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Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3-phosphoadenosine 5-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456 -2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed ϳ29% identity and ϳ48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.Chondroitin sulfate proteoglycans are found in various tissues as molecules having divergent molecular architecture (1, 2). Chondroitin sulfate chains attached to chondroitin sulfate proteoglycans appear to play important roles in the formation and maintenance of cartilage tissue, because undersulfation of chondroitin sulfate resulted from the defective synthesis of PAPS 1 (3, 4) or defective sulfate transport (5) was found to cause underdevelopment of skeleton. Various chondroitin sulfate proteoglycans have been reported to be present in the brain (6, 7) and to function in the regulation of neurite outgrowth and neural cell adhesion (8 -12), neuronal migration (13), and the survival of neurons (14). Chondroitin sulfate chains are also shown to be involved in the interaction with CD44 (15, 16), phospholipase A 2 (17), Plasmodium falciparuminfected erythrocytes (18), and L-selectin (19). Chondroitin sulfates have sulfate group at various positions of the component sugars; position 6 and/or 4 of GalNAc residues and position 2 or 3 of GlcA residues. The pattern of sulfation of chondroitin sulfate chains varies with the source of the proteoglycans, development of animal (20 -22), and malignant change (23), suggesting that sulfate moieties attached to the specific position of the component sugars may be related to ...

Chondroitin 4-sulfotransferase, which transfers sulfate from 3-phosphoadenosine 5-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3,5-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.Chondroitin sulfate proteoglycan is found in various tissues and thought to play important roles in various cellular interactions involving cell adhesion (1, 2), regulation of neurite outgrowth (3-6), migration of neural crest cells (7), binding of phospholipase A 2 (8), and an adherence receptor for Plasmodium falciparum-infected erythrocytes (9). In most of mammalian and avian chondroitin sulfate proteoglycans, the glycosaminoglycan chains bear sulfate at position 4 or 6 of N-acetylgalactosamine residues. The ratio of chondroitin 4-sulfate/chondroitin 6-sulfate has been reported to vary with the development of animals (10 -12), malignant change (13), and leukocyte differentiation (14). Sulfation of positions 4 and 6 of GalNAc residue was shown to be catalyzed by different sulfotransferases (15); chondroitin 4-sulfotransferase (C4ST) 1 and chondroitin 6-sulfotransferase (C6ST). Characterization of the purified sulfotransferases and molecular cloning of these cDNAs are basically important to reveal the functional roles of these chondroitin sulfate isomers. We have purified C6ST from the culture medium of chick chondrocytes (16) and cloned the cDNA (17). Unexpectedly, the purified C6ST was found to catalyze not only chondroitin but also keratan sulfate (18) and sialyl N-acetyllactosamine oligosaccharides (19). We previously observed that C4ST was also secreted to the culture medium of chick chondrocytes (20), but the purification of C4ST from the culture medium of chick chondrocytes has been hampered by the presence of an excess amount of C6ST. Unlike chick chondrocytes, rat chondrosa...

We observed the disassembly of endoplasmic reticulum (ER) exit sites (ERES) by confocal microscopy during mitosis in Chinese hamster ovary (CHO) cells by using Yip1A fused to green fluorescence protein (GFP) as a transmembrane marker of ERES. Photobleaching experiments revealed that Yip1A-GFP, which was restricted to the ERES during interphase, diffused throughout the ER network during mitosis. Next, we reconstituted mitotic disassembly of Yip1A-GFP-labeled ERES in streptolysin O-permeabilized CHO cells by using mitotic L5178Y cytosol. Using the ERES disassembly assay and the anterograde transport assay of GFP-tagged VSVGts045, we demonstrated that the phosphorylation of p47 by Cdc2 kinase regulates the disassembly of ERES and results in the specific inhibition of ER-to-Golgi transport during mitosis. INTRODUCTIONThe first step of anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus is the recruitment of the cytosolic coat complex (COP II) to specialized sites of ER export, called ER exit sites (ERES) or transitional ER (tER), where cargo proteins are actively sorted and concentrated in COP II-coated vesicles (Hobman et al., 1998;Hong, 1998;Kaiser and Ferro-Novick, 1998;Stephens et al., 2000;Muniz et al., 2001;Aridor et al., 2001;Barlowe, 2002). Morphologically, this compartment was originally defined as a ribosome-free ER subdomain that is continuous with the rough ER and contains protrusions resembling budding vesicles (Merisko et al., 1986;Orci et al., 1991;Rossanese et al., 1999;Shugrue et al., 1999). Because ERES are starting points for ER-to-Golgi transport, the balance of which influences Golgi morphology, the biogenesis of ERES is thought to be closely coupled to Golgi biogenesis (Ward et al., 2001;Bevis et al., 2002). Thus, ERES are maintained as an important subdomain of the ER, not only for the sorting/budding of proteins from the ER but also for Golgi biogenesis. However, less is known about the mechanism by which ERES maintain their distinct morphological identity as subdomains within the general ER or how their formation/disassembly is regulated during the cell cycle. Few studies have examined either ERES dynamics within the cell or the biochemical requirements for ERES formation.Time-lapse imaging has been used to study the dynamic behavior of ERES by using fluorescence-tagged cytosolic COP II components as probes, such as in Pichia pastoris or Chinese hamster ovary (CHO) cells by using Sec13-GFP (Hammond and Glick, 2000;Bevis et al., 2002), or in HeLa cells by using Sec23A-YFP (Stephens, 2003). These studies revealed that ERES are long-lived compartments that move slowly on the ER network and with apparently restricted mobility. Despite the limited movement of individual ERES, fusion, fission, or de novo formation of ERES can be seen in interphase HeLa cells (Stephens, 2003). The cell cycle-dependent accumulation of ERES also has been studied in mammalian cells and yeast. Immunofluorescence studies using anti-Sec13 antibodies revealed that ERES grew in number during i...