Vol. 267, No. 3, Iasue of January 25,pp. 1477-1483,1992 Printed in U.S A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology. Inc

Regulated and Constitutive Secretion DIFFERENTIALEFFECTS OF PROTEIN SYNTHESIS ARREST ON TRANSPORT OF GLYCOSAMINOGLYCAN CHAINSTOTHETWOSECRETORYPATHWAYS* (Received for publication, July 19, 1991)

Catherine Brion, StephenG. Miller, and Hsiao-Ping H. Moore From the Department of Cell and Molecular Biology, Divisionof Cell and Developmental Biology, Universityof California, ~~

Berkeley, California 94720

Many neural and endocrine cells possess two path- for theregulated pathway are packaged into densecore secreways of secretion: a regulated pathway and a consti- tory granules which are stored in the cytoplasm until their tutive pathway. Peptide hormonesare stored in gran- release is stimulated by secretagogues. In contrast, plasma ules which undergo regulated release whereas other membrane proteins, extracellular matrix proteins, and other surface-bound proteins are externalized constitutively proteins exported by the constitutive pathway are packaged via a distinct set of vesicles. An important issue is into small, clear vesicles which fuse directly with the plasma whether proper function of these pathways requires membrane without priorstorage. continuous protein synthesis. Wielandet at. (Wieland, The constitutive and regulatedsecretory pathways have F. T., Gleason, M. L., Serafini, T.A., and Rothman, J. been characterizedextensively in themouse anterior pituitary E.(1987) Cell 50, 289-300)have shown thata tripep- cellline,AtT-20. DNAtransfectionstudies have demontide containing the sequence Asn-Tyr-Thr can glybe strated that fusion of a constitutively secreted protein to a cosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 regulated secretory protein redirects it to theregulated pathet al. (1989) injected cells, presumably via the constitutive secretory path- way (Mooreand Kelly,1986).Rosa cells and way. Secretion is not affected by cycloheximide, sug- messenger RNA encoding antibodiesintoPC12 showed that binding of antibodies to a granule component gesting that operationof this pathwaydoes notrequire the components supplied by new protein synthesis.In this diverted them from the constitutive pathway to regulated suggest report we determined the effectsof protein synthesis secretory pathway. Taken together, these experiments actively sorted into dense core inhibitor on membrane traffic to the regulated secre- that peptide hormones are tory pathway in themouse pituitary AtT-20 cells. We secretory granules whereas constitutively secreted proteins examined transportof glycosaminoglycan chains since appear to be transported to the cell surface by a bulk-flow previous studies have shown that these chains enter process (Moore et al., 1987). The intracellular sorting comthe regulated secretory pathways and are packaged partment, in which segregation of constitutive and regulated along with the hormone adrenocorticotropin (ACTH). secretory proteins occurs, has been identified both morphoWe found that cycloheximide treatment severely im- logically and biochemically.Orci et al. (1987) carriedout pairs the cell’s ability to store and secrete glycosami- immunoelectron microscopic studies to examine the distrinoglycan chains by the regulated secretory pathway. bution of insulin, a regulated secretory protein, andinfluenza In marked contrast, constitutive secretionof glycosa- hemagglutinin, a constitutive marker, in AtT-20 cells which minoglycan chains remains unhindered in the absence expressed both proteins. The two proteins are intermixed in of protein synthesis. The differential requirements for protein synthesis indicate differences in the mecha- all compartments of the secretory pathway until they are nismsforsortingand/ortransport of molecules segregated intoconstitutive secretory vesicles or regulated secretorygranules atthetrans-Golgi network.Similarly, through the constitutive and the regulated secretory Tooze and Huttner(1990) used an in vitro budding assay and pathways. We discuss the possiblemechanismsby of constitutive andregulated secretory which protein synthesis may influence trafficking of found that the contents vesicles were segregated upon budding from the trans-Golgi. glycosaminoglycan chains to the regulated secretory Thus, the trans-Golgi network appearsbetoused as a sorting pathway. station. The exact mechanisms controlling secretory vesicle formation and sortingof molecules from the trans-Golgi are still Proteinscan be secreted from animal cells by either a poorly understood. One important question is whether or not constitutive or a regulated secretory pathway (Kelly, 1985; new protein synthesis isrequired to effect sorting and transMoore, 1987). Peptide hormones and other proteins destined port to each of these exocytic pathways. In the case of the does not * This work was supported by National Institutes of Health grant constitutive secretory pathway, protein synthesis GM 35239, National Science Foundation Presidential Young Inves- appear to be required for efficient transport; introduction of cells resultsin its tigator Award DCB 8451636, and American Cancer Society Grant a tripeptideintocycloheximide-treated CD-497 (to H.-P. H. M.), by a 1967 Science and Engineering Schol- glycosylation and rapid secretion in a constitutive manner arship from the Natural Sciences and Engineering Research Council (Wieland et aL,1987). These resultsimply that incorporation of Canada (to C. B.j, and by a Merck Postdoctoral Fellowship of the of the tripeptide into constitutive secretoryvesicles does not Helen Hay Whitney Foundation (to S. G. M.j. The costs of publicaof other newly synthesized cargo proteins tion of this article were defrayed in part by the payment of page require the presence in the trans-Golgi. Moreover, components of the vesicle macharges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate chinery must recycle continuously, and hence no new protein this fact. synthesis is requiredfor vesicle production and/or consump-

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Protein Synthesis

and Constitutive and Regulated Secretion

Immunoprecipitation of [35S]cysteine-labeledinsulin from 9 / ~of~ tion. In thispaper we address the question of whether sorting and transport through the regulated secretory pathway can each sample was carried out as described previously (Moore and Kelly, 1986). 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 cycloheximidefor 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 (0.33mCi/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-BrCAMPwas added to thechase medium a t 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 a t 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-polyacrylamidegels. The chase medium was precipitated in 80% acetone overnight a t -20 "C. The precipitate was collected by centrifugation a t 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 a t -80 "C. GAG Quontitation of GAG Chains by CPC As~ay-[~~S]SO,-Iabeled chains were quantitated by a precipitation/filtration assay as described (Miller and Moore, 1991). The medium was removed, and each well of the 24-well plate was rinsed with 250 pl of phosphatebuffered 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 (pH8.0), 150 mM NaCI, 2 mM M&12,1% Triton X-100 for 5 min a t 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 digested 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 t o 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 a t 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]S04in the range used in the assay (not shown). Metabolic Labeling with [35S]Cysteine, Radioimmunoassay (RIA), and Immunoprecipitation-AtT-20(InsGB) cells stablytransfected 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-CAMPwas added to induce secretion from storage granules. The chase medium was collected, lyophilized in a Speed-Vac, and redissolved in 1ml of NDET (1%Nonidet P-40,0.4% deoxycholate, 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).

RESULTS

GAG Chains Are Synthesized in Cycloheximide-treated Cells To determine whether transport through the regulated secretory pathway can occur in the absence ofnew 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 thedense 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 theregulated 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]sulfateand following the secretion of labeled GAG chains in the presence of cycloheximide. We first verified that synthesis of GAGchains is unaffected by the protein synthesis inhibitor cycloheximide (Fig. 1).AtT20 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 hto allow time for protein turnover and toensure that no newly synthesized peptide hormone would still be CYCLOHEXIMIDE XYLOSIDE

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FIG.1. The synthesis of GAG chains is not affected by cy-

cloheximide. Three identical 10-cm dishes of AtT-20 cells (approximately 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 immediately after labeling, and %5 of each sample was analyzed by SDSThe abbreviations used are: DMEM, Dulbecco's modified Eagle's PAGE (see "Materials and Methods"). The autoradiogram was exminimum essential medium; GAG, glycosaminoglycan;SDS, sodium posed for 3 days a t -80 "C. The sulfated GAG chain staircase (cendodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CPC, tered around 12 kDa) is formed in xyloside-treated cells (lane 2) even cetylpyridinium chloride; RIA, radioimmunoassay; ACTH, adreno- in the presence of cycloheximide ( l a n e 3). Thus, 8-D-xylosideinduces corticotropin; LDL, low density lipoprotein; 8-Br-cAMP, 8-bromo- the synthesis of GAGchains inthe presence or absence of newprotein synthesis. cyclic AMP.

Protein Synthesis Constitutive and Regulated andSecretion present in the Golgi at the time of sulfate labeling. (The transit timefor peptide hormonesfrom endoplasmic reticulum t o mature granules is 60-90 min (Gumbiner andKelly, 1981).) As shown by Burgess and Kelly (1984), treatment with p-Dxyloside induces the synthesisof 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 synthesizea 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). Cycloheximide effectively blocks the synthesisof 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 itallows us toload the Golgi with a non-protein marker.

1479 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 proopiomelanocortinandits processed fragmentsare alsoconstitutively 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 theirabsence, 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 thecell extracts and the media using a precipitation assay (seebelow). Thus, traffic through the constitutive pathway continues in theabsence of cargo proteins, and the machinery for constitutive secretion remains fully functional even during prolonged inhibition of protein synthesis. This is consistent with the observation of Wieland et al. (1987), who showed that constitutive secretionof a tripeptide fluid phase tracer is unaffected by cycloheximide treatment.

Transport of GAG Chains through theRegulated Secretory Constitutive Secretion of Golgi Chains Does Not Require Pathway IsInhibited in theAbsence of Protein Synthesis Newly Synthesized Proteins Inhibition of Stimulated Release-In contrast to constituInhibition of protein synthesis for several hours has no effect on the constitutive secretion of Golgi chains. AtT-20 tive secretion, inhibiting protein synthesis impairs regulated cells pretreated with cycloheximide for 2 h were labeled with secretion. T o analyze regulated secretion of GAG chains, cells ["'S]sulfate for 2 h in the continuous presence of cyclohex- were treated with xyloside and cycloheximide and labeled as imide. The efficiency of constitutive secretion was then as- in Fig. 2 A . All subsequent incubationswere in the continuous sayed by chasing the cells in unlabeled medium containing presence of cycloheximide. Cells were first chased for 3 h to cycloheximide for90min; we have shown previously that deplete GAG chains in the constitutive secretory pathway. Cells were then treated with media containing the secretaconstitutive secretionoccurs with rapid kinetics and that most of the radioactivity incorporated during a pulse label is se- gogue 8-Br-CAMP to induce secretion from the regulated Kelly, 1982). Fig. 2B shows creted within this period (Moore et al., 1983). Fig. 2A shows secretory pathway (Gumbiner and

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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 loGcells/dish) AtT-20 cells were pretreated for 2 h in DMEM, 1mM xyloside and 100 pg/ml cycloheximide. All subsequent incubations DMEM plus1 mM xyloside, or DMEM plus contained thespecified 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-labeledmaterials secreted via the constitutive pathway were collected during a 90-min chase after metabolic labeling. The secreted materials were acetone precipitated, and one-fourthof 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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Protein Synthesis and Constitutive and Regulated Secretion

that cells that had not been treated with cycloheximide reA leased the GAG chain staircase upon stimulation with the CYCLOHEXIMIDE 8-Br-CAMP secretagogue 8-Br-CAMP (Fig. 2B, lanes 3 and 4 ) . By com1 parison, stimulation induced very little secretion of sulfated E staircase from cells that hadbeen 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 byone of two possible models. First, biogenesis of storage granules andassembly of granule contents may require a continuous supply of newly synthesized proteins, such that in the absenceof protein synthesis GAG chains are not stored. Alternatively, cycloheximide may 1 2 3 4 have no effect on granuleassembly but instead may exert its FIG. 3. Stimulated release from preformed granules can oceffect on stimulus release coupling or fusion; that is, storage cur duringcycloheximide treatment. Four identical wells of AtTgranules do form, and GAG chains are properly stored, but 20 (Ins6B) cells, stably transfected with a rat insulin cDNA, were cycloheximide perturbs thesignaling pathway that transduces grown in a six-well dish to semiconfluence (approximately 5 X lo" external stimuli to activate exocytosis. T o distinguish between cells/well). The cells were incubated for 16 h with [""Slcysteine to label insulin that is stored in secretory granules. To test if cyclohexthese possibilities, we firstaskedwhether cycloheximide- imide affects secretion from preformedgranules thelabeled cells were treated cells could store newly synthesized GAG chains prop- first treated for 5 h in DMEM containing 100 pg/ml cycloheximide erly.Wefound that cells treatedwith cycloheximide are and then stimulated in the presence of cycloheximide for 2.5 h in impaired in their ability to store the sulfated staircase. The DMEM containing 5 mM 8-Br-CAMP. The chasemedium during the autoradiogram of cell extract andmedia samples from exper- stimulation period was collected, and the amount of total ACTH in of each samplewas determined by an ACTH radioimmunoassay iments shown in Fig. 2 wasquantitated by scanning. In control 1/40 (panel A ) . The amount of labeled insulin secreted into the medium untreated cells, the percentageof total GAG chains thatwere was determined by immunoprecipitating 9/10 of each sample with secreted into the media were 64, 75, and 77% at the end of anti-insulin antibodies (panel B ) . Panel A , secretion of total immu1.5, 3.0-, and 4.5-h chase, respectively. Incontrast, cells noreactive ACTH detected by a radioimmunoassay. Lanes 1 and 2, treated with cycloheximide secreted 78, 92, and 94% of the secretion from untreated control cells. Lanes 3 and 4, secretion from GAG chains a t 1.5-, 3.0-, and 4.5-h chase, respectively; that 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 is, untreated cells stored 23% of the total GAG chains syn- insulin that had been prelabeled with ["S]cysteine prior to cyclohexthesized after 4.5 h of chase whereas cycloheximide-treated imide treatment. Lanes I and 2,secretion from untreated control cells stored only 6%. Thus, the amount of GAG chains stored cells. Lanes 3 and 4, secretion from cells after treatment with cycloafter 4.5 h of chase is approximately4-fold less in cyclohexi- heximide for 7.5 h. Lanes 2 and 4 are from cells stimulated with 8mide-treated cells compared with controlcells. These results Br-CAMP. The autoradiogram was exposed for 2 days. suggest that cycloheximide treatment inhibits proper storage because of a diminished storagepool size of mature ACTH in of GAG chains within thecell. Preservation of Stimulus-Release Coupling-To eliminate the cell which is notreplenished by the continuousproduction of new ACTH. A secondand more precise method tomeasure directly the possibility that cycloheximide interfereswith stimulus release coupling, we tested whethercells treated with release from preformed granules was to radiolabel the concycloheximide for 5 h could still release granules that had tents of granules before cycloheximide treatment. The stimbeen formed prior to the cycloheximide treatment. A subclone ulated release of granules was then assayed by immunoprecipof AtT-20, InsGB, was used. These cells are stably transfected itation of ["S]cysteine-labeled insulin. As clearly demonwith rat insulin DNA and package insulin as well as ACTH strated in Fig. 3B, the stimulated release of insulin from in their granules (Orci et al., 1987). Thus, stimulation results preformed granules could still occur even after 5-7 h of release in therelease of both ACTH and insulin into the media. Two treatmentwith cycloheximide. Thus,thestimulus assays were used to detect release of granules formed prior to coupling remains intact duringcycloheximide treatment. Quantitation of GAG C h i n Storage and Secretion Using cycloheximide treatment. First, the total amounts of immunoreactive ACTH in the media were determined by RIA. Since CPC Assays-Since quantitation of GAG chains on PAGE storage granules in AtT-20 cells have a long life time in the gels is not very accurate, we sought to confirm these results cytoplasm (half-time of about 7-10 h; Moore and Kelly, 1985), using a quantitative precipitation assayfor GAG chains. GAG most of the ACTHin the media detected by RIA is causedby chains are negatively charged and can be precipitated with secretion from preformed granules. Fig. 3A shows that even CPC (Miller and Moore, 1991). To ensure quantitative precipitation, chondroitin sulfatewas added to bothmedium and after 5 h of cycloheximide treatment, 8-Br-CAMP can still stimulate the release of ACTH; the rate of secretion from extract samples as a carrier (see "Materials and Methods"). stimulated cells was 10-fold higher than from unstimulated Under the conditionsused, the assay is linearover at least a shown).Using this cells. Note that the absolute amount of ACTH secreted from 70-fold concentrationrange(datanot cycloheximide-treated cells was lower than in control cells; assay, we quantitated the effect of cycloheximide treatment this is most likely because of the depletion of newly synthe- on the transport and secretion of labeled GAG chains. We sized precursor ACTH in the constitutive pathway leading to performed two sets of experiments. In onewe determined the a decrease inthe basalrelease of precursor molecules and also effects of pretreatment with cycloheximide onsubsequent (0

Protein Synthesis and Constitutive and Regulated Secretion transport and storage of newly synthesized GAG chains (pretreatment experiment). Fora control, we performed a parallel experiment inwhich the cells were treated withcycloheximide for the same period of time except that the drug was added to the cells only after labeled GAG chains had already accumulated in regulated secretory granules (post-treatment experiment).Theresults for thepretreatmentexperimentsare summarizedinTableI,andthose for the post-treatment experiments are tabulated in Table11; both are from three or four independent determinations. Pretreatment with cycloheximidedecreased 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 duringa 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-hchase(thesum of chases1, 2, and 3); 8-Br-CAMP produced only a slight increase in the rate of secretion from 4.8 to 6.3% (chase 4) (TableI). 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 alsoreduced stimulated release by approximately 4-fold (increment of5.8% uersus 1.5% upon stimulation). These results further confirmed the basic conclusion 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 bythe regulated pathway (Table11). First, the total number of counts recovered in treated and untreated cells were within 10% of each other. Thus, GAG chains were stable incycloheximide-treated cells. This argues against the alternative explanation thatcycloheximide 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 conclui.e. stimulus sion from Fig. 3 by analyses of ACTH and insulin, release coupling was not significantly altered by cycloheximide treatment. Notice that thefold 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 t o constitutive secretion comparedwith the pretreatment experiments (0.5-h label and 1.5-h chase). Taken together,thesedata suggest thatproteinsynthesisisnot 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 paperwe showed that cessationof 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 sensitivity to protein synthesis inhibitor most likely reflect differ-

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Protein Synthesis and Constitutive and Regulated Secretion

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w w +I +I +I

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 tripeptideare secreted by this pathway efficiently in the absence ofnew protein synthesis. This suggests that the formation of constitutive vesiclesfrom 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 oflow density lipoprotein (LDL) receptors from the cell surface (for a review, see Brown et al., 1983). LDL receptors cluster into clathrin-coated pitsat the cell surface where the budding of endocytotic vesicles occurs. The formation of endocytotic vesicles containing the LDL receptor occurs regardless 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 ofnew 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 wouldbe 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 ofGAG chains would be observed. In thisview, the regulated secretory pathway would be regulated at thelevel 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 newprotein synthesis, but packaging ofGAG 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 totalamounts 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 isalso 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 theyfail to “mature” properly in theabsence of newly synthesized protein such that theircontentsare secreted constitutively. One could envisage that maturation of granules requires a short lived protein factor. This factor would normally prevent granules from fusing with the plasma membrane prior to stimulation. Cycloheximide treatment could deplete such a blocking protein, allowing immature granules

Protein Synthesis Constitutive and Regulated andSecretion t o fuse withthe plasma membrane without a signal forrelease. At the present timewe cannot distinguish among these possibilities because markers for granule membranes are not yet available to determine the stagesof granule formation in the absence of hormone contents. Future work will be necessary t o determine the exactrole of protein synthesis on regulated secretion. If the budding of storage granules is indeed triggered by hormones, then the mechanisms underlying the formation of regulatedgranules would befundamentallydifferentfrom those governing the buddingof constitutive vesicles since the latter does not require cargo proteins. Perhaps the budding processes for constitutive andregulated vesicles are drivenby opposite forces: constitutive vesicles driven from the cytoplasmicside by the non-clathrin-protein coat (Orci et al., 1986),and regulated vesicles from the lumenal side by aggregated 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. REFERENCES Brown, M. S., Anderson, R.G. W., and Goldstein, J. L. (1983) Cell 32,663-667

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