Temperature-Sensitive Steps in the Transport of Secretory Proteins Through the Golgi Complex in Exocrine Pancreatic Cells JAAKKO SARASTE, GEORGE E
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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6425-6429, September 1986 Cell Biology Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells JAAKKO SARASTE, GEORGE E. PALADE, AND MARILYN GIST FARQUHAR Department of Cell Biology, Yale University School of Medicine, P.O. Box 3333, New Haven, CT 06510 Contributed by Marilyn Gist Farquhar, May 9, 1986 ABSTRACT The effect of temperature on secretory pro- apply to secretory proteins in situ. In the present study, we tein transport was studied by cell fractionation ofrat pancreatic have examined the effect oflow temperature on the transport lobules, pulse-labeled in vitro with [35S]methionine and chased of secretory proteins in exocrine pancreatic cells. This for 60 min at 16, 20, or 370C. Chase at 370C allowed secretory system was selected because it served as the original model proteins to reach a zymogen granule fraction, whereas chase at in which the kinetics and compartments involved in transport 16 or 20°C led to their extensive retention in a total microsomal along the secretory pathway were worked out (10-13). Our fraction. To pinpoint the sites of transport inhibition, total results define the existence in these cells of a number of microsomes were subfractionated by flotation in a sucrose successive and different temperature-sensitive transport density gradient. Five bands were resolved, of which the steps along the endoplasmic reticulum-plasmalemma path- heaviest or Bi (density = 1.20 g/ml) consisted primarily of way. rough microsomes. The lighter fractions, B2 (1.17 g/ml), B3 (1.15 g/ml), and B4 (1.14-1.13 g/ml), consisted primarily of EXPERIMENTAL PROCEDURES smooth vesicles derived from Golgi elements. B4 had the highest specific activity for galactosyltransferase, a trans Golgi Materials. Aprotininwaspurchasedfrom BoehringerMann- cisternal marker; B2, B3, and B4 are assumed to represent cis, heim. Soybean trypsin inhibitor, benzamidine hydrochloride, middle, and trans Golgi subcompartments, respectively. At the Hepes, Mes, ovalbumin (grade V), UDP-galactose, and ATP end of a 2-min pulse, a single peak of labeled proteins were from Sigma. [35S]Methionine (1375 Ci/mmol; 1 Ci = 37 colocalized with B1. During subsequent 60-min chases, labeled GBq) was obtained from Amersham, UDP-[3H]galactose proteins advanced to B2 at 160C and to B3 at 20°C. At 37C the (10.2 Ci/mmol) was from New England Nuclear, and Eagle's radioactivity remaining in the total microsomal fraction was minimal essential medium (MEM) was from GIBCO. distributed among four peaks (B1-B4). The results indicate Preparation, Incubation, and Radlolabeling of Pancreatic that transport from the endoplasmic reticulum to the Golgi Lobules. Male Sprague-Dawley rats (Charles River Breeding complex is strongly inhibited below 200C. At 16°C, the bulk of Laboratories), 150-300 g, were anesthesized with ether, and the cohort of labeled secretory proteins is still in the rough ice-cold MEM containing 20 mM Hepes, soybean trypsin endoplasmic reticulum, but its advancing front reaches cis inhibitor at 10 ,g/ml, and aprotinin at 100 units/ml (sMEM) Golgi elements. At 200C, the front advances to a middle Golgi was injected into the interstitia of the pancreas to distend the compartment, and at 37PC most of the cohort (,u70%) reaches tissue and facilitate separation ofthe lobules of the gland (14, condensing vacuoles and zymogen granules. It is concluded that 15). The pancreas was removed and placed in ice-cold MEM, transport steps along the endoplasmic reticulum-plasmalem- and individual macroscopic lobules were excised with razor ma pathway have distinct temperature requirements. blades. Each set of lobules (0.5-1 g of tissue) was washed twice with ice-cold sMEM, placed in 25-ml flasks containing Previous work has shown that, in mammalian cells, there are 5 ml of methionine-free sMEM, and incubated for 15 min at specific, low-temperature-sensitive transport steps along 370C in a water bath with agitation and gassing (95% 02/5% both the endocytotic and the exocytotic pathways. Internal- C02). The lobules were pulse-labeled for 2-5 min with ization of ligands bound to cell surface receptors is inhibited [35S]methionine at 25-50 ,uCi/ml in 2 ml of methionine-free below 100C, it proceeds between 15 and 200C, but the sMEM. At the end ofthe pulse the lobules were either placed internalized ligands are not delivered to lysosomes. Instead, directly in ice-cold homogenization buffer or washed once they accumulate in a prelysosomal compartment, suggesting and further incubated in 10 ml ofprewarmed (16-370C) chase that the last fusion steps in the pathway are inhibited below medium (sMEM) containing a 50-fold excess of nonradioac- 200C (1-3). Similarly, exocytosis is inhibited below 200C (4, tive methionine-conditions found to be fully effective. 5) and transport of viral membrane glycoproteins from the Previous work has established that in this system secretory trans Golgi cisternae to the cell surface is arrested (6-8). proteins account for >90% ofthe newly synthesized proteins Moreover, there is evidence that in BHK-21 cells an early, (16). pre-Golgi step in the transport of membrane glycoproteins is Release of Secretory Proteins into the Medium. Rat pancre- also affected by reduced temperature, because these proteins atic lobules were pulse-labeled for 5 min at 370C and chased leave the rough endoplasmic reticulum at 150C but do not for 60 min at different temperatures (16, 20, or 370C) without progress beyond an intermediate compartment located be- stimulation, to allow the secretory proteins to accumulate at tween the rough endoplasmic reticulum and the Golgi com- various intracellular sites. Thereafter medium that had been plex (7, 9). warmed to the appropriate temperature and contained 20 ;kM Up to now, intracellular temperature-sensitive steps along carbamoylcholine (a secretagogue) was added to the lobules, the exocytotic pathway have been described only for the and incubation was continued for 2 hr. Medium samples (200 transport of viral membrane proteins in established cell lines ,ul) were taken at 10- to 15-min intervals and assayed for in culture, raising the question of whether similar effects trichloroacetic acid-insoluble radioactive proteins. Preparation of Cell Fractions. A modification ofthe method The publication costs ofthis article were defrayed in part by page charge previously described (10) for fractionation of guinea pig payment. This article must therefore be hereby marked "advertisement" pancreas was used. All steps were performed at 0-40C. in accordance with 18 U.S.C. §1734 solely to indicate this fact. Unlabeled or [35S]methionine-labeled lobules were washed 6425 Downloaded by guest on September 26, 2021 M"26 Cell Biology: Saraste et al. Proc. Natl. Acad. Sci. USA 83 (1986) (five times) with ice-cold 0.3 M sucrose containing soybean Pasteur pipette), fixed by suspension in 2% glutaraldehyde in trypsin inhibitor at 10 jig/ml, aprotinin at 40 units/ml, and 5 0.1 M cacodylate buffer (pH 7.4) for 30 min at 40C, and mM benzamidine (final pH, =6.2), placed in 4 ml ofthe same pelleted (120 min at 100,000 x g.,g) at 3YC. The resultant medium (tissue concentration, =1.20), and homogenized in a pellets were fixed for 2 hr with 1% OS04 in 0.05 M Brendler-type glass homogenizer (Thomas, size A) with three acetate/Veronal buffer and stained in block with uranyl up-and-down strokes of a Teflon pestle motor-driven at 3000 acetate and processed as described above. rpm. The homogenates were centrifuged for 10 min at 600 x gavg in 12-ml conical Pyrex tubes using a swinging bucket rotor. The ensuing pellets, which contained cell debris and RESULTS nuclei, were washed once (by resuspension-sedimentation) Low Temperature Inhibits the Release ofSecretory Proteins. with 2 ml of0.3 M sucrose. The supernatants were combined, When pancreatic lobules were pulse labeled for 5 min at 370C placed in 15-ml Corex tubes, and centrifuged for 10 min at and then chased for 60 min at 370C, the addition of 3000 X gAvg in a JA20 fixed-angle rotor to pellet zymogen carbamoylcholine to the incubation medium at the end of the granules. The surface of the white zymogen granule pellet chase resulted in rapid release of secretory proteins: 50-60% was rinsed twice with 0.5 ml of 0.3 M sucrose to remove an of the total pulse-labeled proteins of the lobules were dis- overlying green layer of mitochondria. The postgranule charged into the medium over a 2-hr period (Fig. 1). There- supernatant and rinses were and at combined centrifuged fore, under these conditions secretory proteins are transport- 7800 x gavg to yield a pellet enriched in mitochondria. The postmitochondrial supernatant was centrifuged for 60 min at ed to zymogen granules from which they can be discharged by exocytosis upon stimulation. In contrast, when lobules 110,000 X gavg in a Beckman SW41 swinging bucket rotor to pellet a total microsomal fraction. In other experiments the were pulsed at 370C and chased at 16 or 200C, no release of postmitochondrial supernate was subfractionated as de- radioactivity into the medium was observed after stimulation scribed below. with carbamoylcholine (Fig. 1), demonstrating that low Subfractionation of the Total Microsomal Fraction by Flo- temperature effectively inhibits either the intracellular trans- tation in Sucrose Gradients. Postmitochondnral supernatant port or the release of secretory proteins. When separate sets (-6 ml) was layered on top of 5 ml of 0.33 M sucrose/5 mM ofpancreatic lobules chased at 16 or 20'C were shifted to 37TC benzamidine, layered in turn on top of a sucrose cushion in the presence of the secretagogue, there was an efficient consisting of 1 ml of 2 M sucrose/5 mM benzamidine.