The Route of Secretion of Procollagen

Biochem. J. (1976) 156, 81-90Printed in Great Britain

The Route of Secretion of ProcollagenTHE INFLUENCE OF aa'-BIPYRIDYL, COLCHICINE AND ANTIMYCIN A

ON THE SECRETORY PROCESS IN EMBRYONIC-CHICK TENDONAND CARTILAGE CELLS

By RICHARD HARWOOD, MICHAEL E. GRANT and DAVID S. JACKSONDepartment ofMedical Biochemistry, The Medical School, University ofManchester,

Oxford Road, Manchester M13 9PT, U.K.

(Received 30 October 1975)

1. Embryonic-chick tendon cells were pulse-labelled for 4min with ['4C]proline andthe 14C-labelled polypeptides were chased with unlabelled proline for up to 30min. Iso-lation of subcellular fractions during the chase period and their subsequent analysisfor bacterial collagenase-susceptible "4C-labelled peptides demonstrated the transfer ofprocollagen polypeptides from rough to smooth microsomal fractions and thence to theextracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involve-ment of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in theGolgi-enriched fractions demonstrated that the procollagen polypeptides were all presentas disulphide-linked pro-y components. 2. When similar kinetic studies of the intracellulartransport of procollagen were conducted with embryonic-chick cartilage cells almostidentical results were obtained, but the rate of translocation of cartilage procollagen wassignificantly slower than that observed for tendon procollagen. 3. When hydroxylationof procollagen polypeptides was inhibited by aoa'-bipyridyl, the nascent polypeptidesaccumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for4min with [14C]proline and the label was chased in the presence of colchicine, secretion ofprocollagen was inhibited and an intracellular accumulation of procollagen "4C-labelledpolypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence ofthe intracellular transport of procollagen was demonstrated in experiments in whichantimycin A was found to inhibit the transfer of procollagen polypeptides from rough tosmooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classicalroute of secretion taken by other extracellular proteins.

Studies carried out in the last decade on the site ofsynthesis and the secretory route taken by extra-cellular proteins have established a general patternwhich commences with the elaboration of the poly-peptide chains on ribosomes attached to the mem-brane of the rough endoplasmic reticulum. Thesegregation of secretory proteins is achieved by thevectorial release ofthe polypeptides into the cisternaeofthe rough endoplasmic reticulum, from which theyare transported to the plasma membrane via thesmooth endoplasmic reticulum and Golgi elements.The elucidation ofthis pathway has generally involveda combination of morphological analysis of intactcells and tissues and the application of biochemicaltechniques to monitor quantitatively the transportof the polypeptides through the subcellular com-partments (for reviews see Campbell, 1971 ; Jamieson,1972; Scharff, 1975; Palade, 1975).Because of the inherent problems in homogenizing

connective tissues and the consequent difficulties inobtaining homogeneous subcellular fractions, the

Vol. 156

biochemical approach to studying procollagen secre-tion has been somewhat restricted (for review seeGrant & Prockop, 1972a,b,c). Until recently, there-fore, studies on the route of procollagen secretionhave relied almost entirely on morphological andradioautographic approaches. On the basis of suchtechniques several possible routes for procollagentransport have been proposed (Revel & Hay, 1963;Ross & Benditt, 1965; Cooper & Prockop, 1968;Salpeter, 1968; Reith, 1968; Frank & Frank, 1969;Trelstad, 1971; Weinstock, 1972; Hay & Dodson,1973; Weinstock & Leblond, 1974; Scherft &Heersche, 1975), but confirmation of those earlierstudies which indicated that procollagen secretionoccurs via the classical route described above hasbeen obtained in studies using ferritin-conjugatedantibodies against tendon procollagen (Olsen &Prockop, 1974; Olsen et al., 1975; Nist et al., 1975).

Matrix-free cells derived from embryonic-chickleg tendons and sternal cartilages have foundconsiderable application in studies ofthe mechanisms

81

R. HARWOOD, M. E. GRANT AND D. S. JACKSON

involved in procollagen biosynthesis (for reviews seeGrant et al., 1975; Prockop et al., 1976), and theestablishment of conditions for their homogenizationand fractionation (Harwood et al., 1974d, 1975a) hassuggested the suitability of these cell systems fordetailed biochemical analyses of procollagen assemb-ly and secretion at the subcellular level. In the presentstudy the routes of secretion of tendon and cartilageprocollagens have been investigated and evidence ispresented that supports the view that procollagenfollows the secretory route taken by other extra-cellular proteins such as albumin, al-acid glyco-protein, amylase and immunoglobulins.

Experimental

MaterialsFertile eggs obtained from Mytholmroyd Hatch-

eries, Hebden Bridge, W. Yorks., U.K., wereincubated for 17 days at 37°C in a moist atmospherein a Westernette Egg Incubator (Western IncubatorsLtd., Chelmsford, Essex, U.K.). ["4C]Proline(290mCi/mmol) was purchased from The Radio-chemical Centre, Amersham, Bucks., U.K. Chymo-trypsin, trypsin, pancreatic ribonuclease A, colchicineand antimycinA were obtained from Sigma (London)Chemical Co., London S.W.6, U.K. Sephadex G-50was supplied by Pharmacia Ltd., London W.5,U.K., and the preparation ofhighly purified bacterialcollagenase was a generous gift from Dr. V. F.Lee-Own of this department. All other materials andreagents were from sources previously described(Harwood et al., 1974d, 1975a).

Isolation and incubation ofcellsCells were isolated from the leg tendons and sterna

of 17-day-old chick embryos as described previously(Dehm & Prockop, 1971, 1973; Harwood et al.,1974d, 1975a). Cells were incubated at concentrationsof approx. 2 x 107 cells/ml ofmodified Krebs medium(Dehm & Prockop, 1971) and in the experimentsconducted with cartilage cells the medium was alwayssupplemented with 10% (v/v) foetal calf serum.The cells were preincubated for 30min at 37°C thenlabelled for 4min with ["4C]proline, and in experi-ments where the transport through the cell of theprocollagen 'IC-labelled polypeptides was to bestudied, [(2C]proline was added to a concentration oflOO,ug/ml and incubation continued for up to 45min.At various intervals during this chase period portionswere removed from the incubation mixture andrapidly cooled to 4°C. Further protein synthesis wasinhibited by the addition of cycloheximide tolOO,ug/ml. The cells were sedimented at 1200g for5min and resuspended in the buffer appropriate to thesubcellular fractionation procedures to be carriedout.

In studies on the intracellular transport of pro-collagen synthesized under conditions where hydroxy-lation was inhibited, 0.3 mm-aao'-bipyridyl was presentthroughout both the preincubation and pulse-chaseperiods. The fate of procollagen "IC-labelledpolypeptides was also examined when colchicine orantimycin A was added to the incubation medium toa final concentration of 0.1 or 0.01mm respectivelyimmediately after the 4min-labelling period.

Subcellular fractionation of cells

In order to study the secretory route ofprocollagenit was necessary to isolate subcellular fractions ofrough endoplasmic reticulum, smooth endoplasmicreticulum, Golgi elements and cytosol. The homo-genization procedures and fractionation schemesdescribed by Harwood et al. (1974d, 1975b) wereused. Because of the restricted number of cellsavailable it was not possible within one experimentto isolate each of the above fractions for every time-point. Accordingly, tendon cells taken at 5, 10, 15and 30min of the chase period were fractionated toobtain rough and smooth microsomal fractions andthose cells chased for 10, 20 and 25min were fraction-ated to obtain Golgi and cytosol fractions. In asecond identical experiment rough and smoothmicrosomal fractions were isolated from cells chasedfor 5, 20 and 25min and Golgi and cytosol fractionsobtained from cells chased for 5, 10, 15 and 30 min.Thus within the two experiments each fractionationscheme had a common time-point, i.e. 5min for therough microsomal fraction and 10min for the Golgifraction. The value (d.p.m. of collagenase-susceptible"4C-labelled peptides/I x 108 cells) obtained for therough microsomal fraction at 5min in the secondexperiment was 95% of that of the initial experimentwhereas that of the Golgi fraction at 10min was 93%of the corresponding sample in the first experiment.Therefore, by a minor adjustment, it was possible torelate the values obtained in the second experimentto those of the first and hence complete the series oftime-points for the various subcellular fractions.

In similar studies with cartilage cells the methodo-logy described above was followed except that thetime of the chase period was extended to 45minbecause the time for procollagen secretion in cartilagecells is significantly longer than that for tendon cells(Dehm & Prockop, 1973).

Analysis of subcellular fractions for procollagen"C-labelledpolypeptidesThe passage of procollagen "IC-labelled poly-

peptides through the subcellular compartments oftendon and cartilage cells was followed by determiningthe amount of radioactivity in these fractions madediffusible by the action of highly purified bacterial

1976

82

SECRETION OF PROCOLLAGEN

collagenase. The pelleted subcellular fractionsobtained as described above were resuspended inlml of 20mM-Tris/HCl (pH7.6) containing 0.17M-NaCl and 5mM-CaCl2.- The proteins of the cytosolfractions were precipitated by the addition of solid(NH4)2SO4 to 65% saturation and then sedimented bycentrifugation at 21 OOOg for 20min. These cytosolfractions were each resuspended in 1 ml of theTris/HClI/NaCl/CaCI2 buffer. The samples wereincubated at 37°C for 24h with 20,ug of bacterialcollagenase purified as described by Lee-Own &Anderson (1975). This collagenase preparation hadnegligible proteolytic activity towards a [3H]trypto-phan-labelled substrate prepared from chick embryos(Lee-Own & Anderson, 1975). Over 90% of thehydroxy[l.4C]proline was made diffusible under theabove conditions and was recovered by dialysis at4°C for a total of48h against two changes of 50ml ofwater. The diffusate was rotary-evaporated todryness, redissolved in 4ml of water and a portion(lml) counted for radioactivity in 10ml of TritonX-100 toluene scintillant at an efficiency of 72%(Harwood et al., 1974c).

Determination of the direction ofdischarge ofnascentprocollagen '4C-labelled polypeptides synthesized inthe presence of axa'-bipyridylTendon and cartilage cells were preincubated in

the* presence of 0.3mM--aa'-bipyridyl and labelledwith [14C]proline for 4min. Cells were homogenizedin a glass-Teflon motor-driven homogenizer in0.25M-sucrose in 0.05M-Tris/HCl buffer (pH7.5)containing 0.025M-KCI and 0.5mM-MgCI2 andmicrosomal fractions were isolated (Harwood et al.,1974d). The susceptibility ofthe 14C-labelled peptidesto limited proteolysis was examined by using asolution of trypsin and chymotrypsin (3mg ofeach dissolved in 10ml of sucrose/Tris/KCl/MgC12buffer at 4°C) prepared immediately before use.Microsomal pellets resuspended in sucrose/Tris/KCI/MgCI2 buffer (1 ml) were incubated at 4°C in theabsence and presence of the enzyme mixture (finalconcn. 50,ug/ml of each enzyme). These incubationswere also conducted in the presence of a mixture of1 % (w/v) sodium deoxycholate and 2% (v/v)Triton X-100. The reaction mixture was incubatedfor 5h then diluted to 8ml with the above bufferedsucrose solution and centrifuged at 105000g for 4h.The pellets were dissolved in 8M-urea (1 ml), whichstops proteolysis (Harris, 1956), and 0.25mg ofpancreatic ribonuclease was added and the sampleincubated at 37'C for 15min, followed by centrifuga-tion at 10OOg for 10min. The supernatants werechromatographed on a column (60cmx 2cm) ofSephadex G-50 equilibrated with 8M-urea/10mM-2-mercaptoethanol.

Vol. 156

Results

Pathway of procollagen secretion in tendon andcartilage cells

Pulse-chase experiments similar to those carriedout in studies on the secretion of other extracellularproteins (Jamieson & Palade, 1967; Glaumann &Ericsson, 1970; Jamieson & Ashton, 1973) wereundertaken to examine the route of secretion ofprocollagen in tendon and cartilage cells. Tendoncells were incubated for 4min with [1"C]proline,followed by a chase with unlabelled proline for up to30min. During the chase period samples were takenand cells fractionated to yield rough and smoothmicrosomal fractions, Golgi elements, cytosol andextracellular medium. The data presented in Fig. 1(a)indicate that maximum labelling of procollagen with[I4C]proline occurred at 10min in the rough micro-somal fraction and there was a quantitative transferof radioactive label to the smooth microsomalfraction which reached a maximum at 15min.Secretion of '4C-labelled procollagen into themedium commenced at approx. 2Omin and a peak ofradioactivity was observed in this fraction at 25mi.Only a very small proportion of the collagenase-susceptible 14C-labelled polypeptides was recoveredin the cytosol fractions, and the fact that this radio-activity remained constant over the 30min-chaseperiod provides evidence that the cytoplasm is not acompartment in the procollagen transport system.

In experiments conducted with embryonic chickcartilage cells where an extended chase period of45min was used similar results were obtained(Fig. lb). However, in these studies the maximumlabelling of cartilage procollagen with ['4C]prolineoccurred at 1 5mm in the rough microsomal fraction,at 25min in the smooth microsomal fraction, and at40mi in the medium. No significant amount oflabelwas observd in the cytosol fractions at any point inthe chase period.

These- reults are similar to those obtained inanalogous studies on the secretion of amylase(Jamieson & Palade, 1967), albumin (Glaumann &Ericsson, 1970), irnmunoglobulins (Scharff, 1975)and a,-acid glycoprotein (Jamiesoh & Ashton, 1973),and demonstrate the transfer of newly synthesized[14C]procollagen from the rough to the smoothendoplasmic reticulum. In considering the observa-tion that the maxima of labelling of smooth micro-somal and the Golgi-enriched fractions are coincident(see Figs. la and lb), it is im-portant to appreciate thatthese cell types are devoted predominantly to thesynthesis of extracellular protein, and are thereforelikely to have extensive rough endoplasmic reticuilumand well-developed Golgi apparatus [as indicatedin the electron micrographs of tendon cells presentedby Olsen & Prockop (1974) and Olsen et al. (1975)].Consequently application of the method of Dallner

83


a:_4

(A

.8 E 2

- 10x -*

° o 30Chase time (min)

Fig. 1. Translocation ofprocollagen through subcellular compartments oftendon and cartilage cells

(a) Duplicate samples of approx. 18 x 108 tendon cells were incubated at 37°C with 25.uCi of [14C]proline in 125 ml ofmodified Krebs medium. After 4min unlabelled proline was added to a final concentration of lOOpg/ml (final incubation vol.130ml) and incubation was continued for a further 30min. At intervals during the chase period samples (15 ml) were removedfrom the incubation medium, cooled to 4°C, made lOOpg/ml in cycloheximide and the cells collected by centrifugation. Thecells were homogenized and fractionated to yield appropriate subcellular fractions which were analysed for the presence ofprocollagen 14C-labelled polypeptides as described in the text. (b) Approx. 8 x 108 cartilage cells (in duplicate) were incubatedat 37°C with 254uCi of ['4C]proline in 85ml of modified Krebs medium containing 10%4 (v/v) foetal calf serum. After4min, unlabelled proline was added to a final concentration of lOO1g/ml (final incubation vol. 90ml) and the incubationwas continued for a further 45 min. Samples (9 ml) were withdrawn at appropriate intervals and subcellular fractions isolatedand assayed as above. 0, Rough microsomal fraction; o, smooth microsomal fraction; A, Golgi fraction; A, cytosol; *,medium.

(1963) to separate rough- and smooth-endoplasmic-reticulum fractions is likely to result in a large pro-portion of the smooth microsomal fraction from thesecells comprising Golgi-derived vesicles. This problemis possibly accentuated by the relatively vigoroushomogenization conditions required to fracture thesematrix-free cells (Harwood etal., 1974d), but by usingthe procedures of Schachter et al. (1970) it is possibleto separate Golgi-derived vesicles from vesicles ofsmooth endoplasmic reticulum on the basis ofmembrane density. The data obtained by this latterapproach (Figs. la and lb) indicate the involvementofthe Golgi apparatus in the secretion ofprocollagen.The decreased proportion of radioactivity in thisGolgi-enriched fraction can be attributed to thelimited recovery of Golgi elements by this procedure(Schachter et al., 1970).Harwood et al. (1973, 1975a) have demonstrated

that in fractions of rough and smooth microsomalfractions isolated from these cells the major ["C]-proline-labelled species are procollagen polypeptides.In the smooth microsomal fractions these pro-achains are virtually all linked by interchain disulphidebonds to form trimeric triple-helical procollagenmolecules (R. Harwood, A. H. Merry, D. E. Woolley,M. E. Grant & D. S. Jackson, unpublished results)and are glycosylated to an extent almost equivalentto that of the secreted procollagens (Harwood et al.,1975b). Analysis of the Golgi-derived fractions bysodium dodecyl sulphate/polyacrylamide-gel electro-

phoresis (Weber & Osborn, 1969) under both non-reducing and reducing conditions indicated that theGolgi elements of tendon and cartilage cells containpro-a chains, all of which were found to be presentin the pro-y form (Figs. 2a and 2b).

Demonstration of the vectorial release of unhydroxyl-ated procollagen polypeptides into the lumen of theendoplasmic reticulum

To assess whether unhydroxylated nascent pro-collagen polypeptides are directed into the lumen ofthe endoplasmic reticulum or towards the cytoplasm,the susceptibility of the nascent peptides to limitedproteolysis was examined. If the nascent peptideswere being directed across the endoplasmic-reticulummembrane then polypeptides within isolated micro-somal fractions should be protected from proteolysis,whereas if the growing chains were directed towardsthe cytoplasm they would be subject to degradationby added proteinases (Blobel & Sabatini, 1970;Sabatini & Blobel, 1970; Harwood et al., 1974b).The results shown in Fig. 3 indicate that when

microsomal fractions isolated from tendon cellsincubated in the presence of aa'-bipyridyl weretreated with a trypsin/chymotrypsin mixture in theabsence of detergent, minimal proteolysis occurred.Chromatography on Sephadex G-50 revealed a majorpeak at the void volume and a small included peak.By analogy with the studies of Sabatini & Blobel

1976

84


Pro-axlo (a) 1Pr6-ca1

0

0 l0 20 30 40 50 60Gel slice no.

Fig. 2. Electrophoretic analysis ofprocollagen 14C-labelledpolypeptides present in Golgifractions isolatedfrom tendon

and cartilage cells(a) Tendon cells (1570x 106) were incubated for 2h withl5pCi of [14C]proline in I10ml of modified Krebsmedium. The cells were collected by centrifugation and aGolgi fraction was isolated by the method of Schachteret al. (1970). The fraction was resuspended in 2ml of0.02M-sodium phosphate buffer, pH7.4, and a portion(1ml) was alkylated and denatured in sodium dodecylsulphate (1%Y, w/v) as described byHarwood et al. (1975a).A second portion (1nml) was reduced with 2-mercapto-ethanol and denatured with sodium dodecyl sulphate.The samples were analysed on 5%/ (w/v) polyacrylamidegels by the procedure ofWeber & Osbom (1969). Electro-phoresis was carried out for 4h at lOmA/tube andanalysis of the gels for radioactivity was carried out on1mm slices obtained by using a Mickle Gel Slicer (Joyce,Loebl and Co. Ltd., Gateshead, U.K.). The slices wereincubated at 50°C overnight in 0.5ml of 30% (v/v) H202and then 10ml scintillant [5 vol. of toluene containing6g of2,5-diphenyloxazole/litre and 0.12g of 1,4-bis-(5-phenyl-oxazol-2-yl)benzene/litre and 3vol. of 2-methoxyethanol]was added and the sample counted for radioactivity.(b) Cartilage cells (630x 106) were incubated for 2 h with15,uCi of ['4C]proline in 60ml of modified Krebs mediumcontaining 10%4 (v/v) foetal calf serum. A Golgi fractionwas isolated and analysed by gel electrophoresis underreducing and non-reducing cbnditions as described above.0, Unreduced sample; o, reduced sample. The migrationpositions of procollagen standards are indicated.

(1970), the excluded peak corresponds to the largepeptide fragments protected by the microsomalmembranes and the included peak corresponds to theC-terminal segments, which are protected by thelarge ribosomal subunits of the membrane-boundpolyribosomes. In contrast, when the detergent waspresent in the enzyme incubation the high-molecular-weight polypeptides were digested and only the smallpeptides corresponding to the fragments protectedby the ribosomal structure were recovered on gel

Vol. 156

2.

-

80

5.

x 2.

_I

.5

(a)5.0

2.5

(b)

J o.0 (c) d

S

.5 1L -2i

0 40 80 0 40 80

i.o

.5

Fraction no.

Fig. 3. Susceptibility to limitedproteolysis ofnascent tendonprocollagen polypeptides labelled in the presence of

aa'-bipyridyl

Tendon cells (1610x 106) were preincubated for 30minat 37°C in 25ml of modified Krebs medium containing0.3mM-aac'-bipyridyl. After pulse-labelling with 254uCi of1[4C]proline for 4min, cells were homogenized and micro-somal fractions isolated. Portions of the microsomalfractionwere subjected to limited proteolysis witha trypsin/chymotrypsin mixture at 4°C for 5h in the presence andabsence of detergent as described in the text. The sampleswere then centrifuged, treated with urea and ribonuclease,and chromatographed on a column (60cmx2cm) ofSephadex G-50 equilibrated with 8M-urea/lOmM-2-mer-captoethanol; fractions of 3ml were collected. (a) Controlincubation in the absence of detergent; (b) control incu-bation in the presence ofdetergent; (c)enzyme digestion inthe absence of detergent; (d) enzyme digestion in thepresence of detergent.

filtration. Because it was observed that detergenttreatment itself did not affect the elution profile ofthenascent peptides it can be concluded that disruptionof the microsomal vesicles with detergents exposesthe growing unhydroxylated procollagen peptidesto proteolytic digestion.When the above experiments were conducted with

microsomal preparations isolated from cartilagecells that had been pulse-labelled with [14C]prolinein the presence of oa'-bipyridyl identical results (notshown) were obtained.

Studies on the intracellular transport of unhydroxyl-ated procollagen in tendon and cartilage cells

It has been demonstrated that when nascentprocollagen polypeptides growing in the large sub-units of membrane-bound ribosomes emerge fromthe ribosomes they are transferred across the mem-brane into the lumen of the rough endoplasmicreticulum (Harwood et al., 1974b), and the above

85

;F


a~0

x

0 IQ 20 30 40 50

Chase time (min)Fig. 4. Intracellular site ofuaccumlation ofunhydroxylated

procollagen polypeptides

Experiments were conducted as described in Fig. 1, exceptthat during the preincubation (see the text) and pulse-chase periods aa'-bipyridyl was present at a concentrationof 0.3mm. (a) Tendon cells; (b) cartilage cells. 0, Roughmicrosomal fraction; 0, smooth microsomal fraction;A, Golgi fraction; A, cytosol; *, medium.

results indicate that this transfer still occurs evenwhen the process of hydroxylation -is inhibited bydk'-bipyridyl. However, it is well established thatin the absence of hydroxylation, unhydroxylatedprocollagen polypeptides tend to accumulate intra-cellularly and their failure to be secreted may beattributable to their inability to assume a triple-helical conformation at 370C (Prockop et aL,

1976).Studies have' been conducted to ascertain the

subcellular compartment in which unhydroxyatedprocollagen polypeptides accumulate. 'Tendon andcartilage cells were,preincubated in the presence ofxca'-bipyridy 'for 30min and after pulse-labellingwith [14C]proline for 4mi, subcellular fractionswere isolated as described above'at various tunesduring a chase with (12C]proline. The results pre-sented in Fig. 4 demonstrate that when hydroxylati'onis inhibited in both tendon and cartilage cells, un-hydroxylated procollagen "C-labelled polypeptidesaccumulate in the rough microsomal fraction andlittle transfer (less than 15%) to the smooth micro-somal or'Golgi fractions occurs. It should &b ncitedthat the maxima of labelling of the rou h1micr0osoalfractions of tendon and cartilage cells are identicalwith those obtained in experiments on the secretionofthe respective procollagens (ig. 1), indicating thatthe synthesis of the polypeptides and their tirnsfer

into- the lumen of the rough endoplasmic reticulumproceed at a normal rate within the duration of theseexperiments. The findings that unhydroxylatedprocollagen accumulates in the rough microsomalfractions are consistent with the observation ofOlsen et al. (1975) in which ferritin-conjugatedantibodies to procollagen w,ere used to localizeunhydroxylated moleenles' in the 'cisternae of theendoplasmic reticulum of tendon cells treated withaa'-bipyridyl.

Influence ofcolchicine andantimycin A on the secretionofprocollagen

The results presented above are consistent withprocollagen following the secretory route from roughendoplasmic reticulum -* smooth endoplasmic reti-culum -* Golgi apparatus -* extracellular space. Toinvestigate the mechanisms by which the procollagenmolecules are translocated through these cellularcomnpartments we have examined the effects of theantimicrotubular agent, colchicine, and the metabolicinhibitor, antimycin A, on the secretion process incells from embryonic-chick leg tendons and sternalcartilages.

Cells were labelled for 4min with ['4C]proline andchase experiments carried out in the presence of0.1 mM-colchicine. Subcellular fractions were isolatedas described above and the results'of these pulse-chase studie's are presented in Figs. 5(a) and 5(b).The data indicate that in the presence of this'agentquantitative transfer of labelled procollagen fromrough to smooth microsomal fractions occurs inboth, cell types. This transfer is slightly delayed by theaction of colchicine, for in control tendon cells themaximum of labelling of the smooth microsomalfraction occurs at l5min (Fig. la), whereas- in thetreated tendon cells the maximum is observed at20min (Fig. Sa). A similar delay occurs whencartilage cells are treated' with colchicine (compareFigs. ,lb and Sb). The absence of any significanttransfer of labelled procollagen from smooth micro-somal fractions to the extracellular medium (Figs. 5aand Sb) may imply a role for microtubules in thetranslocation of procollagen-containing vesicles tothe plasma membrane. These observations areconsistent with studies suggesting an increasedintracellular accumulation of procollagen in cellsincubated in the presence of antimicrotubular agents(Dehtn & Prockop, 1972; Ehrlich & Bornstein, 1972;Diegelmahn & eterkofsky, 1972) and the localiza-tion Ofthis accumulation in Golgi-associated vacuolesvadesicles (P-hiich et al., 1974; Qlsen & P*ockop,1'974). The coincidence of the maxima of labellihg ofsmooth microsomal and Golgi-enriched fractions inthese experiments(Figs. 5a and 5bJis further evidencethiat sthe smooth mnicrosomal fraction comprisespredominantly Golgi-deriverd vesicles.

1976

86


2 (b)

0U~~~~~~~~~~~~~~~

x

- 0 10 20 30 40 0 10 20 30 40 50

Chase time (min)Fig. 5. Influence ofcolchicine and antimycin A on the transcellular movement ofprocollagen

Experiments were conducted as described in Fig. 1 except that during the chase period either colchicine (0.1 mM) or antimycinA (0.01 inw) were present. (a) Tendon cells pulse-labelled with ["4C]proline and chased in the presence of colchicine; (b)cartilage cells pulse-labelled with [1"C]proline and chased in the presence of colchicine; (c) tendon cells pulse-labelled with[14C]proline and chased in the presence ofantimycin A; (d) cartilage cells pulse-labelled with ['4C]proline and chased in thepresence of antimycin A. *, Rough microsomal fraction; o, smooth microsomal fraction; A, Golgi fraction; A, cytosol;U, medium.

To investigate the energy requirements for intra-cellular transport, experiments were conducted inwhich cells were pulse-labelled as previously describedand the chase experimnts carried out in the presenceof 0.01 mM-antimycin A (Fig. 5c and 5d). Inhibitionof energy production during the chase period gaverise to an accumulation of procollagen '4C-labelledpolypeptides in the rough microsomal preparationsand little transfer to the smooth microsomal fractionswas observed. These results indicate the energydependenwe of the mechanisms involved in thetranslocation of procollagen polypeptides from therough endoplasmic reticulum to the smooth endo-plasmic reticulum and Golgi elements in both tendonand cartilage cells.

Discussion

The suboellular mechanisms imvolved in the trans-port of procollagen from its site of synthesis on therough endoplasmic reticulum to the extracellularspace have been the subject of conflicting reports(Sheldon & Kimball, 1962; Revel & Hay, 1963;Ross & Benditt, 1965; Cooper & Prockop, 1968;Salpeter, 1968; Reith, 1968; Frank & Frank, 1969;Tre}stad, 1971; Hay & Dodson, 1973). In particularthe role of the Golgi complex in the secretion ofprocollagen has until recently been illdefined, prob-ably because in the majority of collagen-synthesizingcels studied there is no distinctive system of con.

Vol. 156

densed secretory granules as seen, in the classicalstudies on exocrine cells of the pancreas (for reviewsee Jamieson, 1972). However, in morphological andradioautographic studies on the highly polarizedodontoblasts (Weinstock, 1972; Weinstock &Leblond, 1974) and in specific imunocytochemicalstudies with chick tendon (Olsen & Prockop, 1974;Olsen et al., 1975) and corneal fibroblasts (Nist et al.,1975) convincing evidence for the involvement of theGolgi apparatus in procollagen secretion has beendescribed. Of course, such studies cannot provideprecise quantitative and kinetic data, but in the workdescribed here we have used a subcellular-fractiona-tion approach which permits a kinetic analysis of thetransfer of procollagen polypeptides through sub-cellular compartments and provides further dataon the differences in the rates of translocation andsecretion of tendon and cartilage procollagens(Dehm & Prockop, 1973; Harwood et al., 1975a).The results of the pulse-chase studies carried out

on subcellular fractions isolated from tendon andcartilage cells (Figs. la and lb) demonstrate aquantitative transfer of procollagen '4C-abelledpolypeptides from rough to smooth microsomalfractions and thence to the extracellular medium and,as discussed above, conconitant analyses of Golgi-eniched fractions indicated the involvement of thisorganelle in the secretory pathway. It should be notedthat the appearance of [(4Cjprocollagen in the tendonand cartilage cell-incubatip mrdii after approx.

87


20 and 35min respectively is consistent with previousestimates from continuous-labelling studies of thesecretion times of these precursor molecules (Dehm& Prockop, 1972, 1973). The longer time required forthe intracellular translocation and secretion ofcartilage procollagen is not attributable to a delay ina particular subcellular compartment (compareFigs. la and Ib), although the extended period spentin the rough endoplasmic reticulum, in which prolinehydroxylase, lysine hydroxylase and the collagenglycosyltransferases are predominantly localized(Harwood etal., 1974d, 1975b), might account for themore extensive hydroxylation and glycosylation ofcartilage collagen (Dehm & Prockop, 1973; Harwoodet al., 1975b).

In early studies concerned with the role of thehydroxylation process in the secretion of collagen itwas found that incubation of connective-tissue cellsunder either anaerobic conditions or in the presenceof aa'-bipyridyl gave rise to an intracellular accumula-tion of unhydroxylated procollagen, termed proto-collagen. The significance of this accumulation andwhether or not protocollagen can be secreted in anundegraded form and follows the same secretorypathway as procollagen have been the subject ofseveral sometimes conflicting studies (for review seeProckop et al., 1976). In the experiments described inFig. 3 the unhydroxylated procollagen 14C-labelledpolypeptides were shown to be directed from thelarge subunits of membrane-bound ribosomes intothe lumen of the endoplasmic reticulum in a mannersimilar to that described in microsomal fractionsfrom untreated cells (Harwood etal., 1974b). Thus noevidence was obtained to support the suggestion(Diegelmann et al., 1973) that inhibition of prolinehydroxylase might result in the redirection of nascentprocollagen polypeptides into the cytoplasm, and theabsence ofsignificant labelling ofthe cytosol fractionsduring the pulse-chase experiments described inFig. 4 is also contrary to this suggestion. It is note-worthy that in these latter experiments aa'-bipyridyldid not influence the times at which the rough micro-somal fractions became maximally labelled, anobservation that is consistent with the report that thischelating agent does not affect the release of nascentprocollagen polypeptides (Lazarides & Lukens,1971).These pulse-chase experiments conducted in the

presence of aa'-bipyridyl provide the first kineticdata showing a failure to transport unhydroxylatedprocollagen polypeptides from the rough to thesmooth endoplasmic reticulum and these observa-tions correlate well with the studies using ferritin-conjugated antibodies against tendon procollagen(Olsen et al., 1975), which demonstrated the localiza-tion of protocollagen in the cisternae of the endoplas-mic reticulum ofchick tendon cells. This inhibition ofprocollagen translocation by aa'-bipyridyl has been

attributed to the formation of stable enzyme-substrate complexes for proline hydroxylase, whichis located almost exclusively in the rough microsomalfractions of these cell types (Harwood et al., 1974d),has a high affinity for protocollagen (for reviews seeGrant & Prockop, 1972b; Prockop et al., 1976).However, since hydroxyproline plays a key role instabilizing the collagen triple helix at 37'C (Jimenezet al., 1973; Berg & Prockop, 1973) and becauseseveral studies have suggested that the critical require-ment for secretion may be the attainment of a triple-helical conformation (Uitto & Prockop, 1974a,b,c;Jimenez & Yankowski, 1975) the exact nature of thisfailure in intracellular transport remains to beevaluated.As discussed above, these biochemical studies on

the secretion ofprocollagen complement many of theprevious morphological, radioautographic and im-munocytochemicalstudies (Sheldon &Kimball, 1962;Revel & Hay, 1963; Frank & Frank, 1969; Trelstad,1971; Weinstock, 1972; Hay & Dodson, 1973;Olsen & Prockop, 1974; Weinstock & Leblond,1974; Olsen et al., 1975; Nist et al., 1975; Scherft &Heersche, 1975) and together this large body ofevidence clearly establishes that the secretion ofprocollagen follows the classical intracellular path-way described for other secretory proteins. However,little information is available onhow proteins destinedfor secretion are transported from one cellularcompartment to another, but some insight into thesemechanisms may possibly be gained by the use ofdrugs that inhibit the secretion process at specificlocations. In our studies of the influence of colchicineon the intracellular transport of procollagen, the rateat which procollagen polypeptides were transferredfrom the rough to the smooth microsomal fractionswas observed to decrease slightly and subsequenttransfer to the extracellular medium was completelyinhibited (Figs. 5a and Sb). This latter observation isin agreement with morphological studies in whichcolchicine was found to induce an accumulation ofprocollagen in Golgi-derived vesicles (Ehrlich et al.,1974; Olsen & Prockop, 1974; Scherft & Heersche,1975), and similar effects have been noted in studieson the secretion of a variety of proteins (Williams &Wolff, 1972; Stein & Stein, 1973; Le Marchand et al.,1974; Redman et al., 1975). These effects havegenerally been interpreted to imply the involvementof microtubules in exocytosis. However, in studieson the influence of colchicine on the release of plasmaproteins from rat hepatocytes, Redman et al. (1975)were unable to ascribe the inhibitory effect of col-chicine on plasma protein secretion to microtubuledepolymerization, since the inhibition was promptand reached its maximum long before the disruptionof microtubules became morphologically detectable.In view of the other known effects of colchicine onmembrane fractions (Wunderlich et al., 1973;

1976

88

SECRETION OF PROCOLLAGEN 89

Stadler & Franke, 1974; Wilson et al., 1974) and therapidity with which this agent influenced the secretionof procollagen in the experiments described here,further studies will be necessary to establish theinvolvement of microtubules in procollagen secre-tion.The possibility that the intracellular transport of

procollagen is dependent on a supply of energy,presumably as ATP, was first suggested by studies ofthe influence of the uncoupler of oxidative phos-phorylation, carbonyl cyanide m-chlorophenylhydra-zone, on procollagen synthesis in chick cranial bones(Ehrlich & Bornstein, 1972; Ehrlich et al., 1974).More recent studies with cultured chick fibroblastshave confirmed a requirement for ATP in the trans-cellular movement and secretion of procollagen(Kruse & Bomstein, 1975), and in the experimentsreported here the accumulation of procollagen 14C-labelled polypeptides in rough microsomal fractionsoftendon and cartilage cells treated with antimycinAsuggests that the first energy-requiring step residesat the point of transfer of procollagen from roughto smooth endoplasmic reticulum. Similar resultshave been obtained in studies of the influence ofmetabolic inhibitors on protein secretion in exocrinecells of the pancreas (Jamieson & Palade, 1968, 1971;Jamieson, 1972) and have led to the concept of anenergy-dependent pre-Golgi lock (or lock gate) alongthe pathway of secretion (Palade, 1975). The observ-ations that analogous mechanisms may exist in tendonand cartilage cells, which unlike the exocrine cells donot exhibit a storage phase during protein synthesisand secretion, suggests that this energy-dependentstep may be a general feature ofthe process ofproteinsecretion. However, the nature and mechanism ofthisinitial energy-requiring process and the possibleexistence of subsequent energy-dependent steps inprocollagen secretion remain to be investigated.

We thank Miss J. Ross for skilled technical assistanceand gratefully acknowledge the financial support of theScience Research Council.

ReferencesBerg, R. A. & Prockop, D. J. (1973) Biochem. Biophys.

Res. Commun. 52, 115-119Blobel, G. & Sabatini, D. D. (1970) J. Cell Biol. 45,

130-144Campbell, P. N. (1971) FEBS Lett. 7, 1-7Cooper, G. W. & Prockop, D. J. (1968) J. Cell Biol. 38,

523-537Dallner, G. (1963) Acta Pathol. Microbiol. Scand. Suppl.

166, 1-94Dehm, P. & Prockop, D. J. (1971) Biochim. Biophys. Acta

240, 358-369Dehm, P. & Prockop, D. J. (1972) Biochim. Biophys. Acta

264, 375-382Dehm, P. & Prockop, D. J. (1973) Eur. J. Biochem. 35,

159-166

Diegelmann, R. F. & Peterkofsky, B. (1972) Proc. Natl.Acad. Sci. U.S.A. 69, 892-896

Diegelmann, R. F., Bernstein, L. & Peterkofsky, B. (1973)J. Biol. Chem. 248, 6514-6521

Ehrlich, H. P. & Bornstein, P. (1972) Nature (London) NewBiol. 238, 257-260

Ehrlich, H. P., Ross, R. & Bornstein, P. (1974)J. Cell Biol.62, 390-405

Frank, R. M. & Frank, P. (1969) Z. Zellforsch. Mikrosk.Anat. 99, 121-133

Glaumann, H. & Ericsson, J. L. E. (1970) J. Cell Biol. 47,555-567

Grant, M. E. & Prockop, D. J. (1972a) N. Engl. J. Med.286, 194-199

Grant, M. E. & Prockop, D. J. (1972b) N. Engi. J. Med.286,242-249

Grant, M. E. & Prockop, D. J. (1972c) N. Engl. J. Med.286,291-300

Grant, M. E., Harwood, R. & Schofield, J. D. (1975) inDynamics of Connective Tissue Macromolecules(Burleigh, P. M. C. & Poole, A. R., eds.), pp. 1-32,North-Holland Publishing Co., Amsterdam

Harris, J. I. (1956) Nature (London) 177, 471-473Harwood, R., Grant, M. E. & Jackson, D. S.

(1973) Biochem. Biophys. Res. Commun. 55, 1188-1196

Harwood, R., Connolly, A. D., Grant, M. E. & Jackson,D. S. (1974a) FEBS Lett. 41, 85-88

Harwood, R., Grant, M. E. & Jackson, D. S. (1974b)Biochem. Biophys. Res. Commun. 59, 947-954

Harwood, R., Grant, M. E. & Jackson, D. S. (1974c)Biochem. J. 142, 641-651

Harwood, R., Grant, M. E. & Jackson, D. S. (1974d)Biochem. J. 144,123-130

Harwood, R., Bhalla, A. K., Grant, M. E. & Jackson,D. S. (1975a) Biochem. J. 148, 129-138

Harwood, R., Grant, M. E. & Jackson, D. S. (1975b)Biochem. J. 152, 291-302

Hay, E. D. & Dodson, J. W. (1973) J. Cell Biol. 57,190-213

Jamieson, J. C. & Ashton, F. E. (1973) Can. J. Biochem.51, 1281-1291

Jamieson, J. D. (1972) in Current Topics in Membranes andTransport (Bronner, F. & Kleinzeller, A., eds.), vol. 3,pp. 273-338, Academic Press, New York

Jamieson, J. D. & Palade, G. E. (1967) J. Cell Biol. 34,577-596



Jimenez, S. A. & Yankowski, R. (1975) Fed. Proc. Fed.Am. Soc. Exp. Biol. 34, 563

Jimenez, S. A., Harsch, M. & Rosenbloom, J. (1973)Biochem. Biophys. Res. Commun. 52, 106-114

Kruse, N. J. & Bornstein, P. (1975) J. Biol. Chem. 250,4841-4847

Lazarides, E. L. & Lukens, L. N. (1971) Science 173,723-725

Lee-Own, V. F. & Anderson, J. C. (1975)Prep. Biochem. 5,229-245

Le Marchand, Y., Patzelt, C., Assimacopoulos-Jeannet,F., Loten, E. G. & Jeanrenaud, B. (1974) J. Clin.Invest. 53, 1512-1517

Vol. 156

90 R. HARWOOD, M. E. GRANT AND D. S. JACKSON

Nist, C., von der Mark, K., Hay, E. D., Olsen, B. RI,Bornstein, P., Ross, R. & Dehm, P. (1975) J. Cell Biol.65,75-87

Olsen, B. R. & Prockop, D. J. (1974) Proc. Natl. Acad.Sc!. U.S.A. 71, 2033-2037

Olsen, B. R., Berg, R. A., Kishida, Y. & Prockop, D. J.(1975) J. Cell Biol. 64,340-355

Palade, G. E. (1975) Science 189, 347-357Prockop, D. J., Berg, R. A., Kivirikko, K. I. & Uitto, J.

(1976) in Biochemistry of Collagen (Ramachandran,G. N. & Reddi, A. H., eds.), Plenum Publishing Co.,New York, in the press

Reciman, C. M., Baneijee, D., Howell, K. & Palade, G. E.(1975) J. Cell Biol. 66, 42-59

Reith, E. J. (1968) J. Ultrastruct. Res. 21, 383-414Revel, J. P. & Hay, E. D. (1963) Z. Zellforsch. Mikrosk.

Anat. 61,110-144Ross, R. & Benditt, E. P. (1965) J. Cell Biol. 27, 83-

106Sabatini, D. D. & Blobel, G. (1970) J. Cell Biol. 45,

146-157Salpeter, M. M. (1968) J. Morphol. 124, 387-422Schachter, H., Jabbal, I., Hudgin, R. L., Pinteric, L.,

McGuire, E. J. & Roseman, S. (1970) J. Biol. Chem.245,1090-1100

Scharff, M. D. (1975) The Harvey Lectures (series 69),pp. 125-142, Academic Press, London

Scherft, J. P. & Heersche, J. N. M. (1975) Cell Tiss. Res.157, 353-365

Sheldon, H. & Kimball, F. B. (1962) J. Cell Biol. 12,599-613

Stadler, 3. & Franke, W. W. (1974) J. Cell Biol. 60,297-303

Stein, 0. & Stein, Y. (1973) Biochim. Biophys. Acta 306,142-147

Trelstad, R. L. (1971) J. Cell Blot. 48,689-694Uitto, J. & Prockop, D. J. (1974a) Biochim. Biophys. Acta

336, 234-251Uitto, J. & Prockop, D. J. (1974b) Eur. 1. Biochem. 43,221-230

Uitto, J. & Prockop, D. J. (1974c) Biochem. Biophys. Res.Commun. 60,414-423

Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244,4406-4412

Weinstock, M. (1972) Z. Zellforsch. Mikrosk. Anat. 129,455-470

Weinstock M. & Leblond, C. P. (1974) J. Cell Biol. 60,92-127

Williams, J. A. & Wolff, J. (1972)J. Cell Biol. 54, 157-165Wilson, L., Bamburg, J. R., Mizel, S. P., Grisham, L. M.& Creswell, K. M. (1974) Fed. Proc. Fed. Am. Soc.Exp. Blot. 33, 158-166

Wunderlich, F., Miller, R. & Speth, V. (1973) Science182, 1136-1139

1976

Documents

The Route of Secretion of Procollagen