Coated vesicles purified from chick tendon fibroblasts contain newly synthesized type I procollagen

Experimental

Cell Research 1.57 (1985) 4149

Coated Vesicles Purified from Chick Tendon Fibroblasts Contain Newly Synthesized Type I Procollagen RICHARD Department

GOLDENBERG*

and RICHARD E. FINE

of Biochemistry, Boston University, Bosron, MA 02118, USA

School of Medicine,

Coated vesicles were purified from embryonic chick tendon tibroblasts pulsed with [‘Hlproline. They were morphologically and biochemically similar to coated vesicles purified from other sources. Furthermore, they contained newly synthesized Type I procollagen which was protected from bacterial collagenase digestion unless detergent was present. The procollagen remained associated with coated vesicles during immune precipitation and agarose gel electrophoresis. Data from pulse-chase experiments demonstrated that the specific activity of the coated vesicle preparations was approx. S-fold higher at the 10 min chase point than at either the 0 or 40 min chase points. These data are consistent with the hypothesis that coated vesicles are intermediates in the intracellular transport of newly synthesized Type I procollagen in chick tendon tibroblasts. 0 1985 Academic press, IX.

The route of intracellular transport of Type I procollagen, the precursor of Type I collagen, has been studied in a variety of secretory cells, using morphological and biochemical techniques (for review, see [ 11). Autoradiographic study of [3H]proline-pulsed odontoblasts demonstrated the sequential appearance of protein in the rough endoplasmic reticulum (RER), spherical Golgi elements and cylindrical Golgi elements, where the protein appeared as parallel threads suggestive of packaged procollagen molecules [2]. Histochemical studies of collagensynthesizing cells using ferritin-linked antibodies directed against procollagen have also demonstrated that procollagen is routed through the Golgi apparatus prior to secretion [3, 41. Furthermore, subcellular fractionation studies have localized Type I procollagen to RER and Golgi fractions [5]. Thus, it appears that procollagen follows an intracellular pathway similar to that reported for pancreatic secretory proteins [6] and membrane glycoproteins [7]. These proteins are synthesized on the rough endoplasmic reticulum (RER) and transported to the cell surface via the Golgi, where maturation of oligosaccharides occurs. Although it is generally accepted that the intracellular transfer of procollagen and other secretory proteins between these compartments is vesiclemediated, the mechanism whereby procollagen is specifically transported in synchrony with its maturation remains obscure. * To whom offprint requests should be sent. Address: Department of Biochemistry, Boston University School of Medicine, Room K-402. 80 East Concord str., Boston, MA 02118, USA. Copyright @ 1985 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/85 $03.00

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Evidence has accumulated that clathrin-coated vesicles 181participate in the intracellular transport of membrane and secretory proteins. For example, coated vesicles have been shown to transport the G protein of vesicular stomatitis virus (VSV), a transmembrane glycoprotein, from the RER to the Golgi, and then to the plasma membrane [9]. In addition, it has been reported that in a B lymphocyte-derived cell line, coated vesicles are involved in the intracellular transport of both secretory and integral membrane proteins [IO]. Finally, preliminary evidence obtained in our laboratory indicates that coated vesicles purified from chick skeletal muscle contain newly synthesized acetylcholinesterase [ 111. In view of this evidence, and the fact that both G protein and procollagen are transported to the cell surface with similar kinetics [12, 131, we investigated the role of coated vesicles in procollagen transport. For our studies, we used embryonic chick tendon fibroblasts, which devote approx. 30% of their protein synthesis to Type I procollagen [ 141.

MATERIALS

AND METHODS

Isolation and Labelling of Tendon Fibroblasts Leg tendon tibroblasts from 300 17-day chick embryos (Spafas, Inc.), were isolated by the method of Dehm & Prockop [14] and resuspended in 140 ml of Krebs II buffer (141 supplemented with 2% fetal bovine serum and 50 udrnl ascorbate. Following a 40 min pre-incubation at 37”C, 7 mCi of [2,3,‘H]r,-proline (New England Nuclear) was added to the cell suspension. After 10 min, 3 ml of 1 M proline was added. At 0, 10 and 40 min following chase, three equivalent volumes of cells were separated from the medium and washed three times with ice-cold Krebs II containing 100 ur$ml cycloheximide to block further protein synthesis.

Purification of Coated Vesicles Coated vesicles were purified from freshly dissociated, radioactively labelled tendon fibroblasts using a modification of the method of Pearse [ 151described by Rothman & Fine [ 161.Each fibroblast cell pellet was thawed in 8.3 ml of 1 mM EGTA, 10 mM Tris-HCl, pH 7.5 and homogenized with ten strokes in a tight fitting Dounce homogenizer (Kontes Co., Vineland, N.J.). After addition of 1110vol 1 M MES buffer [15], each homogenate was centrifuged at 900 g for 5 min. The pellet was re-extracted in 0.1 M MES buffer and the supematants combined. In order to facilitate the purification of coated vesicles from labelled tendon tibroblasts, a homogenate of leg tendons from 600 17-day chick embryos in 150 ml 0.1 M MES buffer was prepared using three IS-set bursts in a Sorvall Omni-mixer (Sorvall, Inc., Newton, Conn.). After centrifuging this homogenate at 900 g for 5 min, the pellet was reextracted and to each labelled fibroblast extract, one third of this extract was added to serve as carrier. This combined post-nuclear supematant was incubated with 10 &ml pancreatic RNase A (Sigma) for 30 min at room temperature and subjected to velocity and equilibrium sucrose gradient centrifugation as described 1171.Agarose gel electrophoresis was carried out according to the method of Rubenstein et al. [18]. Briefly, the coated vesicles were removed from the equilibrium gradients, pelleted and then resuspended in 0.50 ml of 0.05 M MES. The samples were then placed in adjacent wells of a 0.15% agarose gel containing 0.05 M MES. A sample of brain-coated vesicles purified as previously described [17] was placed in adjoining well. Electrophoresis was performed at 15 V for 24 h at 4°C. Turbid bands corresponding to the purified flbroblast-coated vesicles (which migrate about 2.5 cm toward the anode) were excised from the gel. Coated vesicles were separated from the agarose by homogenization and centrifugation for 10 set at 15000 g in the Eppendorf centrifuge (Brickmann Instruments, Inc., Westbury, N.Y.). The coated vesicle containing supematants were assayed for protein by the method of Lowry et al. [ 191 and the amount of radioactivity was determined by Exp CeNRes 157(1985)

Coated vesicles contain type I procollagen

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-II

1

*

1

1. Comparison of polypeptide composition of coated vesicles purified from chick tendon fibroblasts vs. calf brain. Agarose-purified coated vesicles were prepared as described in Materials and Methods and subjected to PAGE on a 7.5 % gel. The Coomassie Blue-stained gel is shown. The migration position of clathrin and the 55 K and 100 K polypeptide components of calf-coated vesicles is indicated. I, 4 ug chick tendon coated vesicles; 2, 5 ug calf brain-coated vesicles. Fig. 2. Negatively stained preparation of agarose-purified coated vesicles from chick tendon tibroblasts. 3 ug of coated vesicles was placed on a carbon-shadowed Formvar-coated EM grid, 400 mesh. After a 1 min of incubation at 25°C the sample was removed from the grid, washed with several drops of 150 mM NaCl, 15 mM NaP04, pH 7.2 and negatively stained with 1% many1 acetate. Samples were examined in a Philips 300 electron microscope. x 158000. Fig.

scintillation counting. The agarose-purified coated vesicles from all three chase points were then pooled for subsequent experiments.

Immune Precipitation For immune precipitation experiments, tibroblasts were pulse-labelled for 10 min with [3H]proline and coated vesicles were purified through two sucrose gradients following a IO-min chase period as described previously [17]. Four ml of a mouse monoclonal IgG antibody directed against bovine brain clathrin light chain LCa with broad tissue and species cross-reactivity [21] (kindly provided by Dr Peter Parham, Stanford University, Calif.) or against dansyl-hapten (kindly provided by Dr V. Oi, Stanford University) was added to 50 ul of a 1 mg/ml coated vesicle suspension. After a 30 mitt incubation at 23”C, rabbit anti-mouse IgG (Miles Laboratory, Inc., Elkhart, Ind.) was added for 30 min and immune complexes were separated by centrifugation (Eppendorf). Immune precipitates were dissolved directly in sample buffer for analysis by SDS-PAGE (see below). The immune supematants were subjected to agarose gel electrophoresis and the appropriate area of the gel was excised in order to recover any coated vesicles.

Collagenase

Assay

For digestion with purified bacterial collagenase (Type III, Advanced Biofactures, Lynnbrook, N.Y.), samples were resuspended in 50 mM Bis-HCl buffer, pH 7.5, 3 uM CaClz and 10 PM Nethylmaleimide. 1% Ttiton X-100 was included where specified. Following addition of 20 units of collagenase, samples were incubated at 37°C for 90 min. The reaction was terminated by the addition of ice-cold EGTA to a final concentration of 2 mM. Exp Cell Res 157 (198s)

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Table 1. Specific activity

of agarose purified labelled chick tendon fibroblasts”

Minutes after chase

Total proteinb (YE9

0 10 40

2.7 3.1 3.0

Total cpm 448 2 876 548

coated vesicles from [3H]proline-

Sp. act. (cpmhg) 166 928 183

a At each chase point, the initial homogenate contained 45 mg protein and 6~10~ TCA-insoluble cpm. b Determined by Lowry assay [19].

SDS-PAGE SDS-PAGE was carried out according to the method of Laemmli [20] using a 7.5 % running gel and a 4% stacking gel.

Fluorography Following impregnation with ENHANCE (New England Nuclear) slab gels were dried on a Model 224 Slab Gel drier (BIO-RAD Laboratories, Richmond, Calif.) under vacuum and heat, and exposed at -70°C to XR-5 film (Eastman Kodak, Rochester, N.Y.). The developed films were scanned with a Transidyne 2955 densitometer (Bansidyne General Corporation, Ann Arbor, Mich.).

RESULTS Characterization

of Coated Vesicles Purified from Chick Tendon Fibroblasts

Coated vesicles were purified from [3H]proline-labelled chick tendon fibroblasts and from calf brain as described in Materials and Methods. The polypeptide composition of coated vesicles from both sources is compared in fig. 1. The major polypeptide in the fibroblast-coated vesicle preparation had the same mobility on SDS gels as clathrin, the major coat protein of brain-coated vesicles [151. The fibroblast preparation also contained characteristic polypeptides with apparent molecular weights of 100000 and 55000 kD, as seen in brain-coated vesicles. A negatively stained preparation of agarose-purified chick tendon fibroblastcoated vesicles is shown in fig. 2. From a count of 500 particles, it was calculated that 95% were coated vesicles, 3 % were empty coats and 2% were smooth vesicles. There was negligible contamination by other membrane fractions. Furthermore, the coated vesicles were comparable in size to those purified from other sources [8, 15, 161, averaging approx. 1000 A in diameter. Pulse-chase experiments were carried out in order to determine whether coated vesicles were intermediates in the intracellular transport of newly synthesized protein. The specific activity of agarose-purified coated vesicles at three different points during the chase period is shown in table 1. The yield of coated Exp Cell Res 157 (1985)

Coated

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vesicles contain type I procollagen

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123

3. SDS-PAGE of [3H]proline-labelled agarose-purified coated vesicles. Samples were prepared for electrophoresis on a 7.5% gel before or after treatment with bacterial collagenase. Following fluorography, radioactive bands were visualized as described in Materials and Methods. Autoradiographs of (A) coated vesicle preparations; (B) [‘4C]proline-labelled calvaria medium proteins. Treated with bacterial collagenase in I, absence of detergent; 2, presence of 1% Triton X-100; 3, untreated medium proteins.

Fig.

vesicles, as determined by assay of total protein, was comparable at each chase point, yet the specific activity at the 10 min chase point was five times greater than either the 0 or 40 min chase point. At each chase point, the initial homogenate contained approx. 45 pg of protein and 6x IO6cpm of TCA-precipitable protein. Association between Coated Vesicles Purified from Chick Tendon Fibroblasts and Newly Synthesized Type I Procollagen

In order to determine whether coated vesicles purified from chick tendon fibroblasts contained procollagen, coated vesicle preparations were treated with bacterial collagenase in the presence and absence of 1% Triton X-100. The coated vesicles were then fractionated on SDS gels and fluorography was performed in order to identify newly synthesized proteins (fig. 3 A). Partially purified [i4C]proline-labelled calvaria medium proteins, including Type I procollagen and its processing intermediates, were also treated with enzyme in order to monitor the specificity of the bacterial collagenase assay and its sensitivity to detergent (fig. 3 B). In the coated vesicle preparation treated with bacterial collagenase, two radioactive polypeptides, having the same mobility as proal chains and proa2(1) chains were identified. Densitometric scanning indicated that the ratio of proal(1) chains to proa2(1) chains was 3 : 1. In the presence of 1% Triton X-100 Exp Cell RPS 157 (1985)

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23412

3

4

Fig. 4. SDS-PAGE of immune precipitates and agarose-purified immune supernatants. Samples were

prepared for PAGE on a 7.5 % gel directly following immune precipitation or agarose purification of the immune supematant as described in Materials and Methods. (A) Coomassie Blue-stained gel; (B) autoradiogram, I, anti-clathrin immune precipitate; 2, anti-dansylhapten immune precipitate; 3, coated vesicle region of agarose purified anti-clathrin immune supematant; 4, coated vesicle region of agarose-purified anti-dansylhapten immune supematant.

and bacterial collagenase, both bands were completely digested, suggesting that the procollagen chains were sequestered within membrane limited vesicles. As shown in fig. 3 B, the presence of detergent had no apparent effect on the specificity or sensitivity of the collagenase assay. In order to determine whether sequestered procollagen was contained within coated vesicles rather than within a previously undetected smooth vesicle contamination of our preparation, the following experiment was performed. A preparation of coated vesicles from [3H]proline-labelled tibroblasts was purified through two sucrose gradients and divided in half. Mouse monoclonal IgG antibody directed against the clathrin light chain, LCa [21] was added to one aliquot and mouse monoclonal IgG antibody directed against dansyl hapten was added to the other aliquot. Rabbit anti-mouse IgG was then added to both and the immune complexes were separated by centrifugation. The supernatants were subjected to agarose gel electrophoresis and the appropriate area of the gel was excised in order to recover coated vesicles. Electrophoretic analysis of the anti-clathrin immune precipitates and the appropriate region of the agarose gel indicated that most of the clathrin was recovered in the Exp Cell Res 157 (1985)

Coated vesicles contain type I procollagen

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immune precipitate and none was detectable in the coated vesicle region of agarose gel. Conversely, analysis of the anti-dansyl hapten immune precipitates failed to reveal any clathrin, most of which was recovered in the agarose gel at its normal migration position (fig. 4A). Fluorography of this gel confirmed the association between newly synthesized procollagen and coated vesicles (fig. 48). [3HlProline-labelled polypeptides, including proal(1) chains, proa2(1) chains as well as other minor species were recovered in the anti-clathrin immune precipitate and were no longer detectable in the coated vesicle region of the agarose gel. Conversely, labelled protein was not detected in the anti-dansyl hapten immune precipitates, but was present in the coated vesicle portion of the agarose gel. The amount of labelled procollagen associated with coated vesicles was greater in this experiment (fig. 4B) than in the previous experiment (fig. 3A). This would be expected, since ten times as much coated vesicle protein was used for immune precipitation. In addition, the specific activity of this coated vesicle preparation was higher because the fibroblasts were all chased for 10 min, which was shown to be optimum for labelling coated vesicles. Thus, the association between newly synthesized procollagen and clathrincontaining coated vesicles is specitic, as evidenced by quantitative immune precipitation, and is maintained during agarose gel purification. DISCUSSION Although coated vesicles have been observed in secretory cells in general (for review, see [22]) and in collagen-synthesizing cells in particular [23], they have not previously been purified from tibroblasts. In this report, we demonstrate that coated vesicles purified from chick tendon tibroblasts are morphologically and biochemically identical to those purified from other sources [S, IS, 231. Furthermore, electrophoretic analysis of agarose-purified coated vesicles showed that they contained Type I procollagen which was protected from digestion with bacterial collagenase. Finally, the association between coated vesicles and newly synthesized Type I procollagen was maintained during immune precipitation and agarose gel electrophoresis. It should be noted that our data do not exclude the possibility that Type I procollagen is present in other clathrin-coated structures such as Golgi stacks which form closed vesicles during our purification protocol. The recovery of [3H]proline-1abelled procollagen in agarose-purified coated vesicles is low. In view of our recoveries of coated vesicles from both brain and cultured cells [17, 161, as well as data from other laboratories [24], we estimate that our yield of coated vesicles after agarose gel electrophoresis represents l-5% of the amount present in the initial homogenate. Since about 30% of the TCA-precipitable protein in the cell homogenate represents Type I procollagen [14], we can estimate that at the 10 min chase point approx. 5-10% of this pool is located within coated vesicles. In conjunction with the fact that the specific 4-858333

Exp Cell Res 157 /I9851

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activity of the coated vesicles peaked at the 10 min chase point, this suggests that coated vesicles may function as intermediates in the intracellular transport of Type I procollagen in chick embryo fibroblasts; however, whether coated vesicles represent the major vehicle for procollagen transport to the cell surface, as has been suggested for G protein [16], is unclear. Since a significant fraction of newly synthesized procollagen is rapidly degraded in tibroblasts [25], it is possible that the newly synthesized procollagen in coated vesicles is en route to the lysosomes. Even though the data presented in this paper are consistent with the involvement of coated vesicles in at least one step in the intracellular transport of newly synthesized procollagen, we do not know the identity of this step(s). Previous work [9, 10, 161is consistent with the involvement of coated vesicles in at least two stages of the intracellular transport of newly synthesized integral membrane and secretory proteins, an early stage involving endoglycosidase H sensitive molecules and a late endoglycosidase H resistant stage. Since Type I procollagen remains in the high mannose, endoglycosidase H sensitive state during its passage through the cell and subsequent secretion [26], we cannot perform analogous experiments in this system. Finally, the ratio of proal chains to proa2(1) chains in coated vesicles was 3 : 1 instead of the 2 : 1 ratio expected for mature Type I procollagen. The excess of proal chains could be due to the presence of either proal trimers or Type III procollagen chains, which have the same electrophoretic mobility as proal(1) chains. The presence of small amounts of both extracellular al(I) trimers and Type III collagen in embryonic chick tendon tibroblasts has been reported [27, 281. REFERENCES 1. Fessler, .I H & Fessler, L I, Ann rev biochem 47 (1978) 129. 2. Weinstock, M & Leblond, C P, J cell biol 60 (1974) 92. 3. Nist, C, Von Der Mark, K, Hay, E D, Olsen, B R, Bornstein, P, Ross, R & Dehm, P, J cell biol65 (1975) 75. 4. Olsen, B R & Prockop, D J, Proc natl acad sci US 71 (1974) 2033. 5. Uchida, N, Smilowitz, H, Ledger, D W & Tanzer, M L, J biol them 255 (1980) 8638. 6. Palade, G, Science 189 (1975) 347. 7. Lodish, H F & Rothman, J E, Sci Am 240 (1979) 48. 8. Pearse, B M F, Proc natl acad sci US 73 (1976) 1255. 9. Rothman, J E, Bursztyn-Pettegrew, H & Fine, R E, J cell biol 86 (1980) 162. 10. Kinnon, C & Owen, M, J biol them 258 (1983) 8470. 11. Porter-Jordan, K, Johnson, R & Fine, R, J cell bio195 (1982) 465 a. 12. Dehm, P J & Prockop, D J, Biochim biophys acta 240 (1971) 358. 13. Knipe, D M, Baltimore, D & Lodish, H F, J virol 21 (1977) 1128. 14. Dehm, P & Prockop, D J, Biochim biophys acta 264 (1972) 375. 15. Pearse, B M F, J mol biol 97 (1975) 93. 16. Rothman, J E & Fine, R E, Proc natl acad sci US 77 (1980) 780. 17. Blitz, A L, Fine, R E & Toselli, P, J cell biol 75 (1977) 735. 18. Rubenstein, J L R, Fine, R E, Luskey, B D & Rothman, J E, J cell biol 89 (1981) 357. 19. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 20. Laemmli, U K, Nature 227 (1970) 680. Exp Ceil Res 1.57 (198s)

Coated vesicles contain type I procollagen 21. 22. 23. 24. 25. 26. 27. 28.

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Brodsky, F M & Parham, P, J mol biol 167 (1983) 197. Kartenbeck, J, Coated vesicles, p. 243. Cambridge University Press, Cambridge (1980). Trelstad, R L, Havashi, K & Toole, B P, J cell biol 62 (1974) 815. Mello, R J, Brown, M S, Goldstein, J L & Anderson, R G W, Cell 20 (1980) 829. Berg, R A, Schwartz, M L & Crystal, R G, Proc natl acad sci US 77 (1980) 4746. Clark, C C, J biol them 254 (1979) 10798. Herrmann, H, Dessau, W, Fessler, L I & Von Der Mark, K, Em j biochem 105 (1980) 63. Jiminez, S A, Bashey, R I, Benditt, M & Rankowski, R, Biochem biophys res commun 78 (1977) 1354.

Received July 26, 1984 Revised September 17, 1984

Printed

in Sweden

Exp Cell Res 157 (1985)