi complex in secretory granules, which do not fuse with
the plasma membrane until the cell receives a specific secretory stimulus. During stimulated secretion the membrane of
the secretory granule fuses with the plasma membrane,
releasing its contents to the extracellular milieu, and the
secretory granule membrane proteins become incorporated
into the plasma membrane. They are rapidly internalized,
and are thought to recycle to newly forming secretory granules, since their rate of turnover is less than that of the
secretory proteins (Winkler, 1977). The secretory granule
membrane proteins are cleared from the plasma membrane
Key words: chromaffin cells, secretion, membrane recycling,
membrane traffic
MATERIALS AND METHODS
Bovine adrenal glands were collected from the local abattoir and
processed as soon as possible after slaughter. Tissue culture and
650
S. M. Hurtley
other reagents were purchased from Gibco or Sigma unless stated
otherwise.
Cell culture
Primary cultures of bovine adrenal chromaffin cells were prepared
using a modification (Cheek et al., 1989) of the method of Greenberg and Zinder (1982) omitting the differential plating step, and
cultured on glass coverslips in a 37°C humidified incubator with 6%
CO2. Plating medium consisted of DMEM + 20 mM Hepes, 2%
foetal calf serum, 25 µg/ml fluorodeoxyuridine, 50 µg/ml ascorbic
acid, 3 µg/ml cytosine arabinoside,100 units/ml penicillin, 100
µg/ml streptomycin, 50 µg/ml gentamycin and 0.25 µg/ml fungizone, and was changed 24-48 hours later to maintenance medium
that lacked serum. Cells were used 3-10 days after plating.
Cell stimulation
Cells were stimulated to secrete at room temperature (20-25°C).
Chromaffin cells grown on glass coverslips were washed with
Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 5.6 mM glucose,
1.2 mM MgCl2, 2.2 mM CaCl2, 5 mM Hepes, pH 7.0), allowed
to equilibrate in Mg-Locke’s (154 mM NaCl, 5.6 mM KCl, 5.6
mM glucose, 3.4 mM MgCl2, 5 mM Hepes, pH 7.0) for 10 minutes and then treated with Ba-Locke’s (154 mM NaCl, 5.6 mM
KCl, 5.6 mM glucose, 5 mM BaCl2, 1.2 mM MgCl2, 5 mM Hepes,
pH 7.0) for 10 minutes; control cells were treated in parallel with
Locke’s buffer. When appropriate, anti-DBH serum was added to
the stimulation medium at a 1:100 dilution. At least 99% of the
cells remained viable throughout the experiment. Cells were either
fixed immediately or returned to culture for various times after
removal of Ba-Locke’s. Approximately 50% of the cells remaining on the coverslips after processing contained DBH and could
respond to barium stimulation.
Immunofluorescence microscopy
Cells were processed for immunofluorescence microscopy using
a method adapted from Timm et al. (1983). Briefly, cells were
fixed for 20 minutes in 3% paraformaldehyde in phosphatebuffered saline (PBS) plus calcium (0.1 mM) and magnesium (0.1
mM), washed with PBS, and aldehyde groups were quenched
with 50 mM ammonium chloride in PBS for 10 minutes, and
then, if internal staining was required, cells were permeabilized
with 0.5% Triton X-100 in PBS for 1 minute. Cells were then
washed with PBS and protein sites were blocked for 30-60 minutes with 5% goat serum (from the Scottish Antibody Production
Unit, Carluke) in PBS. Primary antibodies, if appropriate, were
added at 1:100 to 1:500 dilution in 5% goat serum in PBS for
60 minutes, washed with PBS, reblocked, and the second reagent,
rhodamine-labelled goat anti-rabbit IgG 1:500 (Sigma) in 5% goat
serum in PBS, was applied for 60 minutes; they were then washed
and mounted in Mowiol, left to set overnight at room temperature and observed in a Leitz Ortholux II microscope with a ×63
oil immersion lens and photographed on Ilford HP5+ film. All
negatives and prints of internalized anti-DBH were exposed,
developed and printed under identical conditions to allow direct
comparison of images.
The polyclonal antibody against DBH was provided by D. Apps
and J. Phillips and is described by Hunter and Phillips (1989).
FACS analysis
For FACS analysis freshly isolated cells were stimulated in suspension.
Cells were isolated as usual, but plated onto bacteriological
plastic in order to avoid tight adherence. Cells were scraped from
the dish and resuspended in Locke’s buffer. Treatments were performed in suspension using the same methods as described for the
immunofluorescence experiments. Cells were stimulated for the
first time 2-4 hours post-preparation, allowed to recycle overnight
and then scraped and resuspended for the second stimulation. Samples of cells (approximately 106) were removed after the first stimulation, after overnight incubation and after restimulation were
washed with ice-cold PBS, incubated with anti-DBH for 10 minutes on ice if appropriate, fixed and processed as for immunofluorescence microscopy, omitting the Triton X-100 step. All cells
were stained with a 1:100 dilution of phycoerythrin-conjugated
goat anti-rabbit IgG (Sigma) in 5% goat serum in PBS. The samples were resuspended and examined in a FACS analyser (Becton
and Dickinson FACSCAN).
RESULTS
In order to study membrane recycling after stimulated secretion primary cultures of bovine adrenal chromaffin cells were
used. These cells contain stored catecholamines in their regulated secretory granules, the chromaffin granules, and can
respond in culture to various secretory stimuli.
A major protein of the chromaffin granule membrane is
dopamine-β-hydroxylase (DBH). DBH is a tetramer of 75
kDa subunits associated with the membrane and the lumen
of the chromaffin granule. DBH is a suitable candidate for
following the recycling pathway because it is a major component of the granule membrane and its rate of synthesis
has been shown to be less than that of the granule content
proteins (Winkler, 1977).
DBH is delivered to the cell surface during
stimulation
Cells were stimulated to secrete using 5 mM barium ions.
Barium is one of the most efficient secretagogues, consistently promoting the release of approximately 30% of stored
catecholamines from chromaffin cells (Heldman et al., 1989;
Hunter and Phillips, 1989). Fig. 1 shows the results of indirect immunofluorescence microscopy using a polyclonal
antibody that recognises DBH (Hunter and Phillips, 1989).
After barium stimulation and fixation the cell surface was
labelled with the polyclonal antibody (Fig. 1E), indicating
that chromaffin granules had fused with the plasma membrane to expose DBH at the cell surface. Control cells (Fig.
1A) showed only low levels of surface immuno-reactivity,
suggesting that very few chromaffin granules fused with the
plasma membrane in the absence of a secretory stimulus, as
expected for regulated secretory cells. In this panel one cell
had been permeabilized during fixation and showed punctate
intracellular labelling, serving as an internal control for the
immunofluorescence protocol (Fig. 1A, also seen in Fig.
1G). Inside the cells, both stimulated and control, DBH
showed a typical strong punctate staining characteristic of
chromaffin granules (Fig. 1B, F). In the experiment shown in
Fig. 2 cells were stimulated to secrete in the presence of antiDBH antibodies and fixed, and then surface and internal label
was assessed. The antibody was bound to intact, stimulated
cells (Fig. 2E), whereas unstimulated cells bound very little
antibody (Fig. 2A). This is in good agreement with the results
shown in Fig. 1 and indicates that the polyclonal antibody
could recognise native DBH when delivered to the plasma
membrane in stimulated, unfixed cells. Since the cells were
not fixed before addition of the antibody, the inadvertent per-
Recycling after stimulated secretion
651
Fig. 1. Dopamine-β-hydroxylase is exposed at the cell surface during barium stimulation. Bovine adrenal chromaffin cells were grown on
glass coverslips and stimulated to secrete using barium ions (+Ba2+), control cells were not stimulated (−Ba2+). Indirect
immunofluorescence microscopy was performed, either immediately (t=0) or after reincubation at 37°C for 120 minutes (t=120) on intact
(−Tx) or Triton-permeabilized (+Tx) cells using a polyclonal antibody against DBH to ascertain the distribution of DBH on the surface
versus the interior of the cells. Bar, 10 µm.
meabilization observed in Fig. 1 was not observed. Permeabilized stimulated or unstimulated cells showed very little
additional staining immediately after stimulation (Fig. 2B,
F), confirming that the cells were intact when the antibody
was applied.
DBH is rapidly internalized
Figs 1 and 2 also show the results of reincubation of stimulated cells for 2 hours after the removal of secretagogue.
In Fig. 1 the increase in surface DBH seen at t=0 was
rapidly removed during reculture of stimulated cells in the
absence of secretagogue (Fig. 1G), suggesting that the DBH
delivered to the cell surface during exocytosis had been
internalized. Fig. 2 demonstrates that the polyclonal antiDBH that had been bound to intact cells during barium
stimulation was rapidly cleared from the cell surface after
removal of the secretory stimulus (Fig. 2G), in agreement
with the results for unbound DBH in Fig. 1, and appeared
in punctate intracellular structures (Fig. 2H). This suggested
that the presence of anti-DBH bound to the DBH at the
plasma membrane did not inhibit the internalization of
DBH. Control cells, as expected, showed only low levels
of immuno-reactivity on the surface or inside the cells (Fig.
2C, D). Identical results were observed after only 30 minutes of reculture (not shown).
Thus DBH alone, or when complexed with anti-DBH,
was rapidly internalized after removal of the secretory stimulus. Indeed the increase in amount of punctate label in the
permeabilized, stimulated cells before reincubation in Fig.
2 (compare Fig. 2F with E) suggested that endocytosis had
already been initiated in the presence of stimulus.
Taken together these results indicated that the polyclonal
anti-DBH should be a suitable probe to use for recycling
studies.
Internalized anti-DBH is stable overnight
Cross-linking of surface molecules by antibodies is thought
to cause their internalization and transport to the lysosome
652
S. M. Hurtley
Fig. 2. Binding and internalization of antibody to dopamine-β-hydroxylase after stimulated secretion. Bovine adrenal chromaffin cells
grown on glass coverslips were stimulated to secrete using barium ions (+Ba2+) in the presence of polyclonal antibody to DBH, control
cells were not stimulated (−Ba2+). Cells were either fixed immediately (t=0) or returned to culture for a further 2 hours (t=120). The fixed
cells were processed for immunofluorescence microscopy, either intact (−Tx) or after permeabilization using Triton X-100 (+Tx) to
ascertain the distribution of anti-DBH antibody on the surface versus the interior of the cells. Bar, 10 µm.
(Mellman and Plutner, 1984) where they will be degraded.
Fig. 3 shows that after overnight incubation the bound and
internalized anti-DBH antibody was still easily detectable
inside the barium-stimulated chromaffin cells in a punctate
distribution (Fig. 3F) and, as expected, there was little surface reactivity (Fig. 3E). This suggests that the internalized
antibodies had not been delivered to a degradative compartment such as the lysosome. Control cells showed only
low levels of antibody after overnight incubation inside or
outside the cells (Fig. 3A, B) in agreement with the results
shown in Fig. 2.
These results suggest that the anti-DBH was not transferred to the lysosomes in these cells, but was transferred
to an intracellular compartment where it was stable
overnight. The anti-DBH thus did not appear to cause crosslinking of the DBH and transport to the lysosome, possibly
because DBH is a tetramer, and polyvalency of the antibody would cross-link subunits of DBH without necessarily cross-linking separate oligomers.
Internalized anti-DBH responds to further
stimulation
The functional demonstration of membrane recycling
requires that internalized granule membrane proteins return
to secretory granules that can respond to a further secretory
stimulus. Fig. 3 also shows the effects of a second secretory stimulus on cells that had previously bound and internalized anti-DBH antibodies. Restimulation caused the
appearance of polyclonal anti-DBH at the cell surface (Fig.
3G). This increase in surface antibody in response to stimulation (compare Fig. 3E with G) strongly suggested that
at least some of the antibody-DBH complex internalized 24
hours earlier had been recycled to functional secretory granules. The level of labelling was consistent with the finding
that only approximately 30% of the chromaffin granules
would be expected to be able to respond to barium stimulation. Control cells, as before, showed little antibody either
inside or on the cell surface (Fig. 3C, D).
Thus barium-stimulated cells that had bound and inter-
Recycling after stimulated secretion
653
Fig. 3. Bound, internalized anti-dopamine-β-hydroxylase antibody is stable overnight and can be re-exposed at the cell surface after
further stimulation. Bovine adrenal chromaffin cells were grown on glass coverslips and stimulated to secrete using barium ions (+Ba2+)
in the presence of polyclonal antibody to DBH, control cells were not stimulated (−Ba2+). Cells were returned to culture overnight and
fixed (t=O/N) or stimulated to secrete a second time and then fixed (restim). The fixed cells were processed for immunofluorescence
microscopy either intact (−Tx) or after permeabilization using Triton X-100 (+Tx) to ascertain the distribution of anti-DBH antibody on
the surface versus the interior of the cells. Bar, 10 µm.
nalized polyclonal anti-DBH antibodies could be stimulated
to re-expose antibody at the cell surface after further stimulation. This strongly suggests that the antibody bound to
DBH was recycled to an internal compartment that was
capable of responding to a further secretory stimulus, most
probably the secretory granules.
Recycling is very efficient
The immunofluorescence analysis described so far strongly
suggests that internalized DBH-anti-DBH complex recycles
to a regulated secretory compartment, most probably secretory granules. In order to obtain a quantitative estimate of
recycling efficiency, and to confirm the interpretation of the
immunofluorescence images, FACS analysis was performed. FACS analysis requires single-cell suspensions.
Freshly isolated chromaffin cells were maintained in suspension, or lightly adherent so that stimulation and restimulation could be performed in suspension. Cells were stim-
ulated in the presence or absence of the polyclonal antiDBH antibody and samples were fixed immediately after
stimulation, after overnight incubation or after restimulation. The samples lacking anti-DBH were incubated on ice
for 10 minutes in the presence of anti-DBH after the
required treatment in order to assess the localization of total
DBH.
The results are shown in Fig. 4. Fig. 4A shows that total
DBH behaved as expected. It was exposed at the surface
after stimulation and after restimulation. The restimulation
was just as efficient as the original stimulation, suggesting
that the cells were capable of responding to a second secretory stimulation with the same efficiency as to the first. In
Fig. 4B antibody was applied during the first stimulation to
living intact cells. As in the control condition, DBH was
only exposed in stimulated cells. After restimulation
approximately 30% of the anti-DBH originally bound to the
cells was re-exposed at the plasma membrane. Since cells
654
S. M. Hurtley
A. Antibody applied after experiment to intact cells
Peak fluorescence (arbitrary units)
800
600
400
200
0
L, t=0
L, t=O/N
L, restim
Ba, t=0
Ba, t=O/N
Ba, restim
Treatment
B. Antibody applied to living intact cells at t=0
Peak Fluorescence (arbitrary units)
600
500
400
300
200
100
0
L, t=0
L, t=O/N
L, restim
Ba, t=0
Ba, t=O/N Ba, restim
Treatment
Fig. 4. FACS analysis of stimulated and restimulated cells.
Chromaffin cells were plated on bacteriological plastic to avoid
tight adherence and stimulated to secrete in suspension using
barium ions (+Ba) in the absence (A) or presence (B) of
polyclonal antibody to DBH at 2 hours after preparation. Control
unstimulated cells were treated with Locke’s buffer (L). Cells
were returned to culture overnight, scraped from the plates and
then stimulated to secrete in suspension a second time and then
fixed. The intact, fixed cells were processed for FACS analysis
immediately after the first stimulation (t=0), after overnight
incubation (t=O/N) or after restimulation (restim) as indicated. In
(A) total surface DBH was assessed by addition of antibody to
intact cells on ice after the required treatment prior to fixation. A
single peak of fluorescence was observed during FACS analysis of
each sample, data shown are the mean of two separate
experiments.
respond to barium stimulation by releasing approximately
30% of their stored catecholamine, this suggests that the
recycling of anti-DBH was very efficient.
DISCUSSION
The data presented here represent one of the first demonstrations of membrane recycling to a functional regulated
secretory compartment after stimulated secretion.
In this study primary cultures of bovine adrenal chro-
maffin cells were used. In the past it has been extremely
difficult to demonstrate the recycling of secretory granule
membrane proteins in cell lines that express a regulated
secretory pathway (see e.g. Green and Kelly, 1992). This
is probably a result of the down-regulation of the pathway
due to de-differentiation and to the fact that in growing cells
the rate of synthesis of granule components should be sufficient to maintain the small granule population that is
present.
Here, a secretory granule membrane protein, DBH,
exposed at the plasma membrane during stimulated secretion, was rapidly internalized in the presence or absence of
anti-DBH antibodies. Indeed uptake was already under way
within the 10 minute stimulation period and was complete
within 30 minutes of reincubation. The internalized antiDBH bound to DBH was stable for prolonged periods inside
the chromaffin cells and was present in a compartment that
could respond to further secretagogue stimulation. This
strongly suggested that the majority of the internalized antibody was recycled to the secretory granules and was not
targeted to the lysosomes.
Previous work has shown that certain internalized antibodies bound to plasma membrane or granule membrane
proteins are transported to the lysosomes, but in this case
they are rapidly degraded (Mellman and Plutner, 1984;
Patzak et al., 1987). Since the internalized antibody
remained stable overnight it is assumed that in this study
little transfer to the lysosome had occurred. The precise itinerary of internalized antibody-protein complexes is likely
to depend on several factors, including the valency of the
antibody and the extent of cross-linking of the antigen.
Other work has shown that incubation of the cells at 4°C
immediately after stimulation inhibited recycling and
appeared to stimulate degradation of internalized DBH
(S.M. Hurtley, unpublished data). In both cases, internalization was complete within 30 minutes at 37°C. A working hypothesis is that granule membrane proteins must be
internalized rapidly after delivery to the plasma membrane
and that if they remain at the plasma membrane long
enough to diffuse away from one another they cannot recycle, but are transported to the lysosome and degraded. In
order to compare these two conditions directly it will be
important to examine the sorting between internalized
secretory granule membrane proteins and other endocytic
tracers.
Recycling as measured by FACS analysis appeared to be
very efficient. In response to a 10 minute stimulation with
barium, cells secrete approximately 30% of their stored catecholamines. In the FACS analysis approximately 30% of
internalized anti-DBH could be stimulated to return to the
plasma membrane following restimulation.
Assuming that the DBH-anti-DBH complex returned to
a homogeneous population of granules, recycling would
appear to be extremely efficient. However, the population
of chromaffin granules in cells is known not to be equally
responsive to a stimulus, and newly synthesized granules
are particularly so (Phillips, 1987). The newly synthesized
granules would be most likely to contain recycled DBH and
therefore it is possible that this method will overestimate
recycling efficiency.
These findings should now be extended to other secre-
Recycling after stimulated secretion
tory granule membrane proteins as suitable reagents
become available. The mechanism, rate and route of recycling to a functional compartment that can respond to further stimulation is now under investigation. In order to estimate the efficiency of the recycling pathway it will be
necessary to go on to use a biochemical assay. Hunter and
Phillips (1989) biotinylated the surface of stimulated cells
to look for recycling and produced preliminary evidence
that recycling occurred in their conditions. These findings
will now be extended to look for evidence of return to a
functional compartment, and to monitor the efficiency and
rate of recycling.
The results presented here show that it is possible to
demonstrate recycling of a secretory granule membrane
protein, DBH, to a functional regulated secretory compartment. This contrasts with simple measurements of endocytosis of granule membrane proteins from the plasma membrane, which do not constitute an assay for recycling. This
approach can now be used to define the kinetics and route
of recycling.
The polyclonal anti-DBH was kindly provided by David Apps
and John Phillips. Andrew Sanderson provided excellent technical assistance with the FACS analysis. Many thanks to David Apps
and James Pryde for critical comments on the manuscript. This
work was supported by the Wellcome Trust.
REFERENCES
Cheek, T. R., Jackson, T. R., O’Sullivan, A. J., Moreton, R. B.,
Berridge, M. J. and Burgoyne, R. D. (1989). Simultaneous
measurements of cytosolic calcium and secretion in single bovine adrenal
chromaffin cells by fluorescent imaging of Fura-2 in cocultured cells. J.
Cell Biol. 109, 1219-1227
655
Burgess, T. L. and Kelly, R. B. (1987). Constitutive and regulated
secretion of proteins. Annu. Rev. Cell Biol. 3, 243-293.
Green, S. A. and Kelly, R. B. (1992). Low density lipoprotein receptor and
cation-independent mannose 6-phosphate receptor are transported from
the cell surface to the Golgi apparatus at equal rates in PC12 cells. J. Cell
Biol. 117, 47-55
Greenberg, A. and Zinder, O. (1982). α- and β-receptor control of
catecholamine secretion from isolated adrenal medulla cells. Cell Tiss.
Res. 226, 655-665
Heldman, E., Levine, M., Raveh, L. and Pollard, H. B. (1989). Barium
ions enter chromaffin cells via voltage-dependent calcium channels and
induce secretion by a mechanism independent of calcium. J. Biol. Chem.
264, 7914-7920
Hunter, A. and Phillips, J. H. (1989). The recycling of a secretory granule
membrane protein. Exp. Cell Res. 182, 445-460
Mellman, I. and Plutner, H. (1984). Internalisation and degradation of
macrophage Fc receptors bound to polyvalent immune complexes. J. Cell
Biol. 98, 1170-1177
Patzak, A., Aunis, D. and Langley, K. (1987). Membrane recycling after
exocytosis: An ultrastructural study of cultured chromaffin cells. Exp.
Cell Res. 171, 346-356
Patzak, A. and Winkler, H. (1986). Exocytotic exposure and recycling of
membrane antigens of chromaffin granules: Ultrastructural evaluation
after immunolabelling. J. Cell Biol. 102, 510-515
Phillips, J. H. (1987). Chromaffin granule biogenesis and the
exocytosis/endocytosis cycle. In Stimulus-Secretion Coupling in
Chromaffin Cells, vol. I. (ed. Rosenheck and Lelkes), pp. 31-54. CRC
Press Inc., Boca Raton, Florida.
Timm, B., Kondor-Koch, C., Lehrach, H., Riedel, H., Edström, J.-E.
and Garoff, H. (1983). Expression of viral membrane proteins from
cloned cDNA by microinjection into eukaryotic cell nuclei. Methods
Enzymol. 96, 496-511
Winkler, H. (1977). The biogenesis of adrenal chromaffin granules.
Neuroscience 2, 657-683
Yamashita, S. and Yasuda, K. (1992). Monoclonal antibody to a common
antigen of secretory granule membranes - Intracellular localization and
recycling of the antigen after secretion. J. Histochem. Cytochem.40, 793806.
(Received 8 June 1993 - Accepted 19 July 1993)