Regulated and Constitutive Secretion

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0 1992 by The American Society for Biochemistry and Molecular Biology. Inc Printed in S A .

Regulated and Constitutive Secretion

DIFFERENTIAL EFFECTS OF PROTEIN SYNTHESIS ARREST ON TRANSPORT OF

GLYCOSAMINOGLYCAN CHAINS TO THE TWO SECRETORY PATHWAYS*

(Received for publication, July 19, 1991)

Catherine Brion, Stephen G. Miller, and Hsiao-Ping H. Moore

From the Department of Cell and Molecular Biology, Division of Cell and Developmental Biology, University of California, ~~

Berkeley, California 94720

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et at. (Wieland,

F. T., Gleason, M. L., Serafini,

T.

A., and Rothman, J.

E.

(1987) Cell 50, _289-300)have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be gly- cosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, sug- gesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged

along with the hormone adrenocorticotropin (ACTH).

We found that cycloheximide treatment severely impairs the cell’s ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.

Proteins can be secreted from animal cells by either a constitutive or a regulated secretory pathway (Kelly, 1985; Moore, 1987). Peptide hormones and other proteins destined

* This work was supported by National Institutes of Health grant GM 35239, National Science Foundation Presidential Young Inves- tigator Award DCB 8451636, and American Cancer Society Grant

CD-497 (to H.-P. H. M.), by a 1967 Science and Engineering Schol- arship from the Natural Sciences and Engineering Research Council of Canada (to C. B.j, and by a Merck Postdoctoral Fellowship of the Helen Hay Whitney Foundation (to S. G. M.j. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- rnent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

for the regulated pathway are packaged into dense core secretory granules which are stored in the cytoplasm until their release is stimulated by secretagogues. In contrast, plasma membrane proteins, extracellular matrix proteins, and other proteins exported by the constitutive pathway are packaged into small, clear vesicles which fuse directly with the plasma membrane without prior storage.

The constitutive and regulated secretory pathways have been characterized extensively in the mouse anterior pituitary cell line, AtT-20. DNA transfection studies have demon- strated that fusion of a constitutively secreted protein to a regulated secretory protein redirects it to the regulated pathway (Moore and Kelly, 1986). Rosa et al. (1989) injected messenger RNA encoding antibodies into PC12 cells and showed that binding of antibodies to a granule component diverted them from the constitutive pathway to the regulated secretory pathway. Taken together, these experiments suggest that peptide hormones are actively sorted into dense core secretory granules whereas constitutively secreted proteins appear to be transported to the cell surface by a bulk-flow process (Moore et al., 1987). The intracellular sorting com- partment, in which segregation of constitutive and regulated secretory proteins occurs, has been identified both morpho- logically and biochemically. Orci et al. (1987) carried out immunoelectron microscopic studies to examine the distri- bution of insulin, a regulated secretory protein, and influenza hemagglutinin, a constitutive marker, in AtT-20 cells which expressed both proteins. The two proteins are intermixed in all compartments of the secretory pathway until they are segregated into constitutive secretory vesicles or regulated secretory granules at the trans-Golgi network. Similarly, Tooze and Huttner (1990) used an in vitro budding assay and found that the contents of constitutive and regulated secretory vesicles were segregated upon budding from the trans-Golgi. Thus, the trans-Golgi network appears to be used as a sorting station.

The exact mechanisms controlling secretory vesicle formation and sorting of molecules from the trans-Golgi are still poorly understood. One important question is whether or not new protein synthesis is required to effect sorting and transport to each of these exocytic pathways. In the case of the constitutive secretory pathway, protein synthesis does not appear to be required for efficient transport; introduction of

a tripeptide into cycloheximide-treated cells results in its glycosylation and rapid secretion in a constitutive manner (Wieland et aL, 1987). These results imply that incorporation of the tripeptide into constitutive secretory vesicles does not require the presence of other newly synthesized cargo proteins in the trans-Golgi. Moreover, components of the vesicle machinery must recycle continuously, and hence no new protein synthesis is required for vesicle production and/or consump-

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Protein Synthesis

and Constitutive and Regulated Secretion

tion. In this paper we address the question of whether sorting and transport through the regulated secretory pathway can also occur in the absence of newly synthesized protein.

MATERIALS AND METHODS

Cell Culture and Metabolic Labeling-AtT-20 cells were grown in

15% CO, in Dulbecco's modified Eagle's minimum essential medium (DMEM' H21) supplemented with 10% fetal calf serum. To study the effects of cycloheximide on packaging of glycosaminoglycan (GAG) chains, semiconfluent cells were pretreated in DMEM alone, DMEM with 1 mM xyloside, or DMEM containing 1 mM xyloside and 100 pg/ml cycloheximide for the lengths of time indicated in the figure legends. All subsequent incubations contained the specified concentrations of drugs. The cells were then starved in sulfate-free DMEM and labeled with [JsSS]sulfate (0.33 mCi/ml). To chase, the labeling medium was removed, and cells were rinsed and incubated with DMEM. To stimulate granule release in the final chase, 8-Br- CAMP was added to the chase medium at a concentration of 5 mM.

Analysis of GAG Chains by SDS-PAGE-Cell extracts were prepared either directly after sulfate labeling or at the end of 4.5 h of chase. The cells were rinsed and lifted from the plates by incubation for 10 min with calcium/magnesium-free phosphate-buffered saline containing 5 mM EDTA. Cells were pelleted at 200 X g for 10 min. The pellet was resuspended in 4 volumes of Laemmli sample buffer and boiled for approximately 1 h. Aliquots of the sample were analyzed on 10-18% exponential gradient SDS-polyacrylamide gels. The chase medium was precipitated in 80% acetone overnight at -20 "C. The precipitate was collected by centrifugation at 27,000 X g for 30

min and boiled in Laemmli sample buffer for 5 min. The samples were analyzed by 18% SDS-PAGE. Gels were impregnated with 1 M

salicylate, dried, and exposed to Kodak X-Omat AR film at -80 "C. Quontitation of GAG Chains by CPC As~ay-[~~S]SO,-Iabeled GAG chains were quantitated by a precipitation/filtration assay as de- scribed (Miller and Moore, 1991). The medium was removed, and each well of the 24-well plate was rinsed with 250 pl of phosphate- buffered saline and combined with the chase medium. The samples were then centrifuged for 2 min in an Eppendorf microcentrifuge to remove any cells that may have detached during the chase, and the supernatant was transferred to fresh tubes. Cells were extracted with 100 pl of 50 mM Tris (pH 8.0), 150 mM NaCI, 2 mM M&12,1% Triton X-100 for 5 min at 37 "C, and the wells were rinsed with 0.4 ml of phosphate-buffered saline. The detergent extracts were combined with any cells pelleted from the medium samples. Medium samples or cell extracts (0.5-ml total volume) were then proteolytically di- gested by the addition of 100 pl of 6 pg/ml pronase E and incubation for 4-16 h at 37 "C. 10 pl of 10 mg/ml chondroiton sulfate was added to each sample as a carrier, and sulfated GAG chains were then precipitated by the addition of 150 pl of 10% (w/v) cetylpyridinium chloride (CPC, 2% final). After incubation at 37 "C for an additional 60 min the precipitates were collected by rapid vacuum filtration using Metricel GN-6 filters (2.4 mm, 0.45 pm) followed by four 5-ml washes with 1% CPC, 25 mM Na2S04. The filters were dried and counted in a scintillation counter. The assay was unaffected by the presence of excess free [3sS]S04 in the range used in the assay (not

shown).

Metabolic Labeling with [35S]Cysteine, Radioimmunoassay (RIA), and Immunoprecipitation-AtT-20(InsGB) cells stably transfected with rat insulin DNA (Powell et al., 1988) were labeled for 16 h with

['''SS]cysteine in cysteine-free medium supplemented with 1/20 volume DMEM and 2% fetal calf serum. After labeling, the cells were rinsed and treated with 100 pg/ml cycloheximide for the remainder of the experiment. Cells were first chased in DMEM for two 2.5-h periods, and media from these chases were discarded. During the ensuing 2 h, 5 mM 8-Br-CAMP was added to induce secretion from storage granules. The chase medium was collected, lyophilized in a Speed-Vac, and redissolved in 1 ml of NDET (1% Nonidet P-40,0.4% deoxycho- late, 66 mM EDTA, 10 mM Tris, pH 7.4, and 0.3% SDS) buffer. An RIA for ACTH was performed on %O of each sample as described

previously (Moore et al., 1983).

The abbreviations used are: DMEM, Dulbecco's modified Eagle's minimum essential medium; GAG, glycosaminoglycan; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CPC, cetylpyridinium chloride; RIA, radioimmunoassay; ACTH, adrenocorticotropin; LDL, low density lipoprotein; 8-Br-cAMP, 8-bromo- cyclic AMP.

Immunoprecipitation of [35S]cysteine-labeled insulin from 9 / ~ ~ of

each sample was carried out as described previously (Moore and Kelly, 1986).

RESULTS

GAG Chains Are Synthesized in Cycloheximide-treated Cells To determine whether transport through the regulated secretory pathway can occur in the absence of new protein synthesis, we needed to monitor transport of a granule marker which was not a protein. The AtT-20 cells synthesize a sulfated proteoglycan and sort it into the dense core secretory granules along with the peptide hormone ACTH (Moore et al., 1983; Burgess and Kelly, 1984). Moreover, when cells are treated with the drug 4-methyl umbelliferyl-p-D-xyloside, an acceptor for GAG chains, synthesis of free GAG chains can be induced; some of these GAG chains also enter the regulated secretory granules and are secreted along with ACTH upon stimulation (Matsuuchi and Kelly, 1991). The majority of GAG chains, however, are secreted constitutively. Thus, they serve as convenient markers for both pathways in the absence of protein synthesis. GAG chains are sulfated and can easily be detected when cells are labeled with [35S]sulfate. The basic experimental design involves labeling xyloside-treated cells with [35S]sulfate and following the secretion of labeled GAG chains in the presence of cycloheximide.

We first verified that synthesis of GAG chains is unaffected by the protein synthesis inhibitor cycloheximide (Fig. 1). AtT- 20 cells pretreated for 2 h with 1 mM p-D-xyloside, or 1 mM xyloside plus 100 pg/ml cycloheximide, were labeled with [35S]

sulfate, and cell extracts were analyzed by SDS-PAGE. We found that treatment of AtT-20 cells with 100 pg/ml cycloheximide for 30 min inhibits 97% of their protein synthesis, as measured by trichloroacetic acid-precipitable radioactivity (data not shown). The cells were pretreated with cycloheximide for 2 h to allow time for protein turnover and to ensure that no newly synthesized peptide hormone would still be

CYCLOHEXIMIDE XYLOSIDE _.~_- I 15 kD 4 STAIRCASE

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66 Kd

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4 5

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36

-

2 9

-

2 4

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20.1

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14;2 1 2 3

FIG. 1. The synthesis of GAG chains is not affected by cy- cloheximide. Three identical 10-cm dishes of AtT-20 cells (approx-

imately 3 X lo6 cells/dish) were treated for 2 h in DMEM alone ( l a n e

I ) , DMEM with 1 mM xyloside ( l a n e 2), or DMEM with 1 mM xyloside and 100 pg/ml cycloheximide ( l a n e 3). All subsequent incubations contained the specified concentrations of drugs. The cells were then starved in sulfate-free medium for 30 min and labeled for

1 h with 0.33 mCi/ml [3sSs]sulfate. Cell extracts were prepared im- mediately after labeling, and %5 of each sample was analyzed by SDS-

PAGE (see "Materials and Methods"). The autoradiogram was exposed for 3 days at -80 "C. The sulfated GAG chain staircase (cen- tered around 12 kDa) is formed in xyloside-treated cells (lane 2) even in the presence of cycloheximide ( l a n e 3). Thus, 8-D-xyloside induces the synthesis of GAG chains in the presence or absence of new protein synthesis.

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present in the Golgi at the time of sulfate labeling. (The transit time for peptide hormones from endoplasmic reticulum t o mature granules is 60-90 min (Gumbiner and Kelly, 1981).) As shown by Burgess and Kelly (1984), treatment with

p-D-

xyloside induces the synthesis of a heterogeneous population of sulfated xyloside-GAG chains, which migrate as the low molecular weight "staircase" centering around 12 kDa (Fig. '1,

lane 2). Untreated control cells do not contain detectable levels of the free GAG chains (lane 1 ) but synthesize a variety of sulfated secretory proteins including a major 15-kDa sulfated protein which corresponds to an N-terminal peptide derived from proopiomelanocortin (see lane 1 ). Cyclohexi- mide effectively blocks the synthesis of sulfated proteins but does not affect the production of xyloside-GAG chains (lane

3). Thus, treatment of cells with cycloheximide and xyloside allows us to deplete the Golgi of any transported secretory proteins, as well as other proteins that turn over rapidly. In the meantime it allows us to load the Golgi with a non-protein marker.

Constitutive Secretion of Golgi Chains Does Not Require

Newly Synthesized Proteins

Inhibition of protein synthesis for several hours has no effect on the constitutive secretion of Golgi chains. AtT-20 cells pretreated with cycloheximide for 2 h were labeled with

["'S]sulfate for 2 h in the continuous presence of cycloheximide. The efficiency of constitutive secretion was then assayed by chasing the cells in unlabeled medium containing cycloheximide for 90 min; we have shown previously that constitutive secretion occurs with rapid kinetics and that most of the radioactivity incorporated during a pulse label is secreted within this period (Moore et al., 1983). Fig. 2A shows

that constitutive secretion of the GAG chain staircase occurs efficiently whether cycloheximide is present (Fig. 2 A , lunes 5 and 6 ) , or absent (Fig. 2 4 , lanes 3 and 4 ) . Notice that in control and xyloside-treated cells, sulfated proopiomelanocortin and its processed fragments are also constitutively secreted (Fig. 2 A , lanes I and 2, 3 and 4, bands between 36 and 14 kDa). These proteins are not synthesized or secreted in cells treated with cycloheximide (Fig. 2 A , lanes 5 and 6 ) . Even in their absence, GAG chains are still secreted efficiently through the constitutive secretory pathway. These findings are also confirmed by careful quantitation of the amounts of GAG chains in the cell extracts and the media using a precipitation assay (see below). Thus, traffic through the constitutive pathway continues in the absence of cargo proteins, and the machinery for constitutive secretion remains fully func- tional even during prolonged inhibition of protein synthesis. This is consistent with the observation of Wieland et al. (1987), who showed that constitutive secretion of a tripeptide fluid phase tracer is unaffected by cycloheximide treatment.

Transport of GAG Chains through the Regulated Secretory

Pathway Is Inhibited in the Absence of Protein Synthesis

Inhibition of Stimulated Release-In contrast to constitu-

tive secretion, inhibiting protein synthesis impairs regulated secretion. T o analyze regulated secretion of GAG chains, cells were treated with xyloside and cycloheximide and labeled as in Fig. 2 A . All subsequent incubations were in the continuous presence of cycloheximide. Cells were first chased for 3 h to deplete GAG chains in the constitutive secretory pathway. Cells were then treated with media containing the secretagogue 8-Br-CAMP to induce secretion from the regulated secretory pathway (Gumbiner and Kelly, 1982). Fig. 2B shows

A

_B

CYCLOHEXIMIDE CYCLOHEXIMIDE XYLOSIDE XYLOSIDE 8-Br-CAMP "

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66 kD . 66 Kd

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4 5 . 4 5

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3 6

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3 6

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2 9

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2 9

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2 4

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2 4

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20.1 _. 2 0 . 1 STAIRCASE

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14.2 15 kD r STAIRCASE

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14.2 L 1 2 3 3 5 6 ₁ ₂ 3 4 5 6

FIG. 2. Cycloheximide treatment inhibits release of GAG chains by the regulated secretory pathway but not the constitutive pathway. Panel A , constitutive secretion. Panel B, regulated secretion. Six identical

10-cm dishes of semiconfluent (approximately 3 X loG cells/dish) AtT-20 cells were pretreated for 2 h in DMEM, DMEM plus 1 mM xyloside, or DMEM plus 1 mM xyloside and 100 pg/ml cycloheximide. All subsequent incubations contained the specified concentrations of drugs. The cells were starved in sulfate-free media for 30 min and labeled for 2 h with 0.33 mCi/ml ["S]sulfate. Panel A , [35S]sulfate-labeled materials secreted via the constitutive pathway were collected during a 90-min chase after metabolic labeling. The secreted materials were acetone precipitated, and one-fourth of each sample was analyzed by SDS-PAGE. Lanes 1 and 2, secretion from untreated control cells

Lanes 3 and 4, secretion from xyloside-treated cells. Lanes 5 and 6, secretion from cells treated with xyloside and cycloheximide. Cells treated with xyloside alone or xyloside plus cycloheximide efficiently export the GAG chain staircase by the constitutive pathway. Panel B, after constitutive secretion had been chased out for 180 min, cells were treated with the secretagogue 8-Br-CAMP for 90 min to induce regulated secretion. The secreted materials were acetone precipitated, and one fourth of each sample was analyzed by SDS-PAGE. Lanes 1 and 2, secretion from untreated control cells. Lanes 3 and 4, secretion from cells treated with xyloside alone. Lanes 5 and 6,

secretion from cells treated with xyloside and cycloheximide. Cells shown in lanes 2, 4, and 6 were stimulated with 8-Br-CAMP. All autoradiograms were exposed for 40 h.

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1480

Protein Synthesis and Constitutive

and

Regulated

Secretion that cells that had not been treated with cycloheximide re-

leased the GAG chain staircase upon stimulation with the secretagogue 8-Br-CAMP (Fig. 2B, lanes 3 and 4 ) . By com- parison, stimulation induced very little secretion of sulfated staircase from cells that had been treated with cycloheximide (Fig. 2B, lanes 5 and 6). Control cells that have not been treated or have been treated with xyloside alone stored and released a 15-kDa sulfated N-terminal fragment of proopiomelanocortin by the regulated pathway (Fig. 2B, lanes 1 and 2,3 and 4 ) . Cycloheximide treatment abolished the synthesis and regulated secretion of this protein (Fig. 2B, lanes 5 and 6) as expected.

Reduction of Intracellular Storage-The inability of cycloheximide-treated cells to secrete GAG chains in response to stimulation could be explained by one of two possible models. First, biogenesis of storage granules and assembly of granule contents may require a continuous supply of newly synthesized proteins, such that in the absence of protein synthesis GAG chains are not stored. Alternatively, cycloheximide may have no effect on granule assembly but instead may exert its effect on stimulus release coupling or fusion; that is, storage granules do form, and GAG chains are properly stored, but cycloheximide perturbs the signaling pathway that transduces external stimuli to activate exocytosis. T o distinguish between these possibilities, we first asked whether cycloheximide- treated cells could store newly synthesized GAG chains properly. We found that cells treated with cycloheximide are impaired in their ability to store the sulfated staircase. The autoradiogram of cell extract and media samples from experiments shown in Fig. 2 was quantitated by scanning. In control untreated cells, the percentage of total GAG chains that were secreted into the media were 64, 75, and 77% at the end of 1.5, 3.0-, and 4.5-h chase, respectively. In contrast, cells treated with cycloheximide secreted 78, 92, and 94% of the GAG chains a t 1.5-, 3.0-, and 4.5-h chase, respectively; that is, untreated cells stored 23% of the total GAG chains synthesized after 4.5 h of chase whereas cycloheximide-treated cells stored only 6%. Thus, the amount of GAG chains stored after 4.5 h of chase is approximately 4-fold less in cycloheximide-treated cells compared with control cells. These results suggest that cycloheximide treatment inhibits proper storage of GAG chains within the cell.

Preservation of Stimulus-Release Coupling-To eliminate directly the possibility that cycloheximide interferes with stimulus release coupling, we tested whether cells treated with cycloheximide for 5 h could still release granules that had been formed prior to the cycloheximide treatment. A subclone of AtT-20, InsGB, was used. These cells are stably transfected with rat insulin DNA and package insulin as well as ACTH in their granules (Orci et al., 1987). Thus, stimulation results in the release of both ACTH and insulin into the media. Two assays were used to detect release of granules formed prior to cycloheximide treatment. First, the total amounts of immu- noreactive ACTH in the media were determined by RIA. Since storage granules in AtT-20 cells have a long life time in the cytoplasm (half-time of about 7-10 h; Moore and Kelly, 1985), most of the ACTH in the media detected by RIA is caused by secretion from preformed granules. Fig. 3A shows that even after 5 h of cycloheximide treatment, 8-Br-CAMP can still stimulate the release of ACTH; the rate of secretion from stimulated cells was 10-fold higher than from unstimulated cells. Note that the absolute amount of ACTH secreted from cycloheximide-treated cells was lower than in control cells; this is most likely because of the depletion of newly synthesized precursor ACTH in the constitutive pathway leading to

a decrease in the basal release of precursor molecules and also

A CYCLOHEXIMIDE 8-Br-CAMP 1 E (0 1 2 3 4

FIG. 3. Stimulated release from preformed granules can occur during cycloheximide treatment. Four identical wells of AtT-

20 (Ins6B) cells, stably transfected with a rat insulin cDNA, were grown in a six-well dish to semiconfluence (approximately 5 X lo"

cells/well). The cells were incubated for 16 h with [""Slcysteine to label insulin that is stored in secretory granules. To test if cycloheximide affects secretion from preformed granules the labeled cells were first treated for 5 h in DMEM containing 100 pg/ml cycloheximide and then stimulated in the presence of cycloheximide for 2.5 h in DMEM containing 5 mM 8-Br-CAMP. The chase medium during the stimulation period was collected, and the amount of total ACTH in

1/40 of each sample was determined by an ACTH radioimmunoassay

(panel A ) . The amount of labeled insulin secreted into the medium was determined by immunoprecipitating 9/10 of each sample with anti-insulin antibodies (panel B ) . Panel A , secretion of total immu-

noreactive ACTH detected by a radioimmunoassay. Lanes 1 and 2, secretion from untreated control cells. Lanes 3 and 4, secretion from cells that had been treated with cycloheximide for 7.5 h. Lanes 2 and

4 are from cells stimulated with 8-Br-CAMP. Panel B, secretion of

insulin that had been prelabeled with ["S]cysteine prior to cycloheximide treatment. Lanes I and 2, secretion from untreated control cells. Lanes 3 and 4, secretion from cells after treatment with cycloheximide for 7.5 h. Lanes 2 and 4 are from cells stimulated with 8-

Br-CAMP. The autoradiogram was exposed for 2 days.

because of a diminished storage pool size of mature ACTH in the cell which is not replenished by the continuous production of new ACTH. A second and more precise method to measure release from preformed granules was to radiolabel the contents of granules before cycloheximide treatment. The stimulated release of granules was then assayed by immunoprecipitation of ["S]cysteine-labeled insulin. As clearly demon- strated in Fig. 3B, the stimulated release of insulin from preformed granules could still occur even after 5-7 h of treatment with cycloheximide. Thus, the stimulus release coupling remains intact during cycloheximide treatment.

Quantitation of GAG C h i n Storage and Secretion Using CPC Assays-Since quantitation of GAG chains on PAGE gels is not very accurate, we sought to confirm these results using a quantitative precipitation assay for GAG chains. GAG chains are negatively charged and can be precipitated with CPC (Miller and Moore, 1991). To ensure quantitative precipitation, chondroitin sulfate was added to both medium and extract samples as a carrier (see "Materials and Methods"). Under the conditions used, the assay is linear over at least a 70-fold concentration range (data not shown). Using this assay, we quantitated the effect of cycloheximide treatment on the transport and secretion of labeled GAG chains. We performed two sets of experiments. In one we determined the effects of pretreatment with cycloheximide on subsequent

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transport and storage of newly synthesized GAG chains (pretreatment experiment). For a control, we performed a parallel experiment in which the cells were treated with cycloheximide for the same period of time except that the drug was added to the cells only after labeled GAG chains had already accumu- lated in regulated secretory granules (post-treatment experiment). The results for the pretreatment experiments are summarized in Table I, and those for the post-treatment experiments are tabulated in Table 11; both are from three or four independent determinations. Pretreatment with cycloheximide decreased the amount of label incorporated into GAG chains; therefore, we have presented the data in both raw counts and as percentage of total. Control cells synthesized 12,500 cpm of GAG chains during a 30-min pulse labeling. 78% of these counts were released constitutively during the first 1.5-h chase (the sum of three 0.5-h chases is shown as chases 1

+

2

+

3); as during the next hour, 8-Br-CAMP stimulated the rate of secretion from 3.7 to 9.5% (chase 4) (Table I). In contrast, cells that have been pretreated with cycloheximide synthesized 5,900 cpm of GAG chains during labeling; 87% of these were released constitutively within a 1.5-h chase (the sum of chases 1, 2, and 3); 8-Br-CAMP produced only a slight increase in the rate of secretion from 4.8 to 6.3% (chase 4) (Table I). In summary, pretreatment of cells with cycloheximide decreased the relative amount of GAG chains stored in the cells by 2-3-fold (18.7% uersus 8.0% after a 2.5-h chase); it also reduced stimulated release by approximately 4-fold (increment of 5.8% uersus 1.5% upon stimulation). These results further confirmed the basic con- clusion presented in Fig. 2 and the quantitation of GAG chains in cell extracts by scanning of autoradiograms.

The effects of cycloheximide are specific to newly synthesized GAG chains but not prestored GAG chains. Treating cells with cycloheximide after labeled GAG chains had been packaged in storage granules had little effects on their subsequent release by the regulated pathway (Table 11). First, the total number of counts recovered in treated and untreated cells were within 10% of each other. Thus, GAG chains were stable in cycloheximide-treated cells. This argues against the alternative explanation that cycloheximide treatment caused degradation of secretory granules by crinophagy, leading to an apparent decrease in storage of newly synthesized GAG chains. Second, 8-Br-CAMP increased secretion from 3.2 to 22.4% in cycloheximide-treated cells, compared with 4.0 to 27.5% in untreated cells. These results confirmed the conclu- sion from Fig. 3 by analyses of ACTH and insulin, i.e. stimulus release coupling was not significantly altered by cycloheximide treatment. Notice that the fold of stimulation by secretagogues is much higher in the post-treatment experiments ("-fold) than in the pretreatment experiments (2-3-fold); this is because the labeling and chase conditions (16-h label and 4.5-h chase) in the post-treatment experiments favor the ratio of regulated to constitutive secretion compared with the pretreatment experiments (0.5-h label and 1.5-h chase). Taken together, these data suggest that protein synthesis is not required for the final stage of regulated secretion. However, proper transport of newly synthesized GAG chains to the regulated secretory pathway requires sustained protein synthesis.

DISCUSSION

In this paper we showed that cessation of protein synthesis arrests transport of GAG chains to the regulated secretory pathway but has no effect on their trafficking through the constitutive secretory pathway. The differences in the sensi- tivity to protein synthesis inhibitor most likely reflect differ-

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Protein Synthesis and Constitutive and Regulated Secretion

m m o

C O O N

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ent mechanisms for sorting and/or maintenance of these two secretory pathways. For the constitutive secretory pathway, our evidence with GAG chains corroborates those of Wieland et al. (1987) using a tripeptide; both GAG chains and the tripeptide are secreted by this pathway efficiently in the absence of new protein synthesis. This suggests that the formation of constitutive vesicles from the trans-Golgi is probably not triggered by newly synthesized cargo proteins. This is unlike the budding of virus, in which the binding of viral nucleocapsids to membrane spike proteins is required to trigger the budding of virus particles from the cell surface (for a review, see Simons and Fuller, 1987); without the viral nucleocapsid to trigger budding, no empty virus particles are made. Instead, the situation probably resembles endocytosis of low density lipoprotein (LDL) receptors from the cell surface (for a review, see Brown et al., 1983). LDL receptors cluster into clathrin-coated pits at the cell surface where the budding of endocytotic vesicles occurs. The formation of endocytotic vesicles containing the LDL receptor occurs re- gardless of whether LDL cargo is bound to the LDL receptor or not. Thus, in this case cargo is not required to trigger budding.

In contrast to constitutive secretion, storage of newly synthesized GAG chains fail to occur in the absence of new protein synthesis. Several possibilities exist. One explanation is that formation of storage granules is a triggered process; binding of newly synthesized hormones or other granule components to a region of trans-Golgi membrane may be necessary to initiate granule budding. The situation would be analogous to viral budding, in which binding of nucleocapsids to membrane spike proteins is required to trigger the budding process. According to this model, the cell would only make secretory granules when there was granule content present to be packaged. In the absence of cargos, granules would not form, and therefore no storage of GAG chains would be observed. In this view, the regulated secretory pathway would be regulated at the level of granule formation as well as at the level of granule release from the cell.

A second possibility is that empty granules would continue to form in the absence of new protein synthesis, but packaging of GAG chains is impaired when no granule contents are around. For example, this would occur if GAG chains entered the granules by binding or “hitchhiking” on hormones or other granule contents. We have carried out titration experiments to determine the amount of GAG chains transported to storage granules as a function of total GAG chain synthesized. Within the range permitted by varying xyloside concentrations, the fraction of GAG chains stored in the granules is invariant with respect to total amounts synthesized. Although the lack of saturation argues against transport by binding to granular components, we cannot exclude the possibility that saturation may occur at concentrations higher than what we could reach by maximal doses of xyloside.

A third possibility that is also consistent with our results is that proper formation and/or maturation of secretory granules requires a short-lived protein factor(s). Upon cycloheximide treatment, this factor(s) is rapidly depleted, resulting in impaired storage and secretion from the regulated pathway. A possible scenario is that immature granules continue to bud, but they fail to “mature” properly in the absence of newly synthesized protein such that their contents are secreted constitutively. One could envisage that maturation of granules requires a short lived protein factor. This factor would nor- mally prevent granules from fusing with the plasma membrane prior to stimulation. Cycloheximide treatment could deplete such a blocking protein, allowing immature granules

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to fuse with the plasma membrane without a signal for release. At the present time we cannot distinguish among these possibilities because markers for granule membranes are not yet available to determine the stages of granule formation in the absence of hormone contents. Future work will be necessary t o determine the exact role of protein synthesis on regulated secretion.

If the budding of storage granules is indeed triggered by hormones, then the mechanisms underlying the formation of regulated granules would be fundamentally different from those governing the budding of constitutive vesicles since the latter does not require cargo proteins. Perhaps the budding processes for constitutive and regulated vesicles are driven by opposite forces: constitutive vesicles driven from the cyto- plasmic side by the non-clathrin-protein coat (Orci et al., 1986), and regulated vesicles from the lumenal side by aggre- gated content proteins.

Acknowledgments-We thank Dr. Eve I. B. Briles for helpful discussions, members of the Moore lab for critical reading of the manuscript, and D. Quinn for his assistance in graphic work.

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