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Precursors of Chondroitin Sulfate Proteoglycan Are Segregated Within a Subcompartment of the Chondrocyte Endoplasmic Reticulum Barbara M

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Precursors of Chondroitin Sulfate Proteoglycan Are Segregated Within a Subcompartment of the Chondrocyte Endoplasmic Reticulum Barbara M Precursors of Chondroitin Sulfate Proteoglycan Are Segregated within a Subcompartment of the Chondrocyte Endoplasmic Reticulum Barbara M. Vertel, Angel Velasco, Sherrie LaFrance, Linda Waiters, and Karen Kaczman-Daniel Department of Cell Biology and Anatomy, University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064; and Department of Biology, Syracuse University, Syracuse, New York 13210 Abstract. Immunocytochemical methods were used at chondrocytes, was found homogeneously distributed the levels of light and electron microscopy to examine throughout the ER. The CSPG precursor-containing the intracellular compartments of chondrocytes in- ER compartment exhibits a variable tubulo-vesicular volved in extracellular matrix biosynthesis. The results morphology but is invariably recognized as an elec- of our studies provide morphological evidence for the tronlucent, smooth membrane-bounded region continu- compartmentalization of secretory proteins in the ER. ous with typical ribosome-studded elements of the Downloaded from Precursors of the large chondroitin sulfate proteogly- rough ER. The observation that this ER structure does can (CSPG), the major proteoglycan species produced not stain with antibodies against resident ER proteins by chondrocytes, were present in the Goigi complex. also suggests that the compartment is a specialized re- In addition, CSPG precursors were localized in spe- gion distinct from the main part of the ER. These cialized regions of the ER. Link protein, a separate results support recent studies that consider the ER as gene product which functions to stabilize extracellular a compartmentalized organelle and are discussed in aggregates of CSPG monomers with hyaluronic acid, light of the possible implications for proteoglycan bio- jcb.rupress.org was segregated similarly. In contrast, type II procolla- synthesis and processing. gen, another major secretory molecule produced by on November 6, 2017 ROTEINS destined for secretion are transported from reported, and movement from the ER to the Golgi is fre- p the rough ER (rER) ~ to the Golgi and through the quently the rate-limiting step in the transport of membrane Golgi to the extracellular space (Palade, 1975). The and secretory proteins to the cell surface (Strous and Lodish, polypeptides undergo covalent co- and posttranslational mod- 1980; Fitting and Kabat, 1982; Lodish et al., 1983; Fries et ifications and may become associated with other polypep- al., 1984; Scheele and Tartakoff, 1985; Williams et al., tides as they continue along their route (Farquhar and Palade, 1985). These observations suggest that processes in the ER 1981; Farquhar, 1985). Eventually, secreted proteins are may play a role in the regulation of exocytotic membrane sorted from lysosomal, membrane, and even other secretory trafficking. Other results would suggest that functional sub- proteins (Farquhar, 1985; Pfeffer and Rothman, 1987). Al- compartments may be characteristic of the ER as well (re- though considerable progress has been made in the elucida- viewed by Rose and Doms, 1988). tion of the cellular and molecular bases of these processes, A variety of exocrine (Strous and Lodish, 1980; Lodish et many questions remain concerning the specific sites, se- al., 1983; Fries et al., 1984; Scheele and Tartakoff, 1985; quences of events, and regulatory mechanisms involved in Saraste et al., 1986) and endocrine (Green and Shields, intracellular transport. For example, it is known that the 1984; Orci et al., 1987; Chung et al., 1989) cell systems are Golgi complex functions as a multicompartment organelle being used to investigate the movement of proteins through and is involved in the sorting of proteins into lysosomes and the exocytotic pathway. Studies using viruses and their glyco- in the segregation of secretory products into distinct types of proteins have been particularly informative (reviewed by secretory vesicles, but the mechanisms controlling these Pfeffer and Rothman, 1987; Rose and Doms, 1988). Other events are not known (Farquhar, 1985; Burgess and Kelly, processes amenable to studies of intracellular trafficking in- 1987; Orci et al., 1987; Pfeffer and Rothman, 1987; Chung clude those involved in the synthesis and assembly of ex- et al., 1989). New data indicate that the segregation of mole- tracellular matrix (ECM) molecules. Cells that elaborate cules may begin earlier in the exocytotic pathway (Munro ECM are dedicated to the production of specific, well- and Pelham, 1987; Rothman, 1987; Kabcenell and Atkinson, characterized proteins, often in large quantities. ECM mole- 1986). Differential rates of exit from the ER have been cules undergo numerous processing events and may partici- 1. Abbreviations used in this paper: CSPG, chondroitin sulfate proteogly- pate in a variety of macromolecular interactions as they move can; ECM, extracellular matrix; rER, rough endoplasmic reticulum. through cytoplasmic membrane compartments (reviewed by © The Rockefeller University Press, 0021-9525/89/1011827110 $2.00 The Journal of Cell Biology, Volume 109, October 1989 1827-1836 1827 Hay, 1981). We have used cartilage cells in culture as a model present study we use immunoelectron microscopy to demon- system for the study of protein trafficking. Chondrocytes are strate that this heterogeneity exists within the ER of chondro- committed to the synthesis, processing, and assembly of an cytes, providing morphological evidence for the sorting and ECM composed of abundant amounts of two major macro- segregation of newly synthesized molecules in the ER. These molecules, type II collagen, and chondroitin sulfate pro- results suggest that segregation within the ER can occur dur- teoglycan (CSPG). Large aggregates of CSPG are formed ing the normal course of synthesis and processing of ECM extracellularly by the link protein-stabilized association of molecules and are discussed in the context of subcompart- CSPG monomer and hyaluronic acid. The CSPG monomer mentalization of the ER. itself (Mr 1-5 x 106) has a 300-350-K core protein with many covalently attached chondroitin sulfate and keratan sul- fate glycosaminoglycan chains, N-linked oligosaccharides, Materials and Methods and O-linked oligosaccharides (Hascall, 1981; Heineg~d and Paulsson, 1984; Hassell et al., 1986; Lohmander and Materials Kimura, 1986; Carney and Muir, 1988; Ruoslahti, 1988). Fertile White Leghorn chicken eggs were purchased from Cornell Univer- Link protein is also characterized by the addition and further sity (Ithaca, NY) and Sharp Farms (Glen Ellyn, IL). Trypsin, Ham's F-12 modification of N-linked oligosaccharides. In this case, mul- medium, FCS, antibiotic-antimycotic mixture, Hank's balanced salt solu- tion (HBSS), and goat serum were obtained from Grand Island Biological tiple forms of link protein are generated by differential gly- Supply Co. (Grand Island, NY). Testicular hyaluronidase was a product of cosylation and sulfation (Caterson et al., 1985; Choi et al., Leo (Helsingborg, Sweden). Goat anti-guinea pig IgG, goat anti-rabbit 1985; Hering and Sandell, 1988, manuscript submitted for lgG, and goat anti-mouse IgG coupled to FITC or TRITC, and Fab frag- publication). The biosynthetic modifications of type II colla- ments of goat anti-rabbit IgG coupled to horseradish peroxidase were ob- gen involve the translation of type II procollagen, hydroxyla- tained from Cappel Laboratories (Malvern, PA). Saponin, sodium borohy- dride, diaminobenzidine, potassium ferrocyanide, polyethylene glycol, tion of proline and lysine, glycosylation of asparagine and sodium m-periodate, and BSA were products of Sigma Chemical Co. (St. hydroxylysine, formation of interchain disulfide bonds, and Louis, MO). Glutaraldehyde, osmium tetroxide, hydroxypropyl methacry- Downloaded from assembly of triple helical molecules. The enzymatic conver- late, and Epon were purchased from Electron Microscopy Sciences (Fort sion of procoilagen to collagen and collagen fiber formation Washington, PA); Lowicryl and paraformaldehyde were from Polysciences, Inc. (Warrington, PA); tannic acid was from Mallinckrodt (Paris, KY), pro- occur extracellularly (Prockop et al., 1979; Olsen, 1981; tein A was from Pharmacia Fine Chemicals (Uppsala, Sweden): and Kivirikko and Myllyla, 1984). tetrachloroauric acid was from Fisher Scientific (Pittsburgh, PA). Intracellular sites for the synthesis, processing, and as- sembly of cartilage ECM molecules have been investigated. Cell Culture Ultrastructurai studies have characterized the extensive rER jcb.rupress.org Cartilage cells were prepared from the sterna of 15-d-old chicken embryos and Golgi complex of chondrocytes (Thyberg et al., 1973; as described by Cahn et al. (1967). For immunofluorescence studies, cells Hascall, 1980; Takagi et al., 1981). Functional properties of were cultured in monolayer on gelatinized, carbon-coated coverslips at a chondrocyte organelles have been determined using autora- density of 4 x 105 cells per 60-mm tissue culture dish in 3 ml Ham's F-12 diography (Revel and Hay, 1963; Fewer et ai., 1964; God- medium containing 10% FCS and I% antibiotic-antimycotic mix. For elec- tron microscopy, cells were cultured onto gelatinized 35-ram tissue culture man and Lane, 1964; Kimata et al., 1971), subcellular frac- dishes at the same density in 1.5 ml of the same medium. Cell cultures were on November 6, 2017 tionation (Feilini et al., 1981; Geetha-Habib et al., 1984; fed with fresh medium on days 2 and 3 and fed again several hours before Campbell and Schwartz, 1988),
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