(Received for publication, July 13, 1995; and in revised form, February 16, 1996)
From the
Syntaxin 1A is a nervous system-specific protein thought to
function during the late steps of the regulated secretory pathway by
mediating the docking of secretory vesicles with the plasma membrane.
We have examined the effects of transiently overexpressing syntaxin 1A
on protein secretion in constitutively secreting cell lines that do not
normally express the protein. Syntaxin 1A slowed the constitutive
release of marker proteins human growth hormone (hGH) and vesicular
stomatitis virus glycoprotein from COS-1 cells, increasing the
intracellular half-life of human growth hormone from 90 min to 18 h. A
similar effect was observed in HEK 293 cells. Immunofluorescence
microscopy revealed that these secretory proteins were concentrated in
the periphery of the cell. The effect was specific for the full-length
neuronal protein. Neither a syntaxin 1A variant which lacks a membrane
attachment domain nor syntaxin 2 caused the cells to retain human
growth hormone. The effect of syntaxin 1A was partially reversed by
incubating the cells with botulinum type C neurotoxin,
which specifically cleaves syntaxin 1A. Release of human growth hormone
from syntaxin 1A-expressing cells was maintained during a blockade of
protein synthesis, suggesting that the hormone was being released from
a pool of stored vesicles which accumulated before the addition of
cycloheximide. The existence of a post-Golgi storage compartment in
syntaxin 1A-expressing cells was confirmed using brefeldin A to
collapse the Golgi stacks in both HEK 293 and COS-1 cells. Brefeldin A
rapidly blocked growth hormone release in control cultures while having
no effect on release in cells expressing syntaxin 1A. Reducing the
temperature to 19 °C, which inhibits transport from the trans-Golgi network, also inhibited hGH secretion from cells
without syntaxin 1A but had little effect on hGH secretion from cells
with syntaxin 1A. The present experiments indicate that syntaxin 1A
enables the storage of vesicles which would otherwise be immediately
released.
It has been recognized for many years that there are two pathways, constitutive and regulated, by which secretory proteins are released from cells (reviewed in (1) ). Most cells have an active constitutive pathway, whereby vesicles containing proteins destined for export or for residence on the plasma membrane undergo exocytosis as they are made. The hallmark of constitutive release is that its rate is mainly governed by the rate of synthesis or processing of the protein and not by extracellular stimuli. Proteins which undergo constitutive release do not accumulate to a significant extent inside the cell.
The regulated pathway differs from the constitutive in
several important respects. Vesicles from the trans-Golgi
network (TGN) ()destined for regulated secretion are stored
(sometimes for days), rather than undergoing exocytosis within minutes.
Secondly, release is strongly stimulated by signals originating outside
the cell, usually through a rise in Ca
at release
sites. In addition, cells which express both pathways must possess a
mechanism by which proteins unique to each pathway are sorted to the
appropriate vesicles.
Although the two pathways serve different purposes, they have many similarities. A recent convergence of data has led to a proposed model for the final steps of secretion(2, 3, 4, 5, 6) . Genetic analysis in yeast has led to the identification of specific proteins important for various steps in membrane trafficking in the yeast constitutive secretory pathway. Homologs of these proteins have been identified in mammalian cells including some in secretory vesicle and plasma membranes. Three of these proteins, VAMP-2 (vesicle-associated membrane protein or synaptobrevin-2, in the secretory vesicle membrane), SNAP-25 (synaptosome-associated protein of 25 kDa, in the plasma membrane) and syntaxin (in the plasma membrane) are specific substrates for proteolysis by different clostridial neurotoxins(7, 8, 9, 10, 11, 12, 13, 14, 15) . These toxins are potent inhibitors of regulated exocytosis(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . Their ability to cleave these substrates strongly correlates with their capacity to inhibit exocytosis, thus implicating VAMP-2, SNAP-25, and syntaxin 1A in the late steps in the regulated exocytotic pathway.
Syntaxin seems likely to play a central role in exocytosis. A
neuronal isoform of syntaxin, syntaxin 1A, is predominantly localized
to the cytoplasmic surface of the plasma membrane(28) ,
although there is evidence for its presence on synaptic
vesicles(29, 30) . Syntaxin 1A is found in a 20 S
complex isolated from detergent-solubilized brain membranes, where it
associates with several of the relevant vesicular or plasma membrane
proteins, including VAMP-2 and SNAP-25, and the cytosolic proteins NSF (N-ethylmaleimide-sensitive fusion protein) and - and
-SNAP (soluble NSF attachment proteins)(2) . It can also
exist in at least two other distinct complexes, one which includes
VAMP-2, SNAP-25, synaptotagmin, and the n-type Ca
channel(3, 31, 32, 33) , and
the other with n-Sec1 (also designated munc-18, rbSec1, or mSec1)
alone, from which the other proteins are
excluded(34, 35) . The ability to participate in these
various complexes renders syntaxin 1A a likely candidate for a protein
whose interactions with its neighbors could control important aspects
of secretion. The postulated importance of the interaction of proteins
in donor membranes (vesicular SNAP receptors or v-SNARES) with proteins
in acceptor membranes (target SNAP receptors or t-SNARES) in vesicular
trafficking and exocytosis is the basis for the SNARE hypothesis (2) .
As a t-SNARE, syntaxin 1A, which is normally associated with the regulated secretory pathway, has two postulated functional roles(2, 3) . The first is as a target protein in the plasma membrane, one which interacts with a partner on the approaching vesicle (VAMP-2, a v-SNARE) to ensure specificity of the docking process. The other is as a receptor for soluble NSF-attachment proteins (SNAPs), which may be a necessary step in the pathway leading to fusion. Neither of these putative functions is unique to the regulated pathway. Other more widely distributed members of the syntaxin family (28) may have similar roles in the constitutive pathway.
There is strong evidence implicating syntaxin
1A in relatively late steps in exocytosis. Evoked neurotransmitter
release is abolished in Drosophila mutants lacking syntaxin
1A(30) . Syntaxin 1A is a specific substrate for botulinum type
C neurotoxin(11, 36) , which inhibits
glutamate release from synaptosomes (11) and exocytosis from
chromaffin cells. (
)Injection of the entire cytoplasmic
domain of syntaxin 1A or the cytoplasmic domain immediately adjacent to
the transmembrane domain strongly inhibits exocytosis from PC12
cells(28) . Similarly, Fab fragments derived from
affinity-purified anti-syntaxin 1A also inhibited exocytosis in PC12
cells.
In this study we investigated the effects on constitutive secretion of introducing syntaxin 1A into cells which normally do not express the protein. The motivation for this study was to identify specific functions that syntaxin 1A conveys to the exocytotic pathway. COS-1 and HEK 293 cells have a prominent constitutive pathway, but lack both a regulated secretory pathway and endogenous syntaxin 1A. We adapted to COS-1 and HEK 293 cells a novel approach that was developed to investigate the effects of transiently expressed proteins on the regulated secretory pathway in primary adrenal chromaffin cells. A key part of the technique is the cotransfection of an effector plasmid of interest and a reporter plasmid encoding a protein destined to the regulated pathway. The two proteins are co-expressed in the same cells. In chromaffin cell studies, human growth hormone (hGH) was stored in chromaffin granules (37) and served as a reporter for the regulated secretory pathway in the transfected cells. hGH is normally directed to the regulated secretory pathway(37, 38, 39, 40) , but in cells without a prominent regulated pathway (i.e. COS-1 and HEK 293 cells), it is secreted constitutively.
We examined the effects of expression of members of the syntaxin family on the secretory pathway in COS-1 and HEK 293 cells, using as reporters either hGH or vesicular stomatitis virus glycoprotein (VSV-G), a protein which is constitutively delivered to the plasma membrane. These experiments revealed for the first time the ability of syntaxin 1A to create a storage compartment in secreting cells.
Human GH was measured with either of two (radiometric or luminescent) assay kits from Nichols Institute (San Juan Capistrano, CA). Data are expressed as mean ± S.E. unless indicated otherwise. Error bars smaller than symbols were omitted from figures.
Figure 1: Transiently expressed VSV-G is inserted constitutively into the plasma membrane of COS-1 cells. COS-1 cells were transiently transfected by calcium phosphate precipitation with a plasmid encoding a form of VSV-G which is constitutively inserted into the plasma membrane. After 6 h, cells were shocked with medium containing 10% glycerol, rinsed, and given fresh culture medium containing cytosine arabinoside. Cells were maintained for 3 days in culture before immunofluorescence microscopy as described under ``Experimental Procedures.'' In order to examine VSV-G expression on the exterior surface of the plasma membrane, cells in A were probed with a polyclonal antiserum to VSV-G for 2 h at 4 °C before fixation and permeabilization with methanol. An additional well (B) was probed with anti-VSV-G after permeabilization in order to detect total (external + internal) VSV-G. Calibration bar = 20 µm.
Figure 2: A protein which is normally constitutively delivered to the plasma membrane, VSV-G, is stored when coexpressed with syntaxin 1A in COS-1 cells. In the same experiment as shown in Fig. 1, COS-1 cells were co-transfected with plasmids for VSV-G and syntaxin 1A, a neuronal isoform of syntaxin. Again, VSV-G expression on the exterior surface of the plasma membrane was examined by probing the cells in A with a polyclonal antiserum to VSV-G before fixation and permeabilization with methanol. A separate well (C) was probed with anti-VSV-G after permeabilization to detect total (external + internal) VSV-G. Coexpressed syntaxin 1A was detected with a monoclonal antibody to syntaxin 1A (HPC-1) after permeabilization (B and D). B and D show the same fields as A and C, respectively. Calibration bar = 25 µm.
Figure 3:
hGH
is stored in COS-1 cells which coexpress syntaxin 1A, but not in cells
expressing syntaxin 2, or a truncated syntaxin 1A which lacks a
membrane attachment domain (syn 1AM). COS-1 cells were transiently
transfected by calcium phosphate precipitation with plasmids for hGH,
syntaxin 1A, syntaxin 2 (HA-syn 2), and syn 1A
M as described in
the legend to Fig. 1. After 3 days, cells were prepared for
immunofluorescence microscopy as described. All cells were
permeabilized and probed with a polyclonal antiserum to hGH (shown in A, C, E, and G) and HPC-1 to detect
either syn 1A (B) or syn 1A
M (D), or an antibody
to the HA epitope to detect HA-syn 2 (F). Use of the HA
epitope tag ensures the identification of transfected cells expressing
syntaxin 2 without interference from the endogenous protein. C, E, and G show the same fields as B, D, and F, respectively. Calibration
bar = 25 µm.
These results were confirmed biochemically in parallel
cultures using a sensitive radioimmunoassay for hGH (Fig. 4).
Cells transfected with a plasmid for hGH and the vector pCMVneo stored
only 1% of the hGH produced (41.1 ± 1.3 ng/well), releasing the
rest into the medium. Cells transfected with the syn 1AM or
HA-syntaxin 2 plasmids stored 5-8% of hGH. Cells expressing the
complete syntaxin 1A protein retained half (47%) of the hGH synthesized
during the 72 h following transfection. In some experiments (e.g.Fig. 7B), cells expressing syntaxin 1A stored up
to 70% of the hGH produced.
Figure 4: Effects of syntaxin 1A on storage of hGH in COS-1 cells. In the same experiment as shown in Fig. 3, parallel wells were transfected for biochemical analysis. After 3 days, the amount of hGH in the cells and medium for each group were determined by radioimmunoassay. Data are expressed as the percent of total hGH stored in the cells. n = 4 wells/group.
Figure 7:
Effect of botulinum C neurotoxin on release of hGH from COS-1 cells transfected with
syntaxin 1A. COS-1 cells were transfected with plasmids for hGH and
syntaxin 1A as described for Fig. 5, except that, after 4 h,
half of the wells received botulinum C
toxin (50
nM). After 24 h, the medium was replaced with fresh medium
containing cytosine arabinoside and the toxin. After 2 days, the medium
and cells were harvested and assayed for hGH as before (B).
Aliquots of the cell homogenates were pelleted, and the membrane
fractions and the supernatants were subjected to SDS-polyacrylamide gel
electrophoresis (5-15% gradient) as described (48) and
transferred to Immobilon. Blots (A) were probed with
affinity-purified, polyclonal antisera to syntaxins 1A (lanes
1-3), 2 (not shown), or 3 (lanes 4 and 5),
followed by
I-labeled goat anti-rabbit secondary
antibody. Bands were detected by autoradiography. Preincubating each
antisera with the corresponding fusion protein bound to
glutathione-agarose beads (35) prevented the appearance of
immunoreactive bands (not shown). The blots were first exposed to x-ray
film (A) and then subjected to phosphorimager analysis for
quantitation. Values for syntaxin 1A (arbitrary density units) in the
absence or presence of toxin were 172.9 ± 16.2 or 76.1 ±
5.6, respectively. Values for syntaxin 2 were 183.9 ± 19.0
(-toxin) and 174.5 ± 28.3 (+toxin), and for syntaxin
3 were 207.4 ± 8.1 (-toxin) and 192.8 ± 12.4
(+toxin). n = 4
wells/group.
Figure 5: Cycloheximide blocks constitutive release of hGH but does not block hGH release from COS-1 cells transfected with syntaxin 1A. COS-1 cells were transfected with hGH and syntaxin 1A plasmids as described for Fig. 3, except that the glycerol shock was omitted, and the DNA precipitate remained with the cells for 24 h before being replaced with fresh medium containing cytosine arabinoside. After 3 days, the medium was replaced with fresh medium with or without 5 µg/ml cycloheximide (CHX) for 6 h. Media samples were taken at the indicated times, and the cells were harvested after 6 h. Levels of hGH were determined as above. A shows the time course of inhibition of hGH release by cycloheximide in cells without syntaxin 1A. In B, cells were exposed to medium with or without cycloheximide for 30 min, and hGH secretion was determined during the subsequent 5.5 h in the continuing presence or absence of cycloheximide. C shows the loss of hGH from cells with or without syntaxin 1A during a 6-h incubation with cycloheximide. n = 4 wells/group.
The ability of syntaxin 1A to cause the accumulation of hGH was confirmed in HEK 293 cells. HEK 293 cells transfected with hGH and syntaxin 1A plasmids in ratios of 8:1 and 8:4 stored 29% and 63%, respectively, of the total hGH synthesized.
In order to determine whether the effects of syntaxin in these experiments were due to its overexpression, immunoblotting was used to compare the levels of syntaxin 1A being expressed in COS-1 cells with the endogenous levels in brain synaptosomes. Values (in arbitrary units) from phosphorimager quantitation were 1864 units/µg of protein (bovine brain synaptosomes) and 782 units/µg of protein (COS-1 cell homogenate). Typically, 20-40% of the cells are transfected in the experiments (determined by immunofluorescence). Thus, the effects of syntaxin 1A in transfected COS-1 cells occur at levels of intracellular expression comparable to those found in the nerve ending.
In control cultures, hGH release was indeed strictly dependent on protein synthesis (Fig. 5A). When synthesis was blocked by cycloheximide, release of hGH into the medium slowed dramatically by 1 h. In contrast, inhibition of protein synthesis had little effect on release of hGH when syntaxin 1A was present. Release of hGH into the medium during a 5.5-h incubation of syntaxin 1A-expressing cells was unchanged by the addition of cycloheximide (Fig. 5B). Similar results were seen with HEK 293 cells (data not shown). The continued release of hGH in the absence of continuing protein synthesis indicates that hGH is being released from stored hGH accumulated before the addition of cycloheximide.
If syntaxin 1A is generating a pool
of stored transport vesicles, then the half-life of hGH in
syntaxin-expressing cells should be significantly longer than its
half-life in control cells. We thus compared the half-lives of hGH in
cells transfected with or without syntaxin 1A (Fig. 5C). As expected, intracellular hGH was rapidly
depleted from COS-1 cells lacking syntaxin 1A, with a half-life of
90 min. After the first 30 min, (
)loss of hGH from
cells expressing syntaxin 1A was extremely slow, with a half-life of 18
h. This prolonged half-life is consistent with the observation of a
large intracellular pool of hGH in COS-1 cells expressing syntaxin 1A ( Fig. 3and Fig. 4).
Figure 6:
Effect of brefeldin A and reduced
temperature on release of stored hGH from COS-1 cells transfected with
syntaxin 1A. COS-1 cells were plated at a density of 10 cells/well on 12-well plates and transfected with plasmids
encoding hGH and syntaxin 1A at a ratio of 2:1. After 24 h, the medium
was replaced with fresh medium containing cytosine arabinoside, and the
cells were incubated for an additional 48 h. At the beginning of the
experiments, the medium was replaced by physiological salt solution
± 5 µg/ml brefeldin A (BFA) (A and B) or by physiological salt solution at 19 °C or 30 °C (C and D, a separate experiment). Aliquots of the
medium were taken at the indicated times, and the cells were harvested
after 3 h. Human GH was determined by luminescent assay. Human GH
levels (in ng/well) remaining in the cells after incubation were: A, -BFA, 0.98 ± 0.05; +BFA, 1.61 ±
0.09; B, -BFA, 11.45 ± 0.60; +BFA, 10.97
± 0.74; C, 19 °C, 0.73 ± 0.04; 30 °C,
0.77 ± 0.04; D, 19 °C, 15.17 ± 0.58; 30
°C, 14.74 ± 0.53. n = 4
wells/group.
This conclusion is supported by experiments in which the temperature is reduced to inhibit transport from the TGN(47) . In Fig. 6, C and D, COS cells transfected with or without syntaxin 1A were incubated at 19 or 30 °C for 3 h. Lowering the temperature to 19 °C strongly inhibited hGH release from cells without syntaxin 1A, while hGH release from cells expressing syntaxin 1A was inhibited by less than 20%.
We noted that botulinum C neurotoxin had no
effect on the constitutive release of hGH in the absence of syntaxin 1A (Fig. 7B). This suggests the possibility that a member
of the syntaxin family resistant to cleavage by the toxin may be
involved in the constitutive secretory pathway in COS-1 cells. Using
SDS-polyacrylamide gel electrophoresis followed by immunoblotting with
antibodies specific for syntaxins 2-4, we determined that both
syntaxin 2 and syntaxin 3 are expressed endogenously by COS-1 cells.
Again, membrane fractions were prepared from hGH-transfected COS-1
cells cultured with (lane 5) or without (lane 4)
toxin, and syntaxins 2 and 3 were detected on immunoblots. Neither
syntaxin 3 (Fig. 7A) nor syntaxin 2 (not shown) were
susceptible to cleavage by botulinum C
. Quantitation by
phosphorimager revealed that the toxin reduced the amount of
immunoreactive syntaxins 2 and 3 by 5-7% (n = 4
wells/group).
Figure 8: Effects of expression of VAMP-2 or n-Sec1 in COS-1 cells with or without syntaxin 1A. COS-1 cells were transfected with hGH, syntaxin 1A, and either FLAG-VAMP-2 (A) or n-Sec1 (B) plasmids as described for Fig. 3and Fig. 4. Expression of FLAG-VAMP-2 and n-Sec1 was verified by immunofluorescence microscopy (not shown). After 3 days, the amount of hGH in the cells and medium were determined by radioimmunoassay for each group. n = 4 wells/group.
While there is abundant evidence that syntaxin 1A is important for secretion(28, 30) , the specific functions it serves in the regulated secretory pathway are not well understood. In the present study, we have used a reconstitution approach in continuous cell lines lacking a prominent regulated secretory pathway to investigate whether syntaxin 1A expression modifies the constitutive secretory pathway and/or induces properties characteristic of the regulated secretory pathway.
Two manipulations designed to selectively
interfere with syntaxin 1A function, treatment with botulinum C neurotoxin or coexpression of n-Sec1, lend further support to the
specificity of the observed effects. Botulinum C
toxin
cleaves syntaxin 1A adjacent to the membrane anchor(36) ,
inhibiting neurotransmitter release from neurons (11) and
chromaffin cells.
Thus, the partial reversal of hGH
accumulation following botulinum C
neurotoxin treatment is
most likely due to a reduction in the amount of membrane-anchored
syntaxin 1A.
The neuronal protein n-Sec1 (munc-18, rbSec1, mSec1)
may regulate the availability of syntaxin 1A. n-Sec1 interacts with
syntaxin 1A in vitro with high affinity and prevents its
association with other components required for synaptic vesicle docking
and fusion. Based on this result, n-Sec1 has been proposed to act as a
negative regulator of syntaxin function, a proposal consistent with the
partial suppression of hGH accumulation that we observed. In the
present experiments, n-Sec1 may have sequestered some of the syntaxin
1A, giving a result qualitatively similar to that seen when levels of
syntaxin are reduced by botulinum C neurotoxin action.
How might syntaxin 1A be acting? Since the interactions of syntaxin isoforms with their binding partners, while selective, are not entirely specific, it seems likely that transiently expressed syntaxin 1A substitutes for an endogenous syntaxin of the constitutive pathway. If this is the case, one can envision two possibilities. The accumulation of secretory vesicles may result from the absence of other components of the regulated pathway not present in constitutively secreting cell lines. Alternatively, as a syntaxin isoform normally expressed in tissues with a prominent regulated pathway, syntaxin 1A may be directly involved in the storage of regulated secretory vesicles. Storage may represent a previously unrecognized function of syntaxin 1A in the regulated pathway. Both of these ideas are discussed below.
Given its many reported interactions in vitro(2, 3, 35, 42) , syntaxin 1A would be predicted to associate with a varied group of proteins in vivo. One might thus expect the function of the regulated pathway to depend on a precisely balanced ratio of these proteins. Lack of the appropriate partner(s) for syntaxin 1A might result in an incomplete or partially functional SNARE complex. Thus, the fact that COS-1 and HEK 293 cells are not specialized for regulated secretion may be partially responsible for the effects we observed. These cell lines may lack one or more of the proteins which specifically interact with syntaxin 1A in the neuron. Expressing such proteins might permit syntaxin 1A to act more efficiently or alter its function. However, coexpression of VAMP-2, the v-SNARE partner for syntaxin 1A, did not alter the ability of syntaxin 1A to store hGH (Fig. 8A).
Two pieces of evidence tend to argue against the possibility that storage is a consequence of the absence of a particular factor present only in cells specialized for regulated secretion. First, none of the other SNAREs or SNARE-related proteins we tested caused storage when overexpressed (VAMP-2 and n-Sec1, Fig. 8; synaptotagmin, not shown). Syntaxin 1A was unique in its ability to form a storage compartment. Moreover, a substantial fraction of the stored hGH is competent to undergo release within minutes when medium is removed and replaced (Fig. 6). While we do not understand the signaling mechanisms involved, the ability of vesicles to undergo rapid release from the stored compartment suggests that secretion is not being limited by lack of an essential factor.
These
results lead us to propose an alternative hypothesis: the ability of
syntaxin 1A to create a stored compartment in constitutively secreting
cell lines may reflect a role for syntaxin 1A in storage in the
regulated pathway. By virtue of its interactions with other proteins
(VAMP, SNAP-25, n-Sec1, synaptotagmin), syntaxin 1A may in fact be
uniquely positioned to play several roles in secretion. A storage
function for syntaxin 1A might thus exist in addition to previously
postulated roles (e.g. as a receptor for -SNAP). Such a
storage function may represent a specialized and fundamental difference
between t-SNAREs involved in regulated versus constitutive
secretion. Although the mechanism by which syntaxin 1A is acting cannot
be resolved at this stage, these experiments reveal for the first time
the potential for this protein to create a storage compartment in
secreting cells.
There are a number of functions of the regulated
secretory pathway that the expression of syntaxin 1A did not
reconstitute. Using several experimental approaches, we were unable to
demonstrate Ca-dependent secretion that occurs
because of expression of syntaxin 1A. Syntaxin 1A also did not confer
on the sorting mechanism of COS-1 cells the ability to distinguish
between regulated (hGH) and constitutive (VSV-G) secretory proteins.
Undoubtedly numerous other proteins (e.g. synaptotagmin,
Rab3a, rabphilin3a, p145, other SNAREs) are necessary to constitute a
complete, regulated secretory pathway and to confer
Ca
-dependent secretion. An extension of the present
approach of investigating changes in the secretory pathway in
undifferentiated cells caused by expression of proteins implicated in
the regulated pathway promises further insight into the function of
these proteins and eventual reconstitution of regulated secretion.