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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01191

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Stem domains of heparan sulfate 6-O-sulfotransferase are required for Golgi localization, oligomer formation and enzyme activity

¹ Institute for Molecular Science of Medicine, Aichi Medical University, 21 Yazako, Nagakute, Aichi 480-1195, Japan
² Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA

View larger version (44K): [in a new window]   Fig. 1. Localization of HS6ST-1, -2, and -3 in the trans-Golgi cisternae of CHO-K1 cells. (A) Schematic representation of the C-terminally GFP-tagged murine HS6ST-1, -2, and -3 proteins (m6ST1-EGFP, m6ST2-EGFP, and m6ST3-EGFP). Y denotes the N-glycosylation sites. The pink and blue boxes and the green ovals indicate the stem domain, the PAPS-binding domains and the EGFP protein, respectively. The numbers shown below indicate the amino acid number in the cytoplasmic, transmembrane, lumenal, linker, and EGFP domains. The numbers in parentheses indicate the amino acid number in the stem domains that we defined. The black arrows indicate the position of the conserved Glu-Tyr residues that are detected as the amino-termini of the HS6STs secreted in the culture medium. TM, transmembrane region. (B) Intracellular co-localization of m6ST1-EGFP, m6ST2-EGFP, and m6ST3-EGFP with the Golgi marker GM130. Cells transfected with plasmids encoding m6ST1-EGFP, m6ST2-EGFP, or m6ST3-EGFP were fixed and stained with rabbit polyclonal anti-GM130 antiserum followed by Alexa Fluor 594 goat anti-rabbit IgG secondary antibodies. From left to right, the panels show phase contrast, GM130 immunofluorescence, GFP tag fluorescence, and merged images. (C) CHO-K1 cells transfected with m6ST1-EGFP were pretreated with 100 µM cycloheximide for 30 minutes, then treated with 10 µg/ml BFA for 1 hour or 20 µM nocodazole for 1 or 2 hours in the presence of cycloheximide. Fixation and staining proceeded as in Fig. 1B.

View larger version (36K): [in a new window]   Fig. 2. Localization of HS6ST proteins to the Golgi apparatus in the absence of endogenous HS2ST. (A) In the HS2ST-deficient cell line CHO-F17, the transfected m6ST1-EGFP, m6ST2-EGFP, and m6ST3-EGFP proteins co-localize with the Golgi marker GM130. From left to right, phase contrast, anti-GM130 immunofluorescence, GFP tag fluorescence, and merged images. (B) Left panel, schematic representation of the N-terminally FLAG-Iip33-tagged m2ST, m6ST1, m6ST2 and m6ST3 fusion proteins. Red and blue boxes indicate the FLAG-tag and Iip33-tag respectively. The transmembrane portion of Iip33 is used to express m2ST and m6STs in the ER. Right panel, sulfotransferase activity of FLAG-Iip33-tagged m2ST, m6ST1, m6ST2, and m6ST3 relative to the FLAG-tagged m2ST, m6ST1, m6ST2 and m6ST3 proteins. The sulfotransferase activity of the FLAG-tagged wild-type version was set as 100%. FLAG-Iip33-tagged proteins expressed in the cell showed comparable activity to the FLAG-tagged wild-type protein. The results shown are the mean±s.d. of three experiments. (C) Forced expression of HS2ST or HS6ST1 in the ER does not affect the Golgi localization of HS6ST1 or HS2ST, respectively. Cells were co-transfected with plasmids encoding the FLAG-tagged Iip33/HS2ST (FLAG/p33/m2ST) fusion protein and m6ST1-EGFP (upper panels), or with the Iip33/HS6ST1 (FLAG/p33/m6ST1) fusion protein and m2ST-EGFP (lower panels). The transfected cells were fixed and stained with a mouse monoclonal anti-FLAG antibody followed by Alexa Fluor 594 goat anti-mouse IgG secondary antibodies. From left to right, phase contrast, anti-FLAG immunofluorescence, EGFP tag fluorescence, and merged images. The EGFP fluorescence remained in the Golgi apparatus regardless of the ER localization of the other proteins.

View larger version (133K): [in a new window]   Fig. 3. The stem region of the lumenal domain is necessary for the Golgi localization of HS6ST. (A) The stem region but not the remaining C-terminal domain of HS6ST1 is needed for its Golgi localization. Schematic representations of the EGFP-tagged HS6ST-1 deletion mutants used in the experiment are shown. A mutant lacking the C-terminal region apart from the stem domain (m6ST1C2-EGFP), a mutant lacking the whole C-terminal lumenal domain apart from three residues (m6ST1YQY-EGFP), and mutants lacking 20 membrane-proximal or all 55 amino acids of the stem domain (m6ST1stem20-EGFP and m6ST1stem55-EGFP, respectively) were used for the experiments. The number below m6ST1C2-EGFP indicates the 55-amino-acid length of the putative stem domain. The black boxes and the green ovals show the transmembrane domain and the EGFP protein, respectively. The white N-terminal box indicates the cytoplasmic domain. The fluorescence of the m6ST1 deletion mutants in CHO-K1 cells is also shown. Deletion of most of the C-terminal domain except for the stem region had no effect on the Golgi localization (m6ST1C2-EGFP) but removal of the stem region disturbed the normal Golgi localization pattern of m6ST1. (B) Mutation of the cytoplasmic tail or transmembrane domain does not markedly disturb the Golgi localization of m6ST1. Schematic representations of the EGFP-tagged HS6ST-1 mutants used are shown. In MVAAASA, the amino acids Asp3, Arg4/Lys4, and Lys7 in the cytoplasmic domain that are conserved among m6ST1, m6ST2 and m6ST3 were replaced with Ala. The m6ST1stem20-EGFP mutant is a short-stem version of m6ST1. In m6ST1stem20(AA)-EGFP, the conserved Glu24 and Tyr25 residues were replaced with Ala. In m6ST1TMmut-stem20-EGFP, the putative transmembrane domain of m6ST1 was replaced with that of human pro-TNF. ProTNF(m6ST1stem)-EGFP is a chimeric protein in which the proTNF region that links the transmembrane domain to the mature TNF protein was replaced with the stem domain of HS6ST1. CHO-K1 cells were fixed with 80% ethanol for 1 hour at 4°C to remove the cy tosolic protein (m6ST1Tmmut-stem20-EGFP). The black boxes and the green ovals indicate the transmembrane domains and the EGFP protein, respectively. The hatched boxes denote domains that have been replaced with pro-TNF domains.

View larger version (22K): [in a new window]   Fig. 4. The stem region is important for HS6ST sulfotransferase activity and oligomer formation. (A) Sulfotransferase activities of stem-deleted m6ST1 proteins (m6ST1stem20-EGFP and m6ST1stem55-EGFP) were compared to that of m6ST1-EGFP. To analyze sulfotransferase activity, CHO-K1 cells were transfected with control vector, pm6ST1-EGFP, pm6ST1stem20-EGFP, or pm6ST1stem55-EGFP and 24 hours later cell extracts were prepared with buffer A for at least 1 hour and then centrifuged. Crude cell extracts were used for the sulfotransferase assay as described in the Materials and Methods. The amount of the cell lysate used in the experiment was normalized using the EGFP fluorescence intensity. The sulfotransferase activity of m6ST1-EGFP was set at 100%. The sulfotransferase activities of the stem-deleted proteins were greatly reduced when compared to that of the wild type. The results shown are the mean±s.d. of three experiments. (B) Oligomerization of m6ST involves the stem region. CHO-K1 cells were transfected with pm6ST1YQY-EGFP, pm6ST2LQY-EGFP, pm6ST3YQY-EGFP, pm6ST1C2-EGFP, pm6ST2C2-EGFP or pm6ST3C2-EGFP and 24 hours later the cells were treated with buffer A for at least 1 hour and then centrifuged. The cell lysates were fractionated by HR10/30 SuperoseTM 6 chromatography (Amersham Pharmacia Biotech FPLC system), 0.5 ml fractions were collected, and the GFP fluorescence of each fraction was measured by using a fluorescent spectrophotometer (Hitachi) at the excitation wavelength 488 nm and the emission wavelength 507 nm. The position at which the molecular mass markers eluted, in addition to Vo and Vt, are indicated above the figure.