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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.
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