Xylosylation Is an Endoplasmic Reticulum to Golgi Event

The subcellular site of xylosylation, the first carbohydrate modification of the core protein that initiates glycosaminoglycan chain synthesis, was characterized in situ. Methods were developed to combine electron microscopic (EM) autoradiography and the radiolabeling of semi-intact chondrocytes. In the accompanying paper, Kearns et al. (Kearns, A. E., Vertel, B. M., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11097-11104) presented biochemical and subcellular fractionation studies that utilized semi-intact chondrocytes and radiolabeled UDP sugars to overcome obstacles to the direct analysis of xylosylation. The results suggested that xylosylation begins in the endoplasmic reticulum (ER) and continues in the Golgi. The site of xylosylation was not specified further due to the limitations of subcellular fractionation techniques. The studies described in this report were undertaken to localize these modifications directly in situ. Semi-intact cell preparations were optimized for ultrastructural preservation by modifications of permeabilization methods utilizing nitrocellulose filter overlays. Biochemical analysis demonstrated the exclusive incorporation of UDP-xylose into the cartilage chondroitin sulfate proteoglycan (aggrecan) core protein and 3‘-phosphoadenosine 5‘-phosphosulfate (PAPS) into the highly modified proteoglycan monomer. Immunolocalization studies showed the equivalence of cytoplasmic subcompartments in normal and semi-intact chondrocytes at the levels of light and electron microscopy. Once the biochemical and morphological equivalence of intact and semi-intact cells was established, EM autoradiographic studies were pursued using UDP-[3H]xylose and [35S]PAPS. Based on both qualitative and quantitative data, silver grains resulting from incorporated sulfate were concentrated in the perinuclear Golgi, while those resulting from incorporated xylose were found at the cis or forming face of the Golgi and in vesicular regions of the peripheral cytoplasm associated with the late ER. These data support the view that xylose addition begins in a late ER compartment and continues in intermediate compartments, perhaps including the cis-Golgi

Precursors of chondroitin sulfate proteoglycan are segregated within a subcompartment of the chondrocyte endoplasmic reticulum

Immunocytochemical methods were used at the levels of light and electron microscopy to examine the intracellular compartments of chondrocytes involved in extracellular matrix biosynthesis. The results of our studies provide morphological evidence for the compartmentalization of secretory proteins in the ER. Precursors of the large chondroitin sulfate proteoglycan (CSPG), the major proteoglycan species produced by chondrocytes, were present in the Golgi complex. In addition, CSPG precursors were localized in specialized regions of the ER. Link protein, a separate gene product which functions to stabilize extracellular aggregates of CSPG monomers with hyaluronic acid, was segregated similarly. In contrast, type II procollagen, another major secretory molecule produced by chondrocytes, was found homogeneously distributed throughout the ER. The CSPG precursor-containing ER compartment exhibits a variable tubulo-vesicular morphology but is invariably recognized as an electronlucent, smooth membrane-bounded region continuous with typical ribosome-studded elements of the rough ER. The observation that this ER structure does not stain with antibodies against resident ER proteins also suggests that the compartment is a specialized region distinct from the main part of the ER. These results support recent studies that consider the ER as a compartmentalized organelle and are discussed in light of the possible implications for proteoglycan biosynthesis and processing

Properties of Connexin 46 Hemichannels in Dissociated Lens Fiber Cells

These results verify the existence of Cx46 hemichannels in isolated lens fiber cells. These channels may serve as a pathway for entry of sodium and calcium in the lens

Stable Heparin-Producing Cell Lines Derived from the Furth Murine Mastocytoma

Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line(MST)divides in suspension every 14-18h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 µg of glycosaminoglycan hexuronic acid per 106 cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-0-disulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by \u3e95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells

Sedlin controls the ER export of procollagen by regulating the Sar1 cycle

Newly synthesized proteins exit the endoplasmic reticulum (ER) via coat protein complex II (COPII) vesicles. Procollagen (PC), however, forms prefibrils that are too large to fit into typical COPII vesicles; PC thus needs large transport carriers, which we term megacarriers. TANGO1 assists PC packing, but its role in promoting the growth of megacarriers is not known. We found that TANGO1 recruited Sedlin, a TRAPP component that is defective in spondyloepiphyseal dysplasia tarda (SEDT), and that Sedlin was required for the ER export of PC. Sedlin bound and promoted efficient cycling of Sar1, a guanosine triphosphatase that can constrict membranes, and thus allowed nascent carriers to grow and incorporate PC prefibrils. This joint action of TANGO1 and Sedlin sustained the ER export of PC, and its derangement may explain the defective chondrogenesis underlying SEDT