Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7242-7246, August 1992 Cell Biology Brefeldin A affects early events but does not affect late events along the exocytic pathway in pancreatic acinar cells (Golg complex/membrane trafflcking/) LINDA C. HENDRICKS*t, SUSAN L. MCCLANAHAN*, GEORGE E. PALADEO, AND MARILYN GIST FARQUHARt *Division of Cellular and Molecular Medicine and tCenter for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093 Contributed by George E. Palade, April 30, 1992

ABSTRACT Brefeldin A (BFA) blocks protein export from In rat exocrine pancreatic cells we have seen yet another the (ER) to Golgi complex and causes variation of the effects of BFA on the compartments of the dismanting of the Golgi complex with relocation of resident exocytic pathway: Golgi cisternae disassemble to produce Golgi proteins to the ER in some cultured cells. It is not known persistent clusters ofuncoated Golgi tubules and vesicles and whether later steps in the secretory process are affected. We nonclathrin coated vesicles. Furthermore, no relocation of previously have shown that in BFA-treated rat pancreatic Golgi and antigens into the ER is detected even after lobules, there is no detectable relocation ofGolgi proteins to the prolonged BFA incubation (19). The response of the pancre- ER and, although Golgi clsternae are rapidly ntled, atic acinar cell to BFA treatment warrants further study to clusters of small smooth vesicles consisting of both bona fide determine whether this cell type represents another variant of Golgi remnants and associated vesicular carriers persist even BFA resistance and, more importantly, whether the drug with prolonged BFA exposure. We now report the effects of affects basal and regulated release of secretory proteins, BFA on transport of proteins through the secretory pathway in topics not addressed by previous investigations. In this study exocrine pancreatic cells; we pulse-labeled pancreatic lobules we used pulse-chase analysis ofpancreatic secretory protein with 35Slmethionine and then chased for various times before release to (i) answer the questions posed above, (ii) pinpoint adding BFA. When BFA was added at pulse, treated lobules the site(s) of drug action, and (iii) determine whether single released <10% of radlactive protein in comparison with or multiple steps in the exocytic pathway are affected by controls, regardless of whether or not the lobule cultures were BFA. These inquiries can be ideally addressed in the exo- simulated with carbamoylcholine. However, when lobules crine pancreatic cell because the kinetics ofsecretory protein were pulsed and then chased for 30, 45, or 60 min before BFA transport are well established (20-22) in this cell type which addition, the amount of labeled protein released was compa- provided the original model for transport along the exocytic rable in both BFA-treated and untreated cultures. Further- pathway (23). more, the kinetics and amounts ofbasal and carbamoylcholine- stimulated release of unlbeed a-amylase from storage in MATERIALS AND METHODS zymogen granules were smilar in both control and BFA- treated lobules. Therefore, in the rat pancreas, BFA blocks ER Materials. Animals, reagents, and supplies were obtained to Golgi transport but does not affect later stages along the from the following sources: Male rats (50-125 g) from Har- secretary pathway, includin intra-Golgi transport, exit from lan-Sprague-Dawley, BFA from Epicentre Technologies the Golgi complex, formaton and concentration of secretory (Madison, WI), Ham's F-12 medium from the University of granules, and exocytosis. California, San Diego Core Facility, fetal calf serum from GIBCO, HL-1 from Ventrex Laboratories (Portland, ME), Nutridoma from Boehringer Mannheim, Nu-Serum IV from Brefeldin A (BFA) is a macrocyclic fungal metabolite that Collaborative Research, L-[35S]methionine, cell-labeling rapidly and reversibly blocks the intracellular transport of grade (>28 TBq/mmol) from NEN, Nitex nylon monofila- secretory (1-4), lysosomal (5), and membrane (6-10) proteins ment (100-gnm mesh) from Tetko (BriarcliffManor, NY); and beyond the endoplasmic reticulum (ER) and causes the the Phadebas a-amylase test from Pharmacia. All other concomitant rapid vesiculation of Golgi cisternae and loss of reagents and chemicals were purchased from Sigma. Golgi membranes (2, 8, 11) in most cell types. Recently BFA Pancreas Lobule Preparation. Pancreatic lobules were pre- also has been shown to affect both endocytosis and transcy- pared as described (19). Briefly, the gland was distended by tosis, depending on cell type (12). Thus, in MDCK and PtKj injecting Ham's F-12 medium containing 1% HL-1, Nutri- kidney cell lines, which are "resistant" to the effects of BFA doma, Nu-Serum IV, aprotinin at 0.1 trypsin inhibitor unit/ on Golgi membranes (12-14), the drug induces membrane ml, and soybean trypsin inhibitor at 10 mg/ml into its fusion within the endocytic system (13, 15). In contrast, BFA interstitia to separate lobules. The latter were minced be- affects both endocytic and exocytic pathways in NRK cells tween scalpel blades into individual =2-mm3 lobules (24), (8, 15, 16). Thus, the drug has more pleiotropic effects on which were placed in groups of -20 on Nitex rafts into vials intracellular membranes than originally reported (8). containing 10 ml of medium with or without BFA (7.2 ,uM, 2 Although BFA clearly inhibits vesicular traffic from ER to iug/ml) and allowed to equilibrate in a 95% 02/5% CO2 Golgi complex in many systems, whether it also affects later atmosphere at 370C for 5-10 min with agitation before the steps in the secretory process is unknown. Because BFA beginning of each experiment. apparently generates cation-selective channels in planar lipid Release of Secretory Proteins. To determine the effect of bilayers (17), it may have more nonselective effects on proteins, membranes than reported to date. In fact, BFA has been BFA on release of newly synthesized secretory suggested to be a membrane fusogen (18). Abbreviations: BFA, brefeldin A; LDH, lactate dehydrogenase; ER, endoplasmic reticulum. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Division of payment. This article must therefore be hereby marked "advertisement" Cellular and Molecular Medicine, 0651, 9500 Gilman Drive, Uni- in accordance with 18 U.S.C. §1734 solely to indicate this fact. versity of California, San Diego, La Jolla, CA 92093-0651. 7242 Downloaded by guest on September 24, 2021 Cell Biology: Hendricks et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7243 lobules were pulse-labeled (5 min) with [35S]methionine (100 700- puCi/ml; 1 Ci = 37 GBq), rinsed quickly (five times) with medium containing lOOx excess unlabeled methionine, and 600- then chased for up to 7 hr in the same medium. When BFA was added at 0 min, it was included in both the pulse and 500 chase media. Where indicated, the secretagogue carbamoyl- choline (20 AM) was added 60 min into the chase. In prelim- we400 inary experiments, we established that our enriched medium ~300- allowed retention of acinar cell function for up to 8 hr (data E not shown), which is longer than the experiments reported o here. To define the site of the block in exocytic traffic, BFA 200 was added at 10, 30, 45, or 60 min after the pulse. To examine the time course of recovery from a BFA block, pulse-labeled 100 - | W - lobules were chased for 60 min in the presence ofBFA before I carbamoylcholine addition and further chased for an addi- 0 120 240 360 480 tional 60 min before BFA was washed out by five rapid minutes medium exchanges, and the chase was continued in BFA-free medium containing carbamoylcholine. In these experiments, FIG. 1. BFA blocks basal and stimulated release of newly syn- 0.1-ml samples were removed from the medium at 20-min thesized proteins in rat pancreas. Lobules were pulsed for 5 min with intervals throughout the chase, and release of newly synthe- [35S]methionine and then chased with or without 7.2 AM BFA. In sized proteins was quantitated by trichloroacetic acid pre- some experiments, the secretagogue, carbamoylcholine (20 AuM) was cipitation and scintillation counting of each precipitate. This added 60 min into the chase (arrowhead). Trichloroacetic acid- assay is valid because =95% of newly synthesized proteins in precipitable counts were assayed in medium removed from the lobules at intervals throughout a o7-hr chase. Negligible amounts of the adult pancreas are secretory (25). Data are expressed as radioactive secretory proteins were released from unstimulated cpm per aliquot and normalized to Ag ofdry weight oflobules BFA-treated lobules (0) as compared with controls (o). Radioactive (recovered and air-dried for 48 hr). We found this normal- secretory proteins released by carbamoylcholine-stimulated, BFA- ization more reproducible than normalizing to recovered treated lobules (o) were <10%o ofcontrols (a). Data are expressed as protein (because of the difficulty in obtaining complete sol- cpm//Ag of dry weight of tissue shown as mean ± SD of triplicate ubilization of lobules) or DNA (unreliable due to endogenous samples. The graph is representative of -10 experiments. Cell DNase activity). In all experiments, lactate dehydrogenase integrity as assessed by LDH release was maintained at -98% (LDH) release was also monitored throughout the chase to throughout the experiments. ensure that the radioactive proteins in the medium were from rather than from cell lysis. LDH activity was oylcholine, a secretagogue that stimulates release of pancre- determined by a modification (sodium pyruvate was not atic secretory proteins from zymogen granules (27), resulted added due to adequate levels in the medium) of Sigma in <10% release of radioactive proteins from BFA-treated procedure no. 500. Cell integrity (expressed as percent of lobules past 240 min in comparison with nontreated controls maximal release from detergent-lysed lobules) was typically (Fig. 1). These results indicated that BFA blocked both basal maintained at 98% throughout the chase. and stimulated release of newly synthesized proteins in the a-Amylase Assay. To determine whether BFA had any exocrine pancreas. effect on discharge of secretory protein stored in zymogen BFA Blocks Early but Does Not Block Late Events Along the granules, lobules were prepared and incubated with and Exocytic Pathway. BFA is known to affect ER to Golgi without BFA for 60 min, as described above. Where indi- transport (1, 2, 6, 8) without inhibiting protein synthesis (1, cated, carbamoylcholine (20 ,uM) was added after BFA 3, 9) in this (data not shown) and other cell types. However, treatment. Medium aliquots were assayed for a-amylase it is not known whether BFA affects later events along the activity by using a modification the Phadebas amylase test exocytic pathway. To pinpoint the subcellular site of trans- (26), in which a-amylase hydrolyzes an insoluble dye-starch port arrest, we chased pulse-labeled, stimulated lobules for polymer to a soluble blue dye. activity was ex- various intervals before adding the drug and assessed tri- pressed as dye released (absorbance at 660 nm) normalized to chloroacetic acid-precipitable counts released as a function dry weight of tissue. LDH release was monitored throughout of time in the presence of BFA. Site of the block was inferred the experiments, as above. by fitting the time point at which the effect was lost into the Electron Microscopy. Lobules were fixed (2 hr) with 2.5% known kinetics and compartmental location of newly syn- glutaraldehyde/100 mM cacodylate HCl buffer, pH 7.2/0.1 thesized protein as documented in the literature (20, 22). If mM MgCl2/0.1 mM CaCl2, post-fixed with 1-2% OS04 in the BFA addition were delayed for only 10 min after a 5-min pulse same buffer, stained in block with uranyl acetate, dehydrated when 40-60% of the newly synthesized proteins are still in in graded ethanols, and embedded in Epon. Sections were the ER (24, 27, 28), secretion was greatly diminished (=4- stained with uranyl acetate and lead citrate and then exam- fold) (Fig. 2). However, when the delay after the pulse was ined at 80 kV on a Philips CM-10 electron microscope. for 30 min or longer, release of secretory proteins was identical to controls (Fig. 2). These results indicate that only early events in the exocytic traffic of pulse-labeled secretory RESULTS proteins are affected by BFA. The drug apparently does not BFA Blocks Release of Newly Synthesized Secretory Pro- interfere with either transport through the Golgi complex or teins. To determine the affect of BFA on release of newly formation and discharge of secretory (zymogen) granules. synthesized secretory proteins, we assayed trichloroacetic BFA Does Not Affect Release of a-Amylase from Zymogen acid-precipitable counts in the incubation medium of pulse- Granules. To test this assumption, we assayed a-amylase labeled pancreatic lobules chased in the presence or absence activity in the medium from unlabeled, unstimulated, and of BFA. Small but increasing amounts of radiolabeled secre- carbamoylcholine-stimulated lobules plus or minus BFA. tory proteins were released from control lobules throughout The drug had no effect on stimulated a-amylase release: the chase (Fig. 1). This release represents the well-known half-maximal discharge occurred =7 min after secretagogue basal secretion. In contrast, negligible radioactivity was addition, and total a-amylase activity recovered in the me- released from BFA-treated lobules. Treatment with carbam- dium was identical in both control and BFA-treated lobules Downloaded by guest on September 24, 2021 7244 Cell Biology: Hendricks et al. Proc. Nad. Acad. Sci. USA 89 (1992) 600 pathway by transmission electron microscopy. Zymogen granules were found unaltered in BFA-treated cells by ref- 500 erence to controls (Fig. 4), which indicates that the drug has no readily observed effects on the membranes of these 0 3 400 organelles, in marked contrast to the vesiculation and tubu- lation of Golgi membranes (Fig. 4 and ref. 19). Exocytic Transport Resumes Upon Removal of BFA. To 300 o0 A F determine the time course of recovery from a BFA block, we assessed trichloroacetic acid-precipitable counts released ~200 from pulse-labeled, BFA-treated, and carbamoylcholine- t,2stimulated lobules as a function of time during chase in 100 [11WOK. BFA-free medium. Release of radiolabeled secretory pro- teins resumed 60 min after BFA washout (Fig. 5), which is the 0 time observed for release of secretory proteins pulse-labeled ' 120 180160 240 300 360 in the ER in control tissue (20). Furthermore, discharge 3 hr 3~ after BFA washout was comparable to control levels 3 hr minutes0minutes post-pulse (Fig. 5), indicating that intracellular transport _.+- I~tAADVsAm A FIG. 2. BFA blocks early but does not block later steps along the resumes eincientiy aEer rirA removai. secretory pathway. To determine where along the exocytic pathway BFA blocked secretion, lobules were pulsed-for 5 min with [35SJme- DISCUSSION thionine and then chased for 0 (o), 10 (C), and 30, 45, or 60 (A) min before adding 7.2 AsM BFA. In all cases, carbamoylcholine was We have examined the effect of BFA on release of pulse- added 60 min after the pulse (arrowhead). When BFA was added 10 labeled proteins in rat pancreatic exocrine lobules. We find, min after the pulse, there was a diminished_~~~~~~~~~secretion level. When the consistent with biochemical data reported in a number of addition was delayed until 30, 45, or 60 min into the chase, the different cell types (1, 3, 5, 6, 10), that BFA treatment secretion level was identical and indistinguishable from control levels and (-). Data are shown for 45-min delay; for 30- and 60-min delays reversibly blocks release of newly synthesized proteins both basal and results were identical. The graph shows mean ± SD of triplicate that the block affects carbamoylcholine- samples and is representative of four experiments. Cell integrity as stimulated secretory protein release. Both the block in the assessed by LDH release was maintained at -98% throughout the release of newly synthesized protein and the morphological experiments. effects (10) of BFA were seen at a dose comparable to that used by other investigators (3.6 uM) and were unchanged at (Fig. 3). In unstimulated lobules, a-amylase release was a 10-fold increase in drug concentration. Furthermore, a either unchanged or slightly elevated in BFA-treated lobules single low BFA dose (7.2 ,uM) effectively blocked secretory compared with controls (Fig. 3). These results indicate that protein release >6 hr, indicating that rat pancreatic acinar secretagogue-stimulated release of stored a-amylase from cells, unlike cultured rat hepatocytes (2), do not metabolize zymogen granules is not significantly affected by BFA. BFA to inert products. BFA Does Not Affect the Morphology ofZymogen Granules. We used pulse-chase analysis of secretory protein release Because BFA was recently reported to cause fusion of to pinpoint the time(s) at which the BFA block in exocytic membrane compartments in the endocytic pathway in some transport is established and thereby inferred the site at which cell types (13, 15, 16) and because the drug is assumed to be it occurs. When BFA was added 10 min after a 5-min pulse, a fusogen (18, 29), we decided to check its effect on the when 40-60%o ofthe pulse-labeled proteins are still in the ER morphology of the terminal compartment of the exocytic (25, 27, 28), radioactivity release in the medium decreased, indicating that BFA inhibited vesicular traffic between the *45- ER and the Golgi complex or early in the Golgi complex. The data in Fig. 2 indicate that the inhibition is strong (Q75%) but 140 incomplete because the amounts of radioactivity released increased with time. However, when BFA was added 30, 45, ~35- or 60 min into the chase, when the pulse-labeled proteins are in the Golgi complex (30 min), condensing vacuoles (45 min), ~30- or in zymogen granules beginning to discharge (60 min) (20), the secretion level was comparable to that found in stimu- lated control lobules. These results indicate that BFA does 15- not affect transport through the Golgi complex, packaging of 20 secretory proteins into granules, or exocytosis. Instead, only pre-Golgi events appear influenced. The lack of effect on exocytosis was confirmed by assaying both basal and car- 10 bamoylcholine-stimulated release of nonradioactive amylase co 0 10 20 30 50 from storage in zymogen granules. Kinetics and amounts of minutes release were similar with or without the drug. We do not know where the BFA-induced block occurs FIG. 3. Release of stored a-amylase from zymogen granules is relative to those caused by other known inhibitors of ER-to- unaffected by BFA treatment. a-Amylase activity released into the Golgi transport in the exocrine pancreas, such as incubation incubation medium was assessed at short intervals after preincuba- at 150C (30, 31) or inhibitors of oxidative phosphorylation tion (60 min) of lobules ±7.2 ,uM BFA. After carbamoylcholine (22). However, the BFA-induced block in transport occurs stimulation, enzyme release was identical in control (i) and BFA- before the 200C block (30, 31) or the block induced by the treated (o) lobules. In unstimulated lobules, a-amylase release was unchanged or slightly elevated in BFA-treated (o) compared with ionophore monensin (32), which in the exocrine pancreas controls (o). Activity is given in relative units of solubilized dye from occurs before exit from the Golgi. In BFA-treated pancreatic we amylase substrate (see Methods) and shown as mean ± SD/Ag ofdry acinar cells found persistent clusters ofuncoated vesicles weight tissue. Cell integrity was maintained at -98% throughout the containing Golgi marker proteins as well as nonclathrin experiments. coated transport vesicles surrounded by typical transitional Downloaded by guest on September 24, 2021 Cell Biology: Hendricks et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7245

FIG. 4. Zymogen granule morphology is unaffected by BFA treatment. The cluster of remnant Golgi vesicles (vc) with associated ER transitional elements is typical of BFA-treated (60 min, 7.2 jLM) acinar cells (19). The shape and distribution of zymogen granules (zg) are similar to reported parameters (20) and to our controls (data not shown). n, nucleus. (Bar = 0.2 .&M.) elements of the ER (19). The latter may represent vesicular between successive Golgi subcompartments. Therefore, carriers operating between the ER and Golgi complex or whether the block in transport occurs proximal to or within Golgi remnants remains to be established. 600 The block was reversible, as exocytic transport resumed within 60 min ofBFA washout. Because this is approximately 500 the time for secretory proteins labeled in the ER to be released from normal pancreatic slices (lobules) (20), the 0 400- recovery data suggest that the block in transport of newly 4- synthesized proteins is pre-Golgi. The data also suggest that 2 300- intracellular transport resumed with apparently normal ki- netics after BFA removal. Furthermore, 3 hr after BFA 200- washout discharge was comparable to control levels 3 hr after & pulse, indicating that recovery of Golgi function was com- 100* x plete. 1vvzt We thank David Vansomphone for his volunteer help with the 0 |O secretion assays. This research was supported by National Institutes 0 60 120 180 240 300 36D of Health Grants CA46128 (to M.G.F. and G.E.P.) and DK17780 (to minutes M.G.F.). FIG. 5. Release of proteins blocked in transport resumes after G. removal of BFA. Experimental protocol was the same as for control 1. Misumi, Y., Misumi, Y., Miki, K., Takatsuki, A., Tamura, (-) and BFA-treated (o), carbamoylcholine-stimulated lobules in Fig. & Ikehara, Y. (1986) J. Biol. Chem. 261, 11398-11403. 1. In one set oftreated lobules (A), BFA was washed out 60 min after 2. Fujiwara, T., Oda, K., Yokota, S., Takatsuki, A. & Ikehara, Y. stimulation (white arrowhead). After BFA removal, secretion re- (1988) J. Biol. Chem. 263, 18545-18552. sumed with a =60-min lag, and 3 hr after BFA removal was 3. Magner, J. A. & Papagiannes, E. (1988) Endocrinology 122, comparable to control levels 3 hr after the pulse. Data shown are 912-920. mean + SD and are representative of three experiments. Cell 4. Kato, S., Ito, S., Noguchi, T. & Naito, H. (1989) Biochim. integrity was maintained at -98% throughout the experiments. Biophys. Acta 991, 36-43. Downloaded by guest on September 24, 2021 7246 Cell Biology: Hendricks et al. Proc. Natl. Acad Sci. USA 89 (1992) 5. Oda, K. & Nishimura, Y. (1989) Biochem. Biophys. Res. 18. Heuser, J. E. (1989) J. Cell Biol. 109, 206a (abstr.). Commun. 163, 220-225. 19. Hendricks, L. C., McClanahan, S. L., McCaffery, M., Palade, 6. Takatsuki, A. & Tamura, G. (1985) Agric. Biol. Chem. 49, G. E. & Farquhar, M. G. (1992) Eur. J. Cell Biol., in press. 899-902. 20. Jamieson, J. D. & Palade, G. E. (1967)J. Cel Biol. 34, 577-5%. 7. Doms, R. W., Russ, G. & Yewdell, J. W. (1989) J. Cell Biol. 21. Jamieson, J. D. & Palade, G. E. (1%7) J. Cell Biol. 34, 597- 109, 61-72. 615. 8. Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S. & 22. Jamieson, J. D. & Palade, G. E. (1968) J. Cell Biol. 39, 589- Klausner, R. D. (1989) Cell 56, 801-813. 603. 9. Ulmer, J. B. & Palade, G. E. (1989) Proc. Natl. Acad. Sci. USA 86, 6992-6996. 23. Palade, G. (1975) Science 89, 347-358. 10. Oda, K., Fujiwara, T. & Ikehara, Y. (1990) Biochem. J. 265, 24. Scheele, G. (1983) Methods Enzymol. 98, 17-28. 161-167. 25. Scheele, G. A., Palade, G. E. & Tartakoff, A. M. (1978) J. Cell 11. Ulmer, J. B. & Palade, G. E. (1991) Eur. J. CellBiol. 54, 38-54. Biol. 78, 110-129. 12. Sandvig, K., Prydz, K., Hansen, S. H. & van Deurs, B. (1991) 26. Ceska, M., Hultman, E. & Ingelman, B. G.-A. (1969) Experi- J. Cell Biol. 115, 971-981. entia 25, 555-556. 13. Hunziker, W., Whitney, J. A. & Meliman, I. (1991) Cell 67, 27. Jamieson, J. D. & Palade, G. E. (1971) J. Cell Biol. 50, 135- 617-627. 158. 14. Ktistakis, N. T., Roth, M. G. & Bloom, G. S. (1991) J. Cell 28. Scheele, G. & Tartakoff, A. (1985) J. Biol. Chem. 260, 926-931. Biol. 113, 1009-1023. 29. Orci, L., Tagaya, M., Amherdt, M., Perrelet, A., Donaldson, 15. Wood, S. A., Park, J. E. & Brown, W. J. (1991) Cel 67, J. G., Lippincott-Schwartz, J., Klausner, R. D. & Rothman, 591-600. J. E. (1991) Cell 64, 1-20. 16. Lippincott-Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., 30. Saraste, J., Palade, G. E. & Farquhar, M. G. (1986) Proc. NatI. Orci, L. & Klausner, R. D. (1991) Cell 67, 601-616. Acad. Sci. USA 83, 6425-6429. 17. Zizi, M., Fisher, R. S. & Grillo, F. G. (1991) J. Biol. Chem. 31. Tartakoff, A. M. (1986) EMBO J. 5, 1477-1482. 266, 18443-18445. 32. Tartakoff, A. & Vassalli, P. (1978) J. Cell Biol. 79, 694-707. Downloaded by guest on September 24, 2021