©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence That Syntaxin 1A Is Involved in Storage in the Secretory Pathway (*)

(Received for publication, July 13, 1995; and in revised form, February 16, 1996)

Mary A. Bittner (1)(§) Mark K. Bennett (2) Ronald W. Holz (1)

From the  (1)Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109 and the (2)Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1) 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.


INTRODUCTION

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) (^1)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 alpha- 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(1) neurotoxin(11, 36) , which inhibits glutamate release from synaptosomes (11) and exocytosis from chromaffin cells. (^2)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.


EXPERIMENTAL PROCEDURES

Plasmids

Human GH was expressed with pXGH5 which is under control of the mouse metallothionein I promoter(41) . Incubation with a heavy metal was not necessary to obtain adequate hGH expression. The vector encoding VSV-G, pSPalphaG, was a gift from Dr. John K. Rose, Yale University (New Haven, CT). Plasmids for FLAG-VAMP-2, syntaxin 1A (p35A), syntaxin (HA-syn 2), and the syntaxin variant which lacks the membrane attachment domain of syntaxin 1A (syn 1ADeltaM) have been described previously(28, 42) . A plasmid encoding n-Sec1 (43) from Dr. Jonathan Pevsner (Stanford University) was subcloned into a pCMV vector.

Transfection and Cell Culture

COS-1 and HEK 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 20 mM glutamine, 10% fetal bovine serum (heat-inactivated), penicillin (100 units/ml), streptomycin (100 µg/ml), and gentamicin (25 µg/ml), in 5% CO(2) at 37 °C. Cells were transfected 18-24 h after plating (COS-1 at 2 times 10^5/well; HEK 293 at 4 times 10^5/well) with a total of 6.4 µg of plasmid DNA/35-mm well using a Life Technologies, Inc. Calcium Phosphate Transfection System (Catalog No. 8306SA). Typically, 3.2 µg of the reporter plasmid (hGH or VSV-G) was transfected with 3.2 µg of either the plasmid(s) of interest or pCMVneo as a control(37) . In some experiments, only 0.8 or 1.6 µg/well of the syntaxin 1A plasmid was used, with 1.6 or 0.8 µg/well of pCMVneo. The instructions accompanying the kit were followed closely, with volumes reduced to correspond with the use of 35-mm rather than 100-mm-diameter dishes. In some experiments, the cells were plated in 12-well plates, and the volumes were again reduced proportionally. The procedure involves adding dropwise 0.16 ml of a calcium phosphate/DNA suspension into a 35-mm well containing 2 ml of DMEM (with serum and antibiotics) followed by gentle swirling of the dish. After a 5-h incubation at 37 °C in 5% CO(2), the solution was removed and the cells were incubated for 4 min at room temperature with 10% glycerol in DMEM with antibiotics but without fetal calf serum. Cells were rinsed with media and then incubated in the tissue culture incubator (5% CO(2)) in DMEM with cytosine arabinofuranoside (10 µM). In some experiments (noted in legends to figures), the glycerol shock step was omitted, and the DNA precipitate was left on the cells for 24 h before addition of fresh culture medium containing cytosine arabinofuranoside. Immunofluorescence microscopy and hGH assays were performed 2-3 days after the transfection.

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.

Indirect Immunofluorescence Microscopy

Immunofluorescence microscopy was performed on COS-1 or HEK 293 cells that had been fixed with 4% paraformaldehyde for 30 min, quenched with 50 mM NH(4)Cl for 30 min, and permeabilized with methanol (-20 °C) for 7 min. In some experiments, cells were incubated with the primary antibody to VSV-G at 4 °C for 2 h and rinsed before fixation and permeabilization (noted in legends to Fig. 1and Fig. 2). Typically, cultures were incubated with primary antibodies for 2 h, followed by 4 rinses before a 75-min incubation with secondary antibody. The following primary antibodies were used: hGH, rabbit anti-human pituitary GH polyclonal antibody (1:1000, National Hormone and Pituitary Program, NIDDKD) or mouse anti-hGH (Zymed, San Francisco, CA); syntaxin 1A and syn 1ADeltaM, monoclonal antibody to syntaxin 1A (44) (HPC-1, 1:1000); VSV-G, rabbit polyclonal antibody (gift from Dr. John K. Rose); HA-syntaxin 2, mouse anti-HA1 (monoclonal antibody 12CA5, 1:500, Berkeley Antibody Co); FLAG-VAMP-2, monoclonal anti-FLAGbulletm2 (IBI-Eastman Kodak). Affinity-purified antibody to the 20 C-terminal residues of n-Sec1 was a gift from Dr. Jonathan Pevsner(43) . Affinity-purified secondary antibodies labeled with fluorescein isothiocyanate or lissamine rhodamine were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Cells were observed with a fluorescence Nikon Diaphot microscope at 40 times (Fig. 1Fig. 2Fig. 3) magnification.


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 1ADeltaM). COS-1 cells were transiently transfected by calcium phosphate precipitation with plasmids for hGH, syntaxin 1A, syntaxin 2 (HA-syn 2), and syn 1ADeltaM 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 1ADeltaM (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.



Materials

Reagents were received from the following sources: I-labeled goat anti-rabbit secondary antibody (ICN); cell culture reagents, including trypsin-versene, gentamicin, penicillin/streptomycin, glutamine, and DMEM (BioWhittaker). All other reagents, including fetal bovine serum, were obtained from Sigma.


RESULTS

Expression of VSV-G in COS-1 Cells Cotransfected with Syntaxin 1A

COS-1 cells were transfected with a plasmid which encodes a form of VSV-G which is constitutively inserted into the plasma membrane. The protein can be detected on the surface of intact cells with anti-VSV-G antibody (Fig. 1A). If cells were permeabilized with methanol before incubating with the primary antibody, additional immunoreactive sites were detected intracellularly (Fig. 1B). In contrast, cells which expressed a brain isoform of syntaxin, syntaxin 1A (Fig. 2B), had little or no immunoreactive VSV-G on the external surface of the plasma membrane (Fig. 2A, same field as B). These cells were producing VSV-G, since when parallel cultures were permeabilized before the addition of anti-VSV-G antibody, peripheral staining for VSV-G was readily apparent (Fig. 2C). This suggests that syntaxin 1A substantially prevents the constitutive insertion of VSV-G into the plasma membrane. We saw no evidence that VSV-G was blocked at earlier points in the biosynthetic pathway (e.g. in the endoplasmic reticulum or the Golgi). Rather, as seen in Fig. 2C, VSV-G appears to localize to the inner surface of the plasma membrane.

Expression of hGH in COS-1 Cells Cotransfected with Syntaxin 1A

We next determined the fate of transiently expressed hGH. This protein normally undergoes regulated secretion, but is released constitutively when expressed in cells lacking a regulated pathway. When COS-1 cells were transfected with a plasmid for hGH alone, little immunoreactivity was detected in the cells. Occasional cells did show a bright dense staining located near the nucleus, in a pattern characteristic of the Golgi (Fig. 3A). This was confirmed directly by the colocalization of hGH with a marker for Golgi (not shown). In contrast, cells cotransfected with hGH and syntaxin 1A showed staining for both proteins throughout the cell (Fig. 3, B and C). Antibody to syntaxin 1A labeled the periphery of the cells, but was also present in the Golgi and on vesicles distributed throughout the cytoplasm. Of 133 cells which were strongly positive for syntaxin immunoreactivity, 96% were positive for hGH. The only hGH-containing cells were those with syntaxin 1A. If cells were cotransfected with a syntaxin variant which lacks the membrane attachment domain of syntaxin 1A (syn 1ADeltaM), little storage of hGH was seen (Fig. 3, D and E). Similarly, transfecting COS-1 cells with a plasmid for syntaxin 2, a widely expressed member of the syntaxin family, did not cause significant accumulation of hGH (Fig. 3, F and G).

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 1ADeltaM 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(1) 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(1) 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.

Effect of Cycloheximide on the Rate of Release of hGH from COS-1 Cells Cotransfected with Syntaxin 1A

If VSV-G and hGH are being secreted constitutively from COS-1 cells, then release of these proteins should be linked directly to their synthesis. We examined the rate of release and half-life of hGH in COS-1 cells with or without syntaxin 1A. COS-1 cells were first transfected and allowed to accumulate hGH for 24-48 h. Cultures were then treated with or without 5 µg/ml cycloheximide for 6 h, and aliquots of the culture medium were removed at intervals and assayed for hGH.

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, (^3)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).

Lack of Effect of Brefeldin A on Release of Stored hGH in Syntaxin 1A-expressing Cells

It is possible to prevent the exit of proteins from the TGN by exposing cells to brefeldin A(45, 46) . If syntaxin 1A is causing the accumulation of hGH in a post-Golgi storage compartment, then release of hGH from this compartment should be resistant to inhibition by brefeldin A. This was indeed the case. COS-1 cells transfected with or without syntaxin 1A were incubated for 3 h with 5 µg/ml brefeldin A, and hGH secretion was monitored (Fig. 6, A and B). As expected, brefeldin A caused a profound decrease in secretion of hGH from cells lacking syntaxin 1A (Fig. 6A). (^4)In contrast, secretion from cells expressing syntaxin 1A continued unabated (Fig. 6B). Similar results were seen with HEK 293 cells (not shown). The data confirm that a large fraction of the hGH in cells with syntaxin 1A has reached a compartment beyond the TGN.


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 10^5 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%.

Effects of Botulinum Type C(1) Neurotoxin on hGH Release in COS-1 Cells Transfected with Syntaxin 1A

If syntaxin 1A is acting specifically to cause storage of secreted proteins, then removing syntaxin should reverse its effects. Botulinum type C(1) neurotoxin is a Zn-dependent endopeptidase which specifically cleaves syntaxin 1A and inhibits secretion from synaptosomes (11) and from chromaffin cells.^2 We therefore examined the effect of this toxin on the ability of syntaxin 1A-expressing cells to store hGH. We first verified that the toxin was able to decrease the amount of syntaxin 1A after a 48-h exposure of intact cells to 50 nM botulinum C(1) toxin (Fig. 7A). Membrane fractions were prepared from transfected COS-1 cells that had been cultured with (lane 2) or without toxin (lane 1). Syntaxin was detected by immunoblotting. Quantitation by phosphorimager revealed that the toxin reduced the amount of immunoreactive syntaxin 1A by 56% (n = 4 wells/group). No immunoreactivity was detected in cells not transfected with syntaxin 1A plasmid (lane 3). Fig. 7B shows the amount of hGH released into the medium by these cultures during the 48-h incubation with botulinum C(1) toxin. As seen in previous experiments, syntaxin 1A greatly decreased the percentage of hGH released into the medium, from 99% to 30%. In the presence of the toxin, the percentage of hGH released was almost doubled. The toxin partially relieved the inhibition of secretion by syntaxin 1A by reducing its concentration in the cells.

We noted that botulinum C(1) 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(1). Quantitation by phosphorimager revealed that the toxin reduced the amount of immunoreactive syntaxins 2 and 3 by 5-7% (n = 4 wells/group).

Effects of Coexpression of VAMP-2 or n-Sec1 with Syntaxin 1A on hGH Release from Transfected COS-1 Cells

It seemed possible that inhibition of hGH release by syntaxin 1A might be connected with the absence of its neuronal v-SNARE partner, VAMP-2 (synaptobrevin-2). We thus examined whether overexpression of VAMP-2 altered the ability of syntaxin 1A to induce hGH accumulation. We used a plasmid which was tagged with the FLAG epitope (FLAG-VAMP-2), so that expression of the plasmid could be verified by immunofluorescence microscopy. Overexpression of VAMP-2 itself caused a small increase in hGH storage from 1% to 5% (Fig. 8A). COS-1 cells expressing both syntaxin 1A and FLAG-VAMP-2 stored 49% of hGH synthesized, compared with 47% in cells expressing syntaxin 1A alone. Thus, VAMP-2 did not alter the ability of syntaxin 1A to cause storage of hGH. In contrast, coexpression of n-Sec1, another neuronal protein thought to interact with syntaxin 1A, suppressed by 69% the ability of syntaxin 1A to store hGH (Fig. 8B).


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.




DISCUSSION

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.

Syntaxin 1A Creates a Pool of Stored Secretory Protein

We have demonstrated that expression of syntaxin 1A in COS-1 cells alters the disposition of two markers of the constitutive secretory pathway: VSV-G (a viral protein that is constitutively expressed on the surface of infected cells) and hGH (a protein that is released in a regulated fashion from pituitary cells). As expected, both VSV-G and hGH undergo exocytosis constitutively when expressed alone in COS-1 cells. When either of these markers are coexpressed with syntaxin 1A, their constitutive transport to the cell surface is blocked, resulting in an intracellular accumulation or storage. Storage occurs when cellular levels of syntaxin 1A are comparable to those in brain. The effect can be demonstrated by detection of protein localization by immunofluorescence microscopy and by direct biochemical measurements of hGH storage and release. In the case of hGH, the increased storage is reflected by an increase in intracellular half-life from 90 min in the absence of syntaxin 1A to 18 h in the presence of syntaxin 1A. Furthermore, in cells cotransfected with syntaxin 1A, release of hGH in the presence or absence of cycloheximide remains virtually identical over several hours, indicating that secretion from the intracellular storage pool continues in a constitutive-like manner in the absence of extracellular stimulus or new protein synthesis. Finally, constitutive-like release of hGH from the storage compartment is resistant to inhibition by either BFA treatment or reducing the temperature to 19 °C, indicating that the storage compartment represents a post-Golgi site. These results suggest that syntaxin 1A specifically confers to the cells the ability to accumulate and store a secretion-competent pool of constitutive secretory products.

The Effects of Syntaxin 1A Are Specific

Several lines of evidence demonstrate that the observed effect on the constitutive secretory pathway is specifically due to syntaxin 1A expression. First, expression of a form of syntaxin 1A lacking the hydrophobic C-terminal membrane anchor was without effect. This result suggests that syntaxin must be properly anchored to the membrane to generate the intracellular accumulation of constitutive secretory pathway markers. Another indication of specificity is the lack of effect of expression of syntaxin 2, a syntaxin isoform (63% identical with syntaxin 1A) whose widespread distribution suggests a role in constitutive secretion.

Two manipulations designed to selectively interfere with syntaxin 1A function, treatment with botulinum C(1) neurotoxin or coexpression of n-Sec1, lend further support to the specificity of the observed effects. Botulinum C(1) toxin cleaves syntaxin 1A adjacent to the membrane anchor(36) , inhibiting neurotransmitter release from neurons (11) and chromaffin cells.^2 Thus, the partial reversal of hGH accumulation following botulinum C(1) 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(1) neurotoxin action.

Syntaxin 1A May Have More Than One Function in the Regulated Secretory Pathway

In forming the storage compartment, it seems likely that syntaxin 1A directly alters a step late in the constitutive secretory pathway. Immunofluorescence observations suggest that the constitutive markers VSV-G and hGH accumulate in the cell periphery in a pattern distinct from the perinuclear localization of the Golgi complex. Furthermore, two manipulations which block exit from the TGN, brefeldin A and lowered temperature, inhibited hGH secretion in cells without syntaxin 1A, but had little or no effect on secretion from cells expressing syntaxin 1A. Thus, a large fraction of the hGH that accumulates in syntaxin 1A-expressing cells has passed beyond the early compartments of the secretory pathway and is present in a later compartment. This suggests that storage is the result of an accumulation of post-Golgi vesicles, perhaps constitutive transport vesicles.

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 alpha-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.


FOOTNOTES

*
This study was supported in part by National Science Foundation Grant IBN 9008685 (to M. A. B.) and United States Public Health Service Grant R01 DK27959 (to R. W. H.). 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 correspondence should be addressed: M 1301 MSRB III, Dept. of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632. Tel.: 313-747-2257; Fax: 313-763-4450; mbittner{at}umich.edu.

(^1)
The abbreviations used are: TGN, trans-Golgi network; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; HEK, human embryonic kidney; hGH, human growth hormone; NSF, N-ethylmaleimide-sensitive fusion protein; alpha-SNAP, soluble NSF attachment protein; SNAP-25, synaptosome-associated protein of 25 kDa; t-SNARE, target SNAP receptor; v-SNARE, vesicular SNAP receptor; VAMP-2, vesicle-associated membrane protein or synaptobrevin-2; VSV-G, vesicular stomatitis virus glycoprotein; BFA, brefeldin A.

(^2)
M. A. Bittner and R. W. Holz, unpublished observations.

(^3)
When culture medium was changed at the beginning of the incubation with drug, we observed a brief burst of hGH secretion from syntaxin 1A-expressing cells, apparently as a result of mechanical stimulation (see also Fig. 7). While the significance of this observation is not yet clear, it is consistent with the presence of a pool of stored hGH which is available for release.

(^4)
During a 3-h incubation, cells without BFA secreted 3.9 ng of hGH/well, compared with 0.6 ng/well for cells treated with BFA (Fig. 6A). One might have expected the difference (3.3 ng/well) to accumulate in the BFA-treated cells, yet the actual increase of 0.6 ng/well (-BFA, 0.98 ± 0.05; +BFA, 1.61 ± 0.09) was much less. Interference with vesicular trafficking over this prolonged period may have affected the expression of the protein.


ACKNOWLEDGEMENTS

We thank Dr. Jonathan Pevsner for the gift of n-Sec1 antiserum and plasmid and Dr. John K. Rose for VSV-G antiserum and plasmid.


REFERENCES

  1. Burgess, T. L., and Kelly, R. B. (1987) Annu. Rev. Cell Biol. 3, 243-294 [CrossRef]
  2. Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318-324 [CrossRef][Medline] [Order article via Infotrieve]
  3. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418 [Medline] [Order article via Infotrieve]
  4. Bennett, M. K., and Scheller, R. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2559-2563 [Abstract]
  5. Ferro-Novick, S., and Jahn, R. (1994) Nature 370, 191-193 [CrossRef][Medline] [Order article via Infotrieve]
  6. Pfeffer, S. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1987-1988 [Free Full Text]
  7. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B., and Montecucco, C. (1992) Nature 359, 832-835 [CrossRef][Medline] [Order article via Infotrieve]
  8. Link, E., Edelmann, L., Chou, J. H., Binz, T., Yamasaki, S., Eisel, U., Baumert, M., Sudhof, T. C., Niemann, H., and Jahn, R. (1992) Biochem. Biophys. Res. Commun. 189, 1017-1023 [Medline] [Order article via Infotrieve]
  9. McMahon, H. T., Ushkaryov, Y. A., Edelmann, L., Link, E., Binz, T., Niemann, H., Jahn, R., and Sudhof, T. C. (1993) Nature 364, 346-349 [CrossRef][Medline] [Order article via Infotrieve]
  10. Schiavo, G., Shone, C. C., Rossetto, O., Alexander, F. C. G., and Montecucco, C. (1993) J. Biol. Chem. 268, 11516-11519 [Abstract/Free Full Text]
  11. Blasi, J., Chapman, E. R., Yamasaki, S., Binz, T., Niemann, H., and Jahn, R. (1993) EMBO J. 12, 4821-4828 [Abstract]
  12. Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., De Camilli, P., Sudhof, T. C., Niemann, H., and Jahn, R. (1993) Nature 365, 160-163 [CrossRef][Medline] [Order article via Infotrieve]
  13. Schiavo, G., Rossetto, O., Catsicas, S., Polverino de Laureto, P., DasGupta, B. R., Benfenati, F., and Montecucco, C. (1993) J. Biol. Chem. 268, 23784-23787 [Abstract/Free Full Text]
  14. Binz, T., Blasi, J., Yamasaki, S., Baumeister, A., Link, E., Sudhof, T., Jahn, R., and Niemann, H. (1994) J. Biol. Chem. 269, 1617-1620 [Abstract/Free Full Text]
  15. Yamasaki, S., Baumeister, A., Binz, T., Blasi, J., Link, E., Cornille, F., Roques, B., Fykse, E. M., Sudhof, T. C., Jahn, R., and Niemann, H. (1994) J. Biol. Chem. 269, 12764-12772 [Abstract/Free Full Text]
  16. Bittner, M. A., DasGupta, B. R., and Holz, R. W. (1989) J. Biol. Chem. 264, 10354-10360 [Abstract/Free Full Text]
  17. Bittner, M. A., and Holz, R. W. (1988) J. Neurochem. 51, 451-456 [Medline] [Order article via Infotrieve]
  18. Ahnert-Hilger, G., Bader, M. F., Bhakdi, S., and Gratzl, M. (1989) J. Neurochem. 52, 1751-1758 [Medline] [Order article via Infotrieve]
  19. Ahnert-Hilger, G., and Weller, U. (1993) Neuroscience 53, 547-552 [Medline] [Order article via Infotrieve]
  20. Penner, R., Neher, E., and Dreyer, F. (1986) Nature 324, 76-78 [CrossRef][Medline] [Order article via Infotrieve]
  21. Gansel, M., Penner, R., and Dreyer, F. (1987) Pfluegers Arch. Eur. J. Physiol. 409, 533-539 [Medline] [Order article via Infotrieve]
  22. Dreyer, F., Rosenberg, F., Becker, C., Bigalke, H., and Penner, R. (1987) Naunyn-Schmiedebergs Arch. Pharmakol. 335, 1-7 [Medline] [Order article via Infotrieve]
  23. Poulain, B., Mochida, S., Weller, U., Hogy, B., Habermann, E., Wadsworth, J. D. F., Shone, C. C., Dolly, J. O., and Tauc, L. (1991) J. Biol. Chem. 266, 9580-9585 [Abstract/Free Full Text]
  24. Sanders, D., and Habermann, E. (1992) Naunyn-Schmiedebergs Arch. Pharmakol. 346, 358-361 [Medline] [Order article via Infotrieve]
  25. Simpson, L. L. (1989) in Botulinum Neurotoxin and Tetanus Toxin (Simpson, L. L., ed) pp. 153-178, Academic Press, Inc., San Diego, CA
  26. Simpson, L. L. (1988) J. Pharmacol. Exp. Ther. 245, 867-872 [Abstract]
  27. Halpern, J. L., Habig, W. H., Trenchard, H., and Russell, J. T. (1990) J. Neurochem. 55, 2072-2078 [Medline] [Order article via Infotrieve]
  28. Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, 863-873 [Medline] [Order article via Infotrieve]
  29. Walch-Solimena, C., Blasi, J., Edelmann, L., Chapman, E. R., Fischer von Mollard, G., and Jahn, R. (1995) J. Cell Biol. 128, 637-645 [Abstract]
  30. Schulze, K. L., Broadie, K., Perin, M. S., and Bellen, H. J. (1995) Cell 80, 311-320 [Medline] [Order article via Infotrieve]
  31. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259 [Medline] [Order article via Infotrieve]
  32. Leveque, C., El Far, O., Martin-Moutot, N., Sato, K., Kato, R., Takahashi, M., and Seagar, M. J. (1994) J. Biol. Chem. 269, 6306-6312 [Abstract/Free Full Text]
  33. Sheng, Z.-H., Rettig, J., Takahashi, M., and Catterall, W. A. (1994) Neuron 13, 1303-1313 [Medline] [Order article via Infotrieve]
  34. Garcia, E. P., Gatti, E., Butler, M., Burton, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2003-2007 [Abstract]
  35. Pevsner, J., Hsu, S. C., Braun, J. E. A., Calakos, N., Ting, A. E., Bennett, M. K., and Scheller, R. H. (1994) Neuron 13, 353-361 [Medline] [Order article via Infotrieve]
  36. Schiavo, G., Shone, C. C., Bennett, M. K., Scheller, R. H., and Montecucco, C. (1995) J. Biol. Chem. 270, 10566-10570 [Abstract/Free Full Text]
  37. Wick, P. W., Senter, R. A., Parsels, L. A., Uhler, M. D., and Holz, R. W. (1993) J. Biol. Chem. 268, 10983-10989 [Abstract/Free Full Text]
  38. Schweitzer, E. S., and Kelly, R. B. (1985) J. Cell Biol. 101, 667-676 [Abstract]
  39. Moore, H.-P., and Kelly, R. B. (1985) J. Cell Biol. 101, 1773-1781 [Abstract]
  40. Green, R., and Shields, D. (1984) J. Cell Biol. 99, 97-104 [Abstract/Free Full Text]
  41. Selden, R. F., Howie, K. B., Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986) Mol. Cell. Biol. 6, 3173-3179 [Medline] [Order article via Infotrieve]
  42. Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263, 1146-1149 [Medline] [Order article via Infotrieve]
  43. Pevsner, J., Hsu, S. C., and Scheller, R. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1445-1449 [Abstract]
  44. Inoue, A., Obata, K., and Akagawa, K. (1992) J. Biol. Chem. 267, 10613-10619 [Abstract/Free Full Text]
  45. Strous, G. J., van Kerkhof, P., van Meer, G., Rijnboutt, S., and Stoorvogel, W. (1993) J. Biol. Chem. 268, 2341-2347 [Abstract/Free Full Text]
  46. Huang, X. F., and Arvan, P. (1994) J. Biol. Chem. 269, 20838-20844 [Abstract/Free Full Text]
  47. Matlin, K. S., and Simons, K. (1983) Cell 34, 233-243 [Medline] [Order article via Infotrieve]
  48. Lee, S. A., and Holz, R. W. (1986) J. Biol. Chem. 261, 17089-17098 [Abstract/Free Full Text]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.