Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6425-6429, September 1986 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 , 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 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 -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 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 . 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. release of secretory proteins into the medium; however, in Centrifugation in an SW41 rotor as described above yielded contrast to cells preincubated and stimulated at 37TC, there a total microsome band on top of the cushion. After removal was a 15- to 20-min lag in the appearance of labeled proteins of the supernatant by suction, the total microsome band in the medium (Fig. 1). Therefore, the temperature-induced (=600 ,ul) was collected with a Pasteur pipette, and the inhibition of transport of exocrine proteins is reversible. microsomes were resuspended by vigorous pipetting 30 times When carbamoylcholine was added to the cells immediately through a micropipette tip (Rainin, Woburn, MA; tip diam- after the pulse and secretion was followed at 37°C, the first eter = 0.5 mm). Then, 2.4 ml of 2 M sucrose/5 mM ben- labeled proteins appeared in the medium after a lag of =30 zamidine was slowly added to the sample with repeated min, indicating that this is the minimal time required for Vortex mixing. Microsomes were subfractionated by flota- intracellular transport of secretory proteins from their site of tion using a modification ofthe procedure described by Knipe et al. (17): The sample in 3 ml of 50o (wt/vol) sucrose was placed at the bottom of an SW41 ultracentrifuge tube and 60 overlaid with the following sucrose solutions: 1 ml of45%; 1.5 0 ml each of 40%, 35%, 30%, and 25%, and 2 ml of 20% (all 60~~~~~~~~ solutions contained 5 mM benzamidine). After centrifugation C) at 3°C for 9-10 hr at 170,000 X ga (to reach isopycnic conditions), 25 fractions of 450 ,ul each were collected by cr C- 40 upward displacement or from the bottom of the tube. A Lr_ similar procedure has been used to resolve Golgi elements C-) into subfractions of decreasing density, mostly along the CO cis-trans axis (18-20). Analysis of Gradient Fractions. Protein radioactivity was determined by trichloroacetic acid precipitation from 25- to 20 3 C-) 50-,u aliquots of the gradient fractions followed by scintilla- C,) tion counting in Liquiscint. Proteins were measured by the Bio-Rad assay with ovalbumin as standard, and density was determined by refractometry. RNA was measured as A260, using the method of Blobel and Potter (21). 0- y-Glutamyltrans- 30 60 90 120 ferase activity was determined by the method of Tate and MINUTES Meister (22). Galactosyltransferase activity was assayed by a modification of the method of Brew et al. (23) with ovalbumin FIG. 1. Effect of temperature on discharge of secretory proteins as acceptor. The assay mixture (100 ,ul) contained 50 ,ul of from rat pancreatic lobules pulsed for 5 min at 37°C with sample membranes (diluted 1:1 with water), ovalbumin at 7 [35S]methionine and then chased for 60 min at 370C (o), 20°C (o), or mg/ml, 2 mM ATP, 0.1 mM UDP-galactose, 0.28 ,uCi of 16°C (A); 20 uM carbamoylcholine was added to the medium at the UDP-[3H]galactose, 20 mM MgCl2, 0.5% Triton X-100, and end of the chase (0 min), and incubation was continued for 120 min 50 mM Tris HCl (pH 7.1). at the same temperature as the chase. In contrast to the 370C control, Electron Microscopy. Sets of pancreatic lobules incubated negligible amounts of radioactive secretory proteins were released during stimulation at reduced temperatures (16 or 20°C). However, in vitro for 60 min at 16 or 37°C were fixed overnight at 40C lobules chased at 200C (o) or 160C (A) and then shifted to 370C at the in 1% OS04 in 0.1 M Mes, pH 6.5/1 mM MgCl2/1 mM CaCl2 time of carbamoylcholine addition efficiently discharged proteins (24), stained in block with uranyl acetate, dehydrated, and into the medium, demonstrating the reversibility of the temperature embedded in Epox. Microsomal subfractions obtained by inhibition. The lag (-20 min) is the time needed for transport from sucrose gradient centrifugation were collected (using a the site of the block to the site of discharge. Downloaded by guest on September 26, 2021 Cell Biology: Saraste et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6427

synthesis to their site of exocytotic discharge (data not shown). Secretory Proteins Accumulate in the Microsomal Fraction 1.2 I. at Low Temperatures. To study how reduced temperature = 500 E affects the vectorial movement of secretory proteins through 1.15 '- the various compartments along the transport pathway, we took advantage of procedures available for the fractionation M 300 1.10 tissue (10). The distribution of trichloroacetic of pancreatic 0 CLi acid-precipitated radioactive proteins in fractions isolated 0- from lobules pulse-labeled for 2 min at 37°C with 100 _ [35S]methionine and then chased for 60 min at 16, 20, or 370C

is shown in Table 1. Immediately after the pulse, the majority >- of the radioactivity was associated with the total microsomal 120 fraction, and relatively little was found in the zymogen 70 -_ E granule fraction. The former consists largely of vesicular w -V elements derived from the entire endoplasmic reticulum and '7, 80 50 < the Golgi complex (10), and the latter contains condensing LL-W E vacuoles as well as zymogen granules (11). After a 60-min z 30 9o chase at 37°C there was a 709O decrease in total radioac- 40 I- tivity of the microsomal fraction with a corresponding in- -iE 0(-DC.C. crease in that of the zymogen granule fraction, as expected 10 zymogen from published results (11). The radioactivity ofthe 5 10 15 20 25 was over the postpulse granule fraction increased l1O-fold FRACTION value. This figure is undoubtedly an underestimate since during homogenization there is partial lysis of zymogen FIG. 2. Subfractionation of total pancreatic microsomes from granules and release of radioactive proteins (16), which are pancreatic lobules by flotation in sucrose gradients. Total micro- recovered mostly in the postmicrosomal supernatant (Table somes prepared from homogenized lobules were subfractionated by 1). In contrast to the situation at 37°C, after a 60-min chase centrifugation in 20-50%o sucrose gradients. Arrows indicate loca- at either 16 and 20°C, there was no significant increase in the tions of the five bands detected after centrifugation [density = radioactivity in the zymogen granule fraction, and the secre- 1.19-1.2 (Bi), 1.17 (B2), 1.15 (B3), 1.13-1.14 (B4), and 1.12 (B5) tory proteins remained in the total microsomal fraction. g/ml]. Twenty-five gradient fractions were collected and their Taken together, the cell fractionation data and the kinetics of density (A), protein (A) and RNA content (B), and galactosyl(GAL)- transferase activity (B) were determined. The major protein and secretory protein release indicate that at temperatures RNA peaks mark the location ofrough microsomes (Bi). The highest <20°C, secretory proteins are arrested at some site(s) along specific activity of galactosyltransferase colocalizes with B4, sepa- the transport pathway between the rough endoplasmic rated from rough microsomes by two smooth membrane fractions reticulum and condensing vacuoles. (B2 and B3). Subfractionation of Total Microsomes. Flotation by centrif- ugation oftotal microsomes in sucrose gradients resolved five (25), was associated with B4 (Fig. 2B). y-Glutamyl- distinct bands in the region of the middle two-thirds of the transferase, a marker for acinar cell plasma membrane (26), gradient (Fig. 2A). Most ofthe loaded material (=85% oftotal was detected as a broad peak between densities 1.12 and 1.17 protein) remained as a band or, in some experiments, as a g/ml (data not shown). The distribution of RNA, galactosyl- double band at a density of 1.19-1.20 g/ml in the lower part transferase, and -glutamyltransferase was the same at 16 of the gradient. This band (Bi) consisted of rough micro- and 20°C as at 37°C. somes as shown by electron microscopy and by its RNA When pancreatic lobules were pulse-labeled for 2 min at content (Fig. 2B). Three additional bands were detected 37°C, a single sharp peak containing :=95% of the radioac- above the rough microsomal band at densities of 1.17, 1.15, tivity in the gradient was detected in the rough microsomes and 1.13-1.14 g/ml (B2, B3, and B4, respectively). These (Bi) (Fig. 3A). After a 60-min chase at 37°C, the total consisted predominantly of smooth vesicles as shown by radioactivity recovered from the gradient was decreased by electron microscopy of embedded pellets. B2 also contains =70%, indicating efficient transport of labeled proteins to transitional endoplasmic reticulum elements (part rough/part condensing vacuoles and zymogen granules (Table 1). Under smooth vesicles). When larger amounts of material were these conditions, a small peak of residual radioactivity was loaded on the gradient an additional fifth band (-1.12 g/ml) still observed in the rough microsomes, and there were minor was observed. The major peak of galactosyltransferase peaks of radioactivity associated with each of the three activity, which serves as a marker for trans Golgi elements smooth vesicle (Golgi) fractions (Fig. 3A). Table 1. Distribution of protein radioactivity among cell fractions isolated from pulse-labeled pancreatic cells chased at different temperatures Subcellular fraction Zymogen Postmicrosomal Recovery, Incubation conditions Microsomes granules Mitochondria supernatant % 2-min pulse 68.9 ± 6.8 5.0 ± 1.6 9.7 ± 5.8* 16.3 ± 1.0 81.5 ± 3.0 2-min pulse + 60-min chase at 16'C 64.9 ± 5.0 7.0 ± 2.9 10.2 ± 4.7 17.9 ± 2.6 82.2 ± 8.2 2-min pulse + 60-min chase at 20'C 60.7 ± 1.8 8.0 ± 2.2 15.0 ± 0.8 16.3 ± 3.1 83.2 ± 2.1 2-min pulse + 60-min chase at 370C 19.8 ± 1.0 48.1 ± 3.6 5.6 ± 1.4 26.5 ± 5.2t 92.8 ± 8.5 Pancreatic lobules were pulse-labeled with [35S]methionine for 2 min at 370C and homogenized immediately after the pulse or after a 60-min chase at 16, 20, or 370C in the presence of a 50-fold excess of nonradioactive methionine. Radioactivities measured in the cell fractions are expressed as percentages of total activity recovered in the postnuclear supernatant (column 6). Values represent mean ± SD; n = 3. *The radioactivity in the 7800 x g pellet enriched in mitochondria is due to contaminating microsomes. tThe increase is most probably due to leakage from zymogen granules during homogenization (16). Downloaded by guest on September 26, 2021 6428 Cell Biology: Saraste et al. Proc. Natl. Acad. Sci. USA 83 (1986) After a 60-min chase at 16WC, most of the newly synthe- included (i) a striking increase in the number of small sized proteins remained in the rough microsomal band (Fig. peripheral vesicles located on the cis side ofthe Golgi stacks; 3B), but a peak or shoulder of radioactivity appeared in the (it) swelling and partial fragmentation and disorganization of heaviest ofthe smooth vesicle fractions (B2). B2 (1.17 g/ml), the stacked Golgi cisternae; and (iil) a decrease in the which was well separated from the galactosyltransferase number, size, and density of content of condensing vacuoles peak (1.13-1.14 g/ml, Fig. 2B), typically contained 15-20% on the trans side of the Golgi stacks. Other changes, which of the total radioactivity in the gradient. A minor shoulder were often but not always encountered, included (iv) partial was also noted in association with B3. Thus, at 16WC the loss of the characteristic vesicular protrusions of transitional advancing front of the cohort of secretory proteins reaches a endoplasmic reticulum elements, (v) an increase in the fraction consisting of smooth endoplasmic reticulum and number of small vesicles on the trans side ofthe Golgi stacks, transitional elements. and (vi) the appearance. of small fibrillar deposits on the cis After a 60-min chase at 20'C, another peak ofradioactivity side of the Golgi stacks. These deposits were similar to, but appeared in association with B3 with a minor shoulder in smaller than, those described in anoxic pancreatic acinar association with B4 (Fig. 3B). About 40% of the total cells (24, 27); moreover, they were often surrounded by small radioactivity recovered from the gradient was reproducibly vesicles. In contrast to the situation in anoxic cells, inhibition detected in the smooth membrane fractions. It appears that of protein transport at low temperature did not result in an at 200C, the cohort of secretory proteins can reach a second increase of either coated vesicles or rigid lamellae on the Golgi compartment (B3) beyond the one reached at 16WC. trans side of the Golgi stacks. In some acinar cells, the This compartment probably corresponds to middle Golgi endoplasmic reticulum cisternae appeared locally collapsed elements that fractionate at a slightly higher density than with their cisternal space apparently obliterated by mem- trans Golgi elements (B4), marked by galactosyltransferase brane fusion (Fig. 4). (18-20). Even at this temperature, the majority (60%) of the radioactivity remains in the rough microsomal subfraction. DISCUSSION Morphologic Changes in Cells Incubated at 16'C. Electron microscopy of thin sections was used to compare the orga- We have looked at the effect of reduced temperature on the distribution of pulse-labeled proteins in a series of cell nization of pancreatic acinar cells incubated for 60 min at 37 fractions obtained from pancreatic lobules incubated in vitro. or 16°C. By comparison to the former, the latter showed a The fractions represent the sequential compartments of the number of structural modifications affecting primarily the endoplasmic reticulum-plasmalemma pathway along which Golgi complex (Fig. 4). Changes found in practically all cells newly synthesized secretory proteins are transported (13). The use oftotal protein radioactivity to monitor the transport A of secretory proteins is justified since in pancreatic exocrine cells >90% of the total protein production is destined for secretion (16). Since transport ofindividual pancreatic secre- tory proteins out of the endoplasmic reticulum occurs at different rates (28), it can be assumed that with the approach used we are looking primarily at the advancing front of the 20 cohort of secretory proteins synthesized during the pulse, Our findings show that transport of proteins out of the rough endoplasmic reticulum is severely but not completely inhibited at 16'C: 80-85% of the total protein radioactivity

LAJ remains associated with the rough endoplasmic reticulum fraction. Yet, under our conditions (60-min chase), the advancing front (15-20% of total) reaches a first station 10lo0 consisting predominantly of transitional endoplasmic reticu- lum elements and smooth vesicular elements of relatively high density (1.17 g/ml). At 20TC transport out of the rough endoplasmic reticulum is still inhibited (60% of the protein 20 radioactivity remains associated with the rough microsomal subfraction), but the advancing front (40%o) moves further and reaches a second station consisting of smooth vesicles of intermediate density (1.15 g/ml). At 370C inhibition of trans- 5 10 15 20 25 port through the Golgi is removed, and the major fraction of FRACTION pulse-labeled protein reaches the condensing vacuoles and FIG. 3. Analysis of transport of secretory proteins at reduced zymogen granules. We also obtained results showing that at temperatures. Pancreatic lobules were labeled for 2 min with temperatures below 20'C stimulated exocytosis of radioac- [35S]methionine and chased for 60 min at 37, 20, or 16°C. A total tive secretory proteins stored in zymogen granules is severely microsomal fraction prepared from the labeled cells was subfraction- inhibited or blocked. Thus, progression of secretory proteins ated as in Fig. 2, and aliquots of the 25 fractions collected were from one station to the next the has assayed for protein radioactivity. (A) Distribution of radioactivity at along secretory pathway the end of a 2-min pulse (o) and after a 2-min pulse followed by a different temperature-dependent characteristics, and each 60-min chase at 37°C (e). (B) Distribution of radioactivity at the end step appears to have its own temperature threshold. of a 2-min pulse at 37°C followed by 60-min chase at 16 (o) or 20°C Morphological observations and cell fractionation data (e). Arrows indicate the locations of the visible bands (B1-B5) as suggest that the first site at which temperature inhibition shown in Fig. 2. At the end ofthe pulse the incorporated radioactivity occurs is at the level of transitional (part rough and part was associated with rough microsomes (Bi), whereas after chase at reticulum elements or 16, 20, and 37°C it appeared in one (B2), two (B2 and 3), and three smooth) endoplasmic peripheral (cis) (B2-4) smooth membrane subfractions, respectively. Recovery of Golgi vesicles, shown previously to be involved in the radioactivity from the gradients was 90-100%, and negligible transport of pancreatic secretory proteins from the rough amounts ofradioactivity were associated with the small pellets found endoplasmic reticulum to the Golgi complex (10). Our mor- at the bottom of the tubes after centrifugation. phological findings indicate that at 16TC there is an accumu- Downloaded by guest on September 26, 2021 Cell Biology: Saraste et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6429

CV cv CV cv '

trans .)

I y~~~~~~~~~~~te Gc /7"~~~~~~~'~tte I $~~~~~~~~~~~~~~~~~~~~~~~~ teCS t. ~- *te~~C"-~~~ er ,. ~~. \>~~ .~~ ~ Y A'e

FIG. 4. Morphology of the Golgi complex mn pancreatic exocrmne cells incubated for 60 nun at 16TC This electron nicrograph demonstrates the accumulation of numerous vesicles (ye) on the cis side ofthe Golgi complex in the region between the transitional elements ofthe endoplasmic reticulum (te) and the stacked Golgi cisternae, (Gc), which appear dilated and extensively fragmented. Occasional budding profiles (arrows) are seen on the transitional elements although several lack such protrusions. Small deposits of fibrillar material (*) are seen at the endoplasmic reticulum-Golgi boundary. Condensing vacuoles (cv) on the trans side of the Golgi complex are smaller and lighter than those in control cells. The arrows mark collapsed, apparently fused endoplasmic reticulum cistemnae. (x24,700.) lation of small vesicles, presumably carrier vesicles (11), cis elements on the trans side ofthe Golgi complex at 20°C (7, 8). to the Golgi stacks. The second site reached after 200C In our case, secretory proteins did not progress beyond a probably corresponds to middle elements of the Golgi com- middle Golgi subfraction after 1 hr at 20°C. This finding may plex since the advancing front of protein radioactivity accu- be accounted for by differences in experimental protocols or mulates in smooth vesicles heavier than those associated with differences in the types of cells and proteins studied. the highest specific activity for galactosyltransferase, a trans Golgi marker (18-20, 25). The identification ofthe location of This research was supported by National Institutes of Health these two early (endoplasmic reticulum to Golgi and intra- Grants AM17780 (to M.G.F.) and GM27303 (to G.E.P.). J.S. was Golgi) blocks is by necessity tentative and remains to be supported by European Molecular Biology Organization Long-Term confirmed by further supportive evidence (e.g., electron Fellowship ALTF 270-1986. microscopic autoradiography, immunocytochemistry). 1. Sandwig, K. & Olsnes, S. (1979) Exp. Cell Res. 121, 15-25. Since vesicular transport is known to be involved in most 2. Dunn, W. A., Hubbard, A. L. & Aronson, N. N. (1980) J. Biol. Chem. if not all steps along the secretory pathway-i.e., endoplas- 255, 5971-5978. mic reticulum to cis Golgi subcompartments, between Golgi 3. Marsh, M., Bolzau, E. & Helenius, A. (1983) Cell 32, 931-940. 4. Lagunoff, D. & Wan, H. (1974) J. Cell Biol. 61, 809-811. subcompartments, and from the trans Golgi subcompart- 5. Rotundo, R. L. & Fambrough, D. M. (1980) Cell 22, 595-602. ments to the cell surface (29), we assume that our findings 6. Matlin, K. & Simons, K. (1983) Cell 34, 233-243. define differential temperature inhibition of vesicular trans- 7. Saraste, J. & Kuismanen, E. (1984) Cell 38, 535-549. port between different, successive steps. Either a shortage of 8. Griffiths, G., Pfeiffer, S., Simons, K. & Matlin, K. (1985) J. Cell Biol. 101, 949-964. ATP (10) or changes in membrane viscosity or other (un- 9. Saraste, J. & Hedman, K. (1983) EMBO J. 2, 2001-2006. known) factors could be responsible for the inhibition of 10. Jamieson, J. D. & Palade, G. E. (1967) J. Cell Biol. 34, 577-596. vesicular transport. However, it should be kept in mind that 11. Jamieson, J. D. & Palade, G. E. (1967) J. Cell Biol. 34, 597-615. the morphologic changes found at low (16°C) temperature are 12. Jamieson, J. D. & Palade, G. E. (1968) J. Cell Biol. 39, 589-603. 13. Palade, G. E. (1975) Science 189, 347-358. distinct from those described in anoxic cells that are caused, 14. Scheele, G. A. & Palade, G. E. (1975) J. Biol. Chem. 250, 2660-2670. at least in part, by ATP shortage and involve more extensive 15. Scheele, G. A. (1983) Methods. Enzymol. 98, 17-28. structural changes in the Golgi complex. The latter include 16. Scheele, G. A., Tartakoff, A. M. & Palade, G. E. (1978) J. Cell Biol. 78, replacement of vesicles on the cis side of the Golgi stacks 110-130. 17. Knipe, D. M., Baltimore, D. & Lodish, H. F. (1977) J. Virol. 21, with large fibrillar deposits, a large increase in the number of 1128-1139. coated vesicles, and the appearance of "rigid lamellae" on 18. Dunphy, W. G., Fries, E., Urbani, L. J. & Rothman, J. E. (1981) Proc. the trans side of the stacks (24, 27). Natl. Acad. Sci. USA 78, 7453-7457. 19. Goldberg, D. E. & Kornfeld, S. (1983) J. Biol. Chem. 258, 3159-3165. Our results are in agreement with previous immunocyto- 20. Dunphy, W. G. & Rothman, J. E. (1983) J. Cell Biol. 97, 270-275. chemical studies (7) indicating that at 15°C viral membrane 21. Blobel, G. & Potter, M. (1968) Biochim. Biophys. Acta 166, 48-57. proteins accumulate in an intermediate compartment located 22. Tate, S. S. & Meister, A. (1974) J. Biol. Chem. 249, 7593-7602. and 23. Brew, K., Shaper, J., Olsen, K., Trayer, I. & Hill, R. (1975) J. Biol. between the rough endoplasmic reticulum Golgi stacks. Chem. 250, 1434-1444. They are also in agreement with the results of previous 24. Merisko, E. M., Fletcher, M. & Palade, G. E. (1986) Pancreas 1, studies showing that transport to zymogen granules in pan- 95-109. creatic exocrine cells (30) and transport of secretory (5) and 25. Roth, J. & Berger, E. G. (1982) J. Cell Biol. 93, 223-229. 26. Castle, J. D., Cameron, R. S., Patterson, P. L. & Ma, A. K. (1985) J. membrane (5-7) proteins to the cell surface in other cell types Membr. Biol. 87, 13-26. is inhibited at temperatures <20°C. They differ, however, 27. Merisko, E. M., Farquhar, M. G. & Palade, G. E. (1986) Pancreas 1, from results published on the temperature dependence ofthe 110-123. 28. Scheele, G. A. & Tartakoff, A. M. (1985) J. Biol. Chem. 260, 926-931. transport ofviral membrane glycoproteins that were found to 29. Farquhar, M. G. (1985) Annu. Rev. Cell Biol. 1, 447-488. accumulate in trans Golgi cisternae and vesicular and tubular 30. Tartakoff, A. M. (1985) J. Cell Biol. 101, 220a (abstr.). Downloaded by guest on September 26, 2021