INTRACELLULAR TRANSPORT OF SECRETORY

PROTEINS IN THE PANCREATIC EXOCRINE CELL,

II. Transport to Condensing Vacuoles and Zymogen Granules

JAMES D. JAMIESON and GEORGE E. PALADE

Front The Rockefeller tUniversity, New York 10021

ABSTRACT

In the previous paper we described an in vitro system of guinea pig pancreatic slices whose secretoryproteins can be pulse-labeled with radioactive amino acids. From kinetic experiments performed onsmooth and rough microsoines isolated by gradient centrifugation from such slices, we obtained directevidence that secretory proteins are transported from the cisternae of the rough endoplasmic reticulumto condensing vacuoles of the Golgi complex via small vesicles located in the periphery of the complex.Since condensing vacuoles ultimately become zyniogen granules, it was of interest to study this phaseof the secretory cycle in pulse labeled slices. To this intent, a zymogen granule fraction was isolated bydifferential centrifugation from slices at the end of a 3-min pulse with leucine-1 4C and after varyingtimes of incubation in chase medium. At the end of the pulse, few radioactive proteins were found inthis fraction; after +17 min in chaser, its proteins were half maximally labeled; they became maxi-mally labeled between +37 and +57 min. Parallel electron microscopic radioautography of intactcells in slices pulse labeled with leucine-3H showed, however, that zymogen granules become labeled,at the earliest, +57 min post-pulse. We assumed that the discrepancy between the two sets of resultswas due to the presence of rapidly labeled condensing vacuoles in the zymogen granule fraction. Totest this assumption, electron microscopic radioautography was performed on sections of zymogengranule pellets isolated from slices pulse labeled with leucine-3H and subsequently incubated in chaser.The results showed that the early labeling of the zymogen granule fractions was, indeed, due to thepresence of highly labeled condensing vacuoles among the components of these fractions.

INTRODUCTION

In the preceding paper (Jamieson and Palade,1967), we have described the functional andmorphological characteristics of an in vitro systemof guinea pig pancreatic slices in which the secre-tory proteins produced by the acinar cell can beconveniently labeled with radioactive aminoacids in short, well defined pulses. Because ofsuch labeling, the kinetics of intracellular transportof newly synthesized secretory proteins can beprofitably investigated. Rough and smooth micro-

:L In this paper, in vitro will refer to incubated pan-creatic slices while in vivo will refer to the gland insitu, i.e., in the animal.

somal fractions were isolated from the incubatedslices by isopycnic centrifugation in a continuoussucrose density gradient. The first fraction consistsof healed fragments of the rough endoplasmicreticulum (ER), while the second representsprimarily the peripheral elements of the Golgicomplex. Kinetic experiments performed on thesemicrosomal subfractions after 3 min of pulselabeling and after various times of incubation in achase medium indicated that newly synthesizedsecretory proteins were transported from theirsite of synthesis in the rough ER to cumuli ofsmooth-surfaced vesicles at the periphery of theGolgi complex.

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Since in vivo, secretary proteins are transportedto, and finally stored in zymogen granules (Kellerand Cohen, 1961; Greene, Hirs, and Palade, 1963),experiments were performed on pulse-labeledslices to determine if acinar cells could carry outin vitro the remaining steps of intracellular trans-port, viz., the transfer of secretary proteins to thecondensing vacuoles of the Golgi complex and theirstorage in zymogen granules prior to discharge.The results of combined cell fractionation andradioautographic studies presented in this papershow that the acinar cells of pancreatic slices cancarry out all the operations involved in the secre-tory process up to and including the discharge ofthe zymogen granule content into the ductsystem.

METHODS

Techniques for preparing and pulse-labeling guineapig pancreatic slices, for isolating cell fractions fromhomogenized slices, and for assaying these fractionschemically and radiochemically are described in theprevious paper (Jamieson and Palade, 1967).

Slices to be used for light and electron micro-scopic radioautography were pulse labeled in incuba-tion medium containing 200 pc/ml L-leucine-4,5-3H(40 pM) and incubated post-pulse in a chase mediumcontaining L-leucine-lH at a concentration of 2.0nmM (50X excess). Light and electron microscopicradioautography were performed as described byCaro and van Tubergen (1962) on tissue fixed andembedded in Epon as outlined in the precedingpaper.

LIGHT MICROSCOPIC RADIOAUTOGRAPHY:0.5-p thick Epon sections were mounted on glassslides and coated with Ilford L-4 emulsion. Thepreparations were exposed for 4-7 days at 4°. Afterphotographic processing, the sections were stainedthrough the emulsion with 1 % methylene blue in1% sodium borate (Richardson, Jarett, and Finke,1960), a procedure which does not remove or dis-place the silver grains. The preparations were coatedwith a clear plastic aerosol spray (Jennings, Farqu-har, and Moon, 1959) and were examined and photo-graphed under the oil immersion lens using bright-field illumination. With this technique, a satisfactoryimage of both tissue structure and radioautographicgrains can be obtained.

ELECTRON MICROSCOPIC RADIOAUTOGRA-PHY: Thin sections of cells or cell fractions weremounted on grids covered with a carbon-coatedformvar film. Ilford L-4 emulsion was applied bythe loop method (Caro and van Tubergen, 1962).Exposure times were from 2 4 wk. After photo-graphic processing, the sections were doubly stainedwith uranyl acetate and lead citrate (Venable and

TABLE IChemical Composition of Zymogen Granule Fractions

/~g phos-mg RNA/mg pholipid-P

Source protein mg protein

Guinea pig* pancreatic slice 0.01-0.03 1.3-3.3Guinea pigj 0.16 -Dog§ 0.005 1.1OxJl 0.006 2.2Mouse¶ 0.04

* These values are the range from three experi-ments.tSiekevitz and Palade, 1958 a (isolated by dis-continuous gradient centrifugation).§ Hokin, 1955.

Greene et al., 1963.¶ Van Lancker and Holtzer, 1959.

Coggeshall, 1965). This staining schedule does notproduce any detectable loss or displacement of silvergrains.

MATERIALS: L-leucine-4,5-Hl (specific activity5 c/millimole) was obtained from New EnglandNuclear Corporation, Boston. Ilford Nuclear Re-search emulsion L-4 was obtained from Ilford Limited,Ilford, Essex, England.

RESULTS

Intracellular transport in the slice system wasinvestigated in parallel by cell fractionation andradioautography.

Cell Fractionation StudiesISOLATION AND CHARACTERIZATION OF

THE ZYMOGEN GRANULE FRACTION: So thatwe could follow the transport of secretary proteinsinto zymogen granules, it was necessary to isolatethese granules in a relatively clean cell fraction.The method used is described in the precedingpaper (Jamieson and Palade, 1967) and is based,with minor modifications, on that proposed byHokin (1955) for the isolation of a zymogengranule fraction from the dog pancreas.

CHEMICAL COMPOSITION OF THE ZYMOGENGRANULE FRACTION: Table I gives the RNA/protein ratio of the zymogen granule fractionisolated by us from guinea pig pancreatic slicesand compares it with that of zymogen granulefractions isolated from guinea pig pancreas bydiscontinuous gradient centrifugation, and fromdog, ox, and mouse pancreas by differentialcentrifugation. RNA was determined in all cases

598 TIE JOURNAL OF CELL BIOLOGY VOLUME 84, 1967

FIGURE 1 Low magnification electron micrograph of the zymogen granule fraction. This field is repre-sentative of all levels in the pellet. Zymogen granules (z) comprise the majority of structures. A smallerpopulation ( 5) of condensing vacuoles (arrows), identified by their scalloped profiles, content oflesser electron opacity, or both features, can be recognized in the fraction. A few mitochondlria are seen(m). X 3,000.

by the orcinol reaction (Mejbaum, 1939). Insofaras the RNA content reflects residual microsomalcontamination, the zymogen granule fractionisolated from pancreatic slices compares favorablyto fractions isolated from dog and ox pancreas andis noticeably less contaminated than that isolatedby density gradient centrifugation. The phos-pholipid-phosphorus content of the fractions isgiven for comparison.

MORPHOLOGY OF THE ZYMOGEN GRANULE

FRACTION: Thin sections of fixed and embed-ded pellets of this fraction, cut to include the entiredepth of the pellet in the axis of centrifugation,show that it consists mainly of zymogen granuleswith a few contaminating mitochondria (Fig. 1)and rough microsomes (Fig. 2). The contaminantswere evenly scattered from top to bottom through-out the pellet, which did not show any detectablestratification. Zymogen granules isolated fromincubated slices were identical to those prepared

from fresh pancreatic tissue (Siekevitz and Palade,1958 a; Greene, Hirs, and Palade, 1963) and werecharacterized by their spherical shape, highlydense, homogeneous, and apparently amorphouscontent, and a limiting unit membrane whichmeasures 100 A in thickness, and which can beseen only when sectioned normally.

In addition to zymogen granules, the pelletcontains bodies of comparable size but of irregularshape which gives them angular or scallopedprofiles (Figs. 1, 2). Like zymogen granules, theyare limited by a unit membrane and have anamorphous, homogeneous content of equal orlower density. On account of their morphology,these bodies are identified as condensing vacuolesof the Golgi complex. They represent -5% of thetotal particle population of the zymogen granulefraction.

KINETICS OF LABELING OF THE ZYMOGEN

GRANULE FRACTION: To determine the ki-

J. D. JAMIESON AND G. E. PALADE Intracellular Transport of Secretory Proteins. II 599

FIGITIrE Higher magnification field from the zynlo-genl granule fraction. A few conltallinating rough micro-somes (rm) are seen. c, colldensing vacuoles; z, zymo-gen granule. X 23,000.

netics of labeling of the zymogen granule fraction,we pulse labeled sets of slices as before (Jamiesonand Palade, 1967) with leucine-14 C and fraction-ated them at the end of the pulse and after varioustimes of incubation in chase medium. For theseexperiments, the fractionation scheme was sim-plified to the extent that only a zymogen granulefraction and a total microsomal fraction wereisolated by differential centrifugation.

1. LABELING OF CELL FRACTIONS DURING

1-HR INCUBATION: The results of a typicalshort-term experiment are presented in Fig. 3. Thespecific radioactivity of the proteins of the zymo-gen granule fraction is negligible initially (i.e.,compared to that of microsomal proteins); after+17 min of incubation in chase medium, 2 itsproteins are half-maximally labeled and becomemaximally labeled between +37 and +57 minof incubation. In other cell fractionation experi-ments not shown, a small but significant amount oflabeled protein appeared in the zymogen granulefraction as early as +7 min following the pulse.

2 The times of incubation in chase medium following3-min pulse labeling will be referred to hereafter as+17 min of incubation, +37 min of incubation, etc.

The specific radioactivity curve of microsomalproteins declines rapidly after the pulse. Theunchanging specific activity of total homogenateproteins indicates an effective chase. The specificactivity of the proteins of the post-microsomalsupernate remains constant throughout incuba-tion, signifying negligible transfer of labeled pro-teins from cell particulates to the cytoplasmicmatrix, i.e., the cell compartment which is themain contributor to the post-microsomal sper-nate. After +17 min of incubation, labeled pro-teins begin to appear in the incubation medium.

2. LABELING OF CELL FRACTIONS DURING

3 HR OF INCUBATION: Fig. 4. shows the ki-netics of labeling of proteins in cell fractionsisolated from sets of pulse-labeled slices incubatedin chase medium for +1, +2, and +3 hr. Asbefore, the specific radioactivity curve of micro-somal proteins falls rapidly during the chase.Labeling of proteins of the zymogen granulefraction is essentially complete after +1 hr ofincubation, rises slightly during the next hr, andthen begins to fall. The specific radioactivity ofproteins in the incubation medium shows a slowprogressive rise during 3 hr. The specific radio-

12,000

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Homogenote:........--

-A _ o____, ___----- P-M sup./ ½__-.-------- - _""

_ / _____End Smin pulse 17 +37 +57

f Minutes incubation in chose

FIGtoIrE 3 Specific radioactivity of proteins in cellfractions isolated front pancreatic slices pulse labeledfor 3 min and incubated up to +57 nin i chase. Pulsemedium: Krebs-Ringer bicarbonate, supplemented withamino acids (inus leucine-12 C) and containing 1 c/nl(4 AM) L-leucine- 1 4C. Chase medium: as above with L-leucine 12 C, 0.4 IM. P-M supt., post microsoimalsupernate.

600 THE JOURNAL OF CELL BIOLOGY VOLUME 34, 1967

Mlirosome

F

reported that in vivo longer times elanse (-150min) before labeled proteins appear in the pan-creatic juice.

Radioautographic StudiesIntracellular transport was also studied by

radioautography taking advantage of the factthat the slice system allows heavy labeling ofsecretory proteins during a short (3-min) pulsewith L-leucine-4,5-3H. The conditions are dis-tinctly better than in the whole animal in whichneither an equally high dose of labeled amino

-AJ ... . _. ___:_A .... .I I.J __1_ + 1-End 3-min pulse +1

f+2

Hours incubation in cho

FIGotRE 4 Labeling of cell fractionslabeled slices during 3 hr of incubation. Foof Illedia, see Fig. 3.

activity of proteins in both the totaland the post-microsomal supernate rstant throughout incubation.

These cell fractionation data indiczymogen granule fraction begins toradioactive proteins as early as +pulse labeling and that it becomeslabeled after +37- +57 min of incwas demonstrated in the previous papeand Palade, 1967), secretory proteinferred in time from the cisternae of thto the small vesicles at the peripherycomplex: the labeled proteins whichin the zymogen granule fraction have 'fore, from these small vesicles. Thelabeling of zymogen granule fractionssimilar to those of labeling of the satin vivo, as shown by data obtained iipig (Siekevitz and Palade, 1958 b) anpancreas (Morris and Dickman, 196(1959) found, however, a somewhavivo labeling of zymogen granuleguinea pig pancreas.

After +20 min of incubation, Iteins begin to appear in small amcincubation medium. Although sonprobably represent leakage from dalespecially at the early times, the radiostudies reported later in this paper iexocrine cells in vitro are able to dischproteins in the acinar lumina beginhr after pulse-labeling. Junqueira,Rothschild (1955) and Hansson

+3 acius can De amnistered nor an equany enec-tive chase can be achieved.

The results obtained on slices can be used tofrom pulse- complete and correct-if necessary-previouscoiiiposition findings on the intact animal. In addition, they

represent an independent line of evidence neededfor a reliable interpretation of the kinetic data

homogenate obtained from cell fractionation studies.emains con- INCUBATION CONDITIONS: Slices were

pulse labeled for 3 min with L-leucine- 3 H (200ate that the gc/ml; 40 UM) and incubated post-pulse foraccumulate further +7, +17, +37, +57, and +117 min

7 min after in chase medium containing 2.0 mM unlabeled; maximally L-leucine (50x excess). Determinations of TCA-ubation. As soluble and insoluble radioactivity on homoge-:r (Jamieson nates of these slices at the end of the pulse andIs are trans- after chase incubation indicated that even with.e rough ER this relatively massive dose of tracer, a true pulseof the Golgi label was obtained.accumulate To ascertain that the incorporated label is

come, there- retained in the tissue during processing for elec-kinetics of tron microscopy, we treated specimen blocks

in vitro are from slices at the end of the pulse and at variousme fractions times during the chase as follows. After fixation,n the guinea dehydration, and infiltration with Epon, thed the mouse blocks were hydrolyzed in 1 N NaOH (with heat-0). Hansson ing) and aliquots assayed for protein and radio-.t slower in activity. It was found that the specific radioac-fractions in tivity of proteins from these blocks was identical

to that of proteins from slices labeled under thelabeled pro- same conditions but homogenized, precipitatedmunts in the with TCA, and assayed for incorporated radio-ne of them activity in the usual way. These results indicatermaged cells, that the tissue-processing schedule extracts negli-)autographic gible protein-associated radioactivity although itndicate that effectively washes out soluble, nonincorporatedarge labeled label (cf. Caro and Palade, 1964). We assume,iing about 1 then, that the radioautographic grains mark onlyHirsch, and label incorporated into protein.1959) have LIGHT MICROSCOPIC RADIOAUTOGRAPHY:

J. D. JAMIESON AND G. E. PALADE Intracellular Transport of Secretory Proteins. II 601

rE

q)

... _ _ , .. .

FIGURES 5-9 Light microscopic radioautograph of slices pulse labeled for 3 min with L-leucine-3 H (Fig.5) and incubated in chase for further +17 min (Fig. 6), +37 min (Fig. 7), +57 min (Fig. 8), and +117min (Fig. 9). B, base of cell; C, centrosphere region (Golgi complex); L, acinar lumen; D, collecting duct;A, cell apex with zymogen granules. Exposure time 7 days. X 1,750.

Epon sections, 0.5 thick, from tissue at the endof the pulse (3-min) and after +7, +17, +37,+57, and + 117 min of incubation in chase me-dium were prepared for radioautography as de-scribed under Methods.

At the end of the pulse (Fig. 5), radioauto-graphic grains were generally localized over thebasal cytoplasm of the exocrine cell. No grainswere seen to overlie either the centrosphereregion (Golgi complex) or zymogen granules.

The few grains among zymogen granules prob-ably marked rough ER elements scattered in theapical regions of the cell. After +7 min, and es-pecially after + 17 min (Fig. 6), however, fewgrains were seen over the basal cytoplasm; themajority were located centrally in the cell andwere often seen to outline the pale-staining cen-trosphere region. After +37 min, the grains wereeven more concentrated over the centrosphereregion of the cells (Fig. 7) with few grains re-

602 THE JOURNAL OF CELL BIOLOGY . VOLTME 34, 1967

maining over the basal region. At this time,zymogen granules in the apical region of the cellswere generally unlabeled. At +57 min (Fig. 8),the zymogen granules in the cell apex began toaccumulate label and after + 117 min werehighly labeled. At this time, numerous grainswere also found over acinar lumina and largercollecting ducts of the gland (Fig. 9).

From these results, it is clear that the initialsite of incorporation of label in the pancreaticacinar cell is the basal ergastoplasm. During post-pulse incubation in chase medium, the labeledproteins move as a well defined wave through thecells and eventually appear in large amounts inthe duct system after 2 hr of incubation.

It should be noted that acini were not uniformlylabeled throughout the thickness of the slice. Agradient of labeling from the periphery to thecenter of the slice was found, though only a fewacini, farthest from the surface of the slice, werefree of label. Apparently, the thickness of theslice does, to some extent, present a barrier to thepenetration of label. Nevertheless, at any time thelocation of grains over cell structures was thesame throughout the depth of the slice, indicatingthat the penetration barrier merely affected the

intensity of the radioautographic response with-out introducing asynchronous labeling. Thus, thegradient does not alter the intracellular trans-port schedule.

ELECTRON MICROSCOPIC RADIOAUTOG-

RAPHY: Since the resolution achieved by lightmicroscopic radioautography does not allow usto determine the relationship of silver grains tothe individual elements of the centrosphere andapical regions, electron microscopic radioautog-raphy was performed on thin sections cut fromthe same material used for light microscopicradioautography.

At the end of the pulse (Fig. 10), the radio-autographic grains were localized exclusivelyover elements of the rough ER. No grains wereseen over elements of the Golgi complex or zymo-gen granules.

After +7 min (Fig. 11), although the majorityof the label still marked the rough ER, radio-autographic grains began to appear over theclusters of small, smooth-surfaced vesicles locatedat the periphery of the Golgi complex. Little ofthe label progressed as far as condensing vacuolesat this time.

At + 17 min, the situation was intermediate

J. D. JAMIESON AND G. E. PALADE Intracellular Transport of Secretory Proteins. II 603

FIGIIHE 10 Electron microscopic radioautograph of an acinar cell at the end of pulse labeling for 3min with L-leucine-31I. The radioautographic grains are located almost exclusively over elements of therough ER (rer). A few grains partly overlie mitochondria but, for reasons discussed in the text, mostlikely label the adjacent rough ER. m, nmitochondrion; N, nucleus. X 17,000.

604

FIGURE 11 Electron microscopic radioautograph of an acinar cell after +7 min of incubation post-pulse. The majority of the label marks the periphery of the Golgi complex (arrows) with little remainingin the rough ER (rer). X 17,000.

J. D. JAMIESON AND G. E. PALADE Intracellular Transport of Secretory Proteins. II 605

between that at +7 and +37 min: most of thelabel had migrated from the basal rough ER andwas localized over the periphery of the Golgicomplex, while part of it had already reachedcondensing vacuoles.

After +37 min (Fig. 12), few grains were stillfound at the periphery of the Golgi complex;most of them now were preferentially and highlyconcentrated over the condensing vacuoles of thecomplex. At this time, a small number of grainswas already located over zymogen granules.

After +57 min, a larger proportion of the labelwas found over zymogen granules, especially thoseadjacent to the Golgi region. Some label re-mained over the condensing vacuoles though fewgrains still labeled the periphery of the Golgicomplex. By + 117 min (which was the longesttime point examined by radioautography), thegrains heavily labeled zymogen granules groupednear the cell apex and numbers of them werepresent over fibrous material (presumably dis-charged secretory protein) in the acinar luminaand collecting ducts of the gland (Fig. 13). Thecondensing vacuoles of the Golgi complex werenow practically devoid of label.

At all times longer than + 17 min, a smallnumber of grains persisted over the regions of thecell occupied by rough ER. No significant labelingof centroacinar cells, islet cells, or duct cells wasobserved at any time. Background label wasnegligible in all experiments.

Quantitation of radioautographic grain distri-bution over various components of acinar cellswas made at each time point by counting grainson a series of low magnification electron micro-graphs taken at random (Table II). Analysis ofthese data gives information about the quantityof labeled material in the compartment repre-sented by the corresponding cell componentrather than about the specific radioactivity of itscontent.3 Thus, grain counts cannot be compareddirectly to specific radioactivity data derivedfrom cell fractionation experiments, althoughsimilar patterns of labeling occur.

At the end of the pulse, radioautographicgrains were associated almost exclusively withthe rough ER of the acinar cell; during chase

3 It should be noted that the method of analyzingradioautographic data reported here (Table II) isvalid since the total amount of label remains con-stant in the tissue during the times of observation(efficient chase incubation).

incubation, the numbers of grains over the roughER progressively decreased. This is consistentwith the kinetics of labeling of the rough micro-somes which are derived from the rough ER (cf.Jamieson and Palade, 1967). At the end of + 117min, -20%, of the grains were still located overthe rough ER. This label probably identifies non-exportable proteins with a long turnover time(see Siekevitz and Palade, 1960; Warshawsky,Leblond, and Droz, 1963).

Concurrently with the decrease in grains overthe ER, there was a progressive rise in grainsover elements of the Golgi complex. Thus, at +7min, -477% of the grains were over this region,with 43c%/ located over the small vesicles at theperiphery of the complex. By +17 min, -58%of the grains were located over the Golgi complex,with the majority still clustered over peripheralelements of the complex. Following +37 min ofincubation, the majority of the label (-49,%)) hadmigrated into condensing vacuoles. At this time,a small but significant number of zymogen gran-ules was labeled. Labeling of the zymogen gran-ules progressively increased during the next twotime points examined (+57 and +117 min)with an over-all decrease over condensing vacu-oles. During the same times, a significant per-centage of the grains was located in the acinarlumina.

At all time points, the labeling of mitochondriaand nuclei was low and generally decreasedduring the chase.

From radioautography we can conclude thatpancreatic acinar cells incubated in vitro areable to perform the entire sequence of operationsinvolved in the secretory cycle and that the time-table of these events is similar to that establishedin the whole animal by the radioautographicstudies of Caro and Palade (1964).

According to our radioautographic data, at+37 min only a small proportion of the labelhad progressed as far as zymogen granules, themajority being located over condensing vacuoles.From the labeling kinetics of the zymogen granulefraction (cf. Fig. 3), however, protein radioactiv-ity appears in this fraction as early as + 17 minand the fraction is maximally labeled between+37 and +57 min. Because zymogen granulefractions are known (this work and Greene et al.,1963) to contain a small population of condensingvacuoles, we assumed that this discrepancy wasonly apparent and reflected the presence of con-

606 THE JOURNAL OF CELL BIOLOGY VOLurMFE 34, 1967

FIGURE 12 Portion of an acinar cell pulse labeled as before and incubated post-pulse for +37 min.Arrows indicate the periphery of the Golgi complex. Radioautograplhic grains are highly concentratedover condensing vacuoles (cv) of the Golgi complex. Zymogen granules (z) are unlabeled. X 13,000.

J. D. JAMIESON AND G. E. PALADE Intracellular Transport of Secretory Proteins. II 607

FIGTRE 13 Electron microscopic radioautograph of several acinar cells incubated post-pulse for +117min. Radioautographic grains are located primarily over zymogen granules near the cell apex. Con-densing vacuoles (cv) are practically devoid of label. Note residual labeling of the rough ER (arrows).Some label marks secretory material in the acinar lumen (L). X 13,000.

608

TABLE II

Distribution of Radioautographic Grains over Cell Components

% of radioautographic grains

3-min Chase incubation(pulse) +7 min +17 min +37 min +57 min +117 min

Rough endoplasmic reticulum 86.3 43.7 37.6 24.3 16.0 20.0Golgi complex*

Peripheral vesicles 2.7 43.0 37.5 14.9 11.0 3.6Condensing vacuoles 1.0 3.8 19.5 48.5 35.8 7.5

Zymogen granules 3.0 4.6 3.1 11.3 32.9 58.6Acinar lumnen 0 0 0 0 2.9 7.1Mitochondria 4.0 3.1 1.0 0.9 1.2 1.8Nuclei 3.0 1.7 1.2 0.2 0 1.4

No. of grains counted 300 1146 587 577 960 1140

The boldfaced numbers indicate maximum accumulation of grains over the corre-sponding cell component.* At no time were significant numbers of grains found in association with the flat-tened, piled cisternae of the complex.

FIGURE 14 Low magnification electron microscopic radioautograph of the zymogen granule pelletderived from slices pulse labeled for 3 min with leucine-3H and incubated +37 min in chase. The arrowsindicate highly labeled condensing vacuoles. The majority of the zymogen granules is unlabeled. X3,000.

J. . JAMIESON AND G. E. PALADE Intracellular Transport of Secretory Proteins. 11 609

FIGURE 15 Higher magnification view of the same pellet seen in Fig. 14, showing the localization ofradioautographic grains over condensing vacuoles (cv). z, zymogen granules; rm, rough microsomes. X13,000.

densing vacuoles highly labeled at early timesafter the pulse. Since at present no satisfactorymethod exists for the separation of condensingvacuoles from zymogen granules, this assumptionwas tested by performing electron microscopicradioautography on thin sections of zymogengranule pellets derived from slices pulse labeledwith leucine-3H for 3 min and incubated for + 17and +37 min in chase medium.

For these studies, the conditions of pulse label-ing and incubation in chase medium were identi-cal to those used for tissue radioautography. Thinsections of zymogen granule pellets from each ofthe three time points were cut through the entiredepth of the pellet in the direction of the centrifu-gal field and mounted on bar-grids which weresubsequently processed for electron microscopicradioautography as described under Methods.Grids from the three time points were coated withemulsion at the same time (in order to ensure equaldistribution of photographic grains over the speci-mens), exposed for 34 wk, and finally processedphotographically. Sequential micrographs were

taken in the electron microscope at low magnifica-tion (X 750) so as to include the entire depth ofthe pellet.

At the end of the pulse, no radioautographicgrains were found over any component of thezymogen granule pellet. After +17 min, and es-pecially after +37 min of incubation, numerousgrains were localized over recognizable con-densing vacuoles in the zymogen granule pellets.A typical low magnification field, taken for illus-tration from the +37-min time point, is shown inFig. 14. Even at this magnification, radioauto-graphic grains are seen to be preferentially locatedover condensing vacuoles which are identified inthe pellet, as in the cell, by their angular profiles.This is seen to advantage in Fig. 15, which is ahigher magnification view of the same pellet.

To quantitate these results, we enlarged photo-graphically the low magnification micrographs to16 x 20 in. A grid of this size, subdivided into10- x 10-cm squares, was drawn on a thin plasticsheet and overlaid on the photographic prints. Thenumbers of labeled condensing vacuoles and

610 THE JOURNAL OF CELL BIOLOGY VOLUME 34, 1967

TABLE IIILabeled Condensing Vacuoles and 7ymogen Granules

in Zymogen Granule Fraction

Pulse + chaseincubation

-rin 3 + 17 3 + 37(pulse) min min

Condensing vacuoles la- 0 7.3 14.0beled (%)

Zymogen granules la- 0 0.15 0.53beled (%)

Labeled condensing vac- 0 73 65uoles as % total labeledcondensing vacuoles +zymogen granules

Condensing vacuoles as 4.95 4.06 6.60% total condensingvacuoles + zymogengranules

zymogen granules were recorded within each 10-cm square (Table III).

No labeled condensing vacuoles or zymogengranules were found in the micrographs from the3-min zymogen granule pellet. By + 17 min, 7.3 %of all condensing vacuoles were labeled and at+37 min the number had increased to 14.0%. Atthis time, the number of radioautographic grainsper vacuole was also considerably greater than at+17 min. At +17 min, and especially at +37min, a small but significant number of zymogengranules was labeled, though at these time pointsthe majority of labeled particles was condensingvacuoles. Contaminating rough microsomes andmitochondria were not labeled at any time.

These data thus support the original assump-tion that early labeling of the zymogen granulefraction is accounted for by highly labeled con-densing vacuoles and satisfactorily explain theapparent discrepancy between cell fractionationdata and electron microscopic radioautography.

DISCUSSION

General Premises Used to Interpret theExperimental Data

In interpreting the results of the experimentsreported in this and the previous paper, we haveassumed that the label identifies newly synthesizedsecretory proteins in all cell compartments exam-ined, including smooth microsomes. This assump-

tion is justified by the data of Siekevitz and Palade(1960) who showed that the rate of labeling ofsecretory proteins in the guinea pig pancreas isfive to seven times faster than that of proteinsretained in the exocrine cell; it is compatible withour radioautographic data which indicate that themajority of the label moves as a single wave fromone cell compartment to another; and it is sup-ported by the results of experiments which showthat a large and comparable amount of labeledproteins can be extracted by saline-bicarbonatefrom all fractions examined (rough and smoothmicrosomes, zymogen granule fraction). Thistreatment is known to solubilize digestive enzymesand enzyme precursors from zymogen granules(Greene et al., 1963). Incorporation of label intononexportable protein undoubtedly takes place,but can be considered negligible within the timelimits involved in these experiments.

Results of Present StudySYNTHESIS AND SEGREGATION OF SECRE-

TORY PROTEINS: In the present study, short(2 - 3-min) pulse labeling of secretory proteinscoupled with efficient chase incubation has al-lowed us to demonstrate unequivocally by elec-tron microscopic radioautography and cell frac-tionation that the single site of incorporation oflabel into proteins is in the rough ER. The generallocation of this site has been identified by radio-autography, and the structure involved-roughmicrosomes derived from the rough ER-has beenpinpointed by cell fractionation. The existence ofa second site of synthesis in the Golgi complex(Sj6strand, 1962) is excluded since, by the timelabeled proteins appear in the complex, the in-tracellular pool of soluble precursors has effec-tively been diluted. Further, the label whichappears in Golgi elements can be accounted forby label lost from the rough ER (Jamieson andPalade, 1967). The existence of a second sitecould not be ruled out by previous radioauto-graphic work on intact animals (Caro and Palade,1964). Thus, the present results put on a factualbasis the hypothesis of Siekevitz and Palade(1958 b) according to which secretory proteins aresynthesized only in the rough ER and subse-quently transported to other cell compartments.

Our radioautographic studies indicate that atthe end of the pulse only -3% of the label waslocated over mitochondria and -30% over nuclei;during incubation in the chase medium, the per-

J. D. JAMIESON AND G. E. PAI.ADE Intracellular Transport of Secretory Proteins. II 611

centage of grains located over these structuresprogressively decreased in parallel with the lossof label from the rough ER. It is, therefore, likelythat the labeling of mitochondria and nucleireflects labeling of the adjacent rough ER. Ac-cordingly, the data do not support the assumptionof Straub (1958) that mitochondria participate inthe synthesis of pancreatic secretory proteins.Similarly, the limited amount of label locatedover elements of the Golgi complex and zymogengranules at the end of the pulse should be takenas a maximum figure which reflects to a largeextent labeling of adjacent rough ER elements.

Our results do not provide further insight intoeither the actual site of protein synthesis in therough ER, or the fate of the protein immediatelyafter synthesis. Earlier evidence (Siekevitz andPalade, 1960) had indicated that -chymotryp-sinogen is synthesized on attached ribosomes, andrecently Redman, Siekevitz, and Palade (1966)have shown that another secretory protein, a-amylase, is synthesized in the attached ribosomesof pigeon pancreatic microsomes incubated invitro. In addition, these investigators have dem-onstrated that newly synthesized amylase ispreferentially transported to the content of themicrosomal vesicles, which is the in vitro equiva-lent of the content of the cisternal space. Usingliver microsomes, Redman and Sabatini (1966)have also shown that peptides labeled in vitro anddischarged by puromycin from attached ribo-somes are preferentially transported into the cis-ternal space, indicating that from the inception ofits synthesis, the peptide chain is destined to reachthis space. It can be tentatively assumed thatthese findings (Redman et al., 1966; Redman andSabatini, 1966) apply to all secretory proteinsproduced by the exocrine cell.

TRANSPORT OF SECRETORY PROTEINS

THROUGH THE PERIPHERY OF THE GOLGI

COMPLEX: The results of kinetic experimentsperformed on smooth microsomal fractions(Jamieson and Palade, 1966, 1967), isolated frompancreas slices at the end of the pulse and aftervarious times of incubation in the chase medium,provide direct evidence that secretory proteinspass through the small peripheral vesicles of theGolgi complex in transit from their site of synthe-sis and segregation in the rough ER to their siteof concentration in condensing vacuoles. Thecell fractionation data also indicate that the spe-cific radioactivity of proteins in the post-micro-somal supernate (i.e., of proteins located mainly

in the cytoplasmic matrix or cell sap) remainsconstant at all times examined, indicating negli-gible equilibration between labeled proteins ofthe particulate components of the cell and thecytoplasmic matrix.4 These results do not supportthe assumption (Laird and Barton, 1958; Red-man and Hokin, 1959; Morris and Dickman,1960) that secretory proteins leave the micro-somes and pass, in soluble form, through thecytoplasmic matrix to zymogen granules or theacinar lumen.

Cell fractionation findings are in good agree-ment with radioautographic data obtained onintact cells labeled either in slices or in the wholeanimal (Caro and Palade, 1964). The data showthat, at the time labeled protein becomes concen-trated in the smooth microsomes (between +-7and + 17 min), radioautographic grains are con-centrated over a region of the Golgi complexcontaining numerous small, smooth-surfacedvesicles; at this time, no other smooth-surfacedvesicles in the cell (e.g., small clusters of vesicleslocated among the rough ER cisternae and underthe plasmalemma, and small apical vacuoles) orpotential sources of smooth vesicles (cell mem-brane or mitochondria) are labeled. The resultsof cell fractionation and radioautography thussupport and complement each other: radio-autography indicates that the smooth micro-somal fraction must contain peripheral elementsof the Golgi complex while cell fractionationshows that the label is localized to the smallvesicles of this region rather than to the surround-ing cytoplasmic matrix. The majority of the radio-active protein in the smooth microsomes can beextracted by mild alkaline treatment, indicatingthat the label is located within the cavity of thesmall Golgi vesicles and not in their limitingmembrane.

From these combined data, it is clear that thesmall vesicles of the Golgi complex mediate thetransport of secretory proteins from the roughER to condensing vacuoles. This implies transportin bulk or in mass since each vesicle must containand carry a large number of protein molecules.

4 Froln recovery experiments, not shown in thispaper, it could be calculated that if the total labeledproteins of the microsomal fraction were transferredcompletely to the post-microsomal supernate duringchase incubation, then the specific radioactivity ofproteins in the supernate should increase 2.5- to3-fold. This did not occur (see Fig. 3).

612 TIIE JOURNAL OF CELL BIOLOGY VOLUME 34, 1967

The transport must also be discontinuous; other-wise, concentration of the solution of secretoryproteins at the next step (condensing vacuoles)would not be possible. With these restrictions inmind, we can inquire into cellular mechanismspossibly involved in this operation. It should berecalled that the transitional elements of therough ER adjacent to the periphery of the Golgicomplex are limited by part rough- and partsmooth-surfaced membranes. The latter are fre-quently in contact with smooth-surfaced vesiclesor protrude as smooth-surfaced blebs toward theGolgi complex. These blebs are comparable insize to the small, smooth-surfaced vesicles of theregion. Likewise, the smooth-surfaced membranebounding the condensing vacuoles is often incontact with small vesicles or is thrown into smallsurface blebs. These images suggest repeatedfission of, and fusion with, vesicles at both ter-minals. It can be assumed, then, that secretoryproteins are transported from the transitionalelements of the rough ER to condensing vacuolesthrough intermittent tubular connections, or,alternatively, that transport is effected by discretevesicles that shuttle between the two compart-ments. Both possibilities are compatible with thedata, but the second is more consistent with thefine structural details of the Golgi complex.

The model proposed for the transport of secre-tory proteins within the periphery of the Golgicomplex is reminiscent of the transport of macro-molecules in bulk across the vascular endotheliumby pinocytic vesicles (Palade and Bruns, 1964;Karnovsky, 1965), though in the case of the pan-creatic exocrine cell, transport in bulk operatesbetween two intracellular compartments, therough ER and the condensing vacuoles.

TRANSPORT OF LABELED PROTEINS TO

CONDENSING VACUOLES, ZYMOGEN GRAN-

ULES, AND THE ACINAR LUMEN: The radi-oautographic studies of Caro and Palade (1964)have firmly established that proteins are trans-ported to condensing vacuoles of the Golgi com-plex where they are intensively concentrated, thecondensing vacuoles being converted in theprocess into zymogen granules. We have con-firmed the position of the condensing vacuoles inthe secretory cycle by electron microscopicradioautography of pancreatic slices and havedemonstrated that the time taken to accumulatelabeled proteins within condensing vacuoles isapproximately the same in vitro as in vivo. Fur-ther, because of the short, well defined label which

passes through the cell as a single wave, the arrivaland concentration of radioactive proteins in con-densing vacuoles are more strikingly demonstratedthan was possible in the whole animal.

Earlier cell fractionation (Siekevitz and Palade,1958 b) and radioautographic data (Caro andPalade, 1964) were not in agreement concerningthe rate of transport of labeled secretory proteinsinto zymogen granules. According to the former,the zymogen granule fraction accumulates radio-active protein as early as 10 min after the injectionof tracer, whereas radioautography indicated thatzymogen granules were not labeled until -1 hrafter injection of tracer. In the present experi-ments, a similar discrepancy was observed. Sincethe zymogen granule pellet contains a smallpopulation of condensing vacuoles, we assumedthat the discrepancy was due to the presence ofearly and intensive labeling of condensing vacu-oles. We tested this possibility by electron micro-scopic radioautography of sections of the zymogengranule pellet at various times after pulse labeling.The results substantiated the assumption andpointed out the important contribution made bycondensing vacuoles to the early labeling of thezymogen granule fraction. Evidently, a correc-tion factor must be applied to previous cell frac-tionation data which indicated that zymogengranules become labeled shortly after the admin-istration of the tracer (Siekevitz and Palade,1958 b; Hansson, 1959; Morris and Dickman,1960).

It should be noted here that Schramm andBdolah (1964) have concluded, on the basis ofcell fractionation studies carried out on pulse-labeled rat parotid slices incubated in vitro, thatnewly synthesized amylase moves from the micro-somal fraction sequentially into fractions contain-ing granules of increasing size or density. Sincethe pancreatic and salivary exocrine cells aresimilar in organization (cf. Parks, 1961, 1962) itis probable that here, too, early labeling of thegranule fractions is due to the presence of labeledcondensing vacuoles.

Radioautographic observations on intact cellsof slices after 1 hr and especially after 2 hr post-pulse incubation conclusively demonstrate thetransformation of condensing vacuoles into zymo-gen granules and, finally, the discharge of proteininto the acinar lumen. The terminal steps in thesecretory cycle are events well documented byprevious work (Palade, 1959; Caro and Palade,1964). Hence, we now have a reasonably com-

J. D. IAMIESON AND G. E. PALADE Intracellular Transport of Secretory Proteins. II 613

plete elucidation of the entire intracellular path-way of secretory proteins from their site of syn-thesis on attached ribosomes to their ultimatedischarge into the duct system of the gland. Ac-cording to the radioautographic data, our inter-pretations probably apply to the pathway andtimetable of transport of all digestive enzymes inthe guinea pig pancreas. Exceptions for exportableproteins which represent a small fraction of thetotal output in the guinea pig or for any proteinin other species are improbable but not excluded(cf. Redman and Hokin, 1959; Morris and Dick-man, 1960).

We should mention that our radioautographicstudies indicate that the piled Golgi cisternaelocated at the periphery of the complex do notseem to play a direct role in the transport andconcentration of labeled secretory proteins.Nevertheless, in the exocrine pancreas of otherspecies (rat, mouse), condensing vacuoles appearto form at the expense of the innermost cisternaeof the piles. A comparable situation exists inother secretory cells where the function of thecondensing vacuoles is taken over by the piledcisternae of the Golgi complex (Kurosumi, 1963,Bainton and Farquhar, 1966; Smith and Far-quhar, 1966; Essner and Novikoff, 1962).

To what extent does the well established se-quence of operations involved in the pancreaticsecretory cycle apply to secretory processes inother cells? The same sequence seems to operatein glandular cells specialized in the synthesis ofproteins for export, provided the secretory productis quantized and stored temporarily in the cell in

concentrated form. For example, radioautographicevidence suggests that the model applies to themammary gland (Wellings and Philp, 1964), andto myelocytes (Fedorko and Hirsch, 1966). In cellsin which there is no intracellular accumulation ofsecretory product or in which the latter accumu-lates within the ER cisternae (Ross and Benditt,1965; Revel and Hay, 1963; Rifkind et al., 1962),the extent to which the Golgi complex is involvedis not clear.

In conclusion, we now have a satisfactorygeneral understanding of the secretory cycle of thepancreatic exocrine cell based on experimentallyestablished facts from a number of studies. Inaddition, these studies provide direct or indirectevidence that several types of intracellular trans-port, some of them new, participate in the cycle.These include: vectorial transport of newly syn-thesized proteins across the membranes of therough ER (Redman et al., 1966; Redman andSabatini, 1966); transport in bulk of materialsbetween cell compartments; concentration of cellproducts within membrane-bounded structurespossibly by intracellular ion pumps; and finally,transport of secretory proteins from zymogengranules to the acinar lumina (Palade, 1959). Theforces, molecular events, and control mechanismsoperating at each step remain to be elucidated byfuture work.

Part of this work was supported by a United StatesPublic Health Service Research Grant AM 10928-01.

Received for publication 20 February 1967.

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