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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 (m6ST1{Delta}C2-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 (m6ST1{Delta}stem20-EGFP and m6ST1{Delta}stem55-EGFP, respectively) were used for the experiments. The number below m6ST1{Delta}C2-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 (m6ST1{Delta}C2-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{alpha}(m6ST1stem)-EGFP is a chimeric protein in which the proTNF{alpha} region that links the transmembrane domain to the mature TNF{alpha} 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.