TY - JOUR
T1 - Human DRA functions as a sulfate transporter in Sf9 insect cells
AU - Byeon, Mee Kyeong
AU - Frankel, Arthur
AU - Papas, Takis S.
AU - Henderson, Kelly W.
AU - Schweinfest, Clifford W.
N1 - Funding Information:
The authors are grateful to Dr. Mark C. Willingham for use of his immuno¯uorescence microscope. The authors also thank Dr. Adam Smolka for his help with Sf9 cultures, for critical review of the manuscript, and for many helpful discussions. This work was supported by Department of Energy Grant DoE DE-FG05-94ER61893 and Grant CB-187 from the American Cancer Society (to C.W.S.)
PY - 1998/2
Y1 - 1998/2
N2 - DRA is a gene that is down-regulated in colon adenomas and adenocarcinomas in humans. We have previously shown that DRA proteins are found as various forms in tissue due to differential glycosylation. This study has focused on the function of DRA related to its subcellular localization. We used the baculovirus expression system and overexpressed a nearly full-length DRA driven by a polyhedrin promoter in Sf9 insect cells. DRA protein expressed in this cell was underglycosylated relative to normal colon mucosa, but uniformly targeted to the cell membrane. It also appears to undergo posttranslational cleavage, removing about 100 amino acids from its amino terminus. This membrane localization is similar to what we observed in the colon mucosa. An ion transport assay demonstrated that DRA functions as a sulfate transporter. When DRA was expressed, sulfate import was increased more than threefold compared to the control. Sulfate import was inhibitable by the anion transporter inhibitor, DIDS, in a dose-dependent fashion. Given that (1) DRA has high similarity to other identified sulfate transporters and the proposed structure of DRA polypeptide is characteristic of those transporters, (2) DRA localization is limited to the cell membrane, and (3) DRA expression correlates with intestinal differentiation in mouse, we suggest that DRA represents a tissue-specific member of the sulfate transporter family.
AB - DRA is a gene that is down-regulated in colon adenomas and adenocarcinomas in humans. We have previously shown that DRA proteins are found as various forms in tissue due to differential glycosylation. This study has focused on the function of DRA related to its subcellular localization. We used the baculovirus expression system and overexpressed a nearly full-length DRA driven by a polyhedrin promoter in Sf9 insect cells. DRA protein expressed in this cell was underglycosylated relative to normal colon mucosa, but uniformly targeted to the cell membrane. It also appears to undergo posttranslational cleavage, removing about 100 amino acids from its amino terminus. This membrane localization is similar to what we observed in the colon mucosa. An ion transport assay demonstrated that DRA functions as a sulfate transporter. When DRA was expressed, sulfate import was increased more than threefold compared to the control. Sulfate import was inhibitable by the anion transporter inhibitor, DIDS, in a dose-dependent fashion. Given that (1) DRA has high similarity to other identified sulfate transporters and the proposed structure of DRA polypeptide is characteristic of those transporters, (2) DRA localization is limited to the cell membrane, and (3) DRA expression correlates with intestinal differentiation in mouse, we suggest that DRA represents a tissue-specific member of the sulfate transporter family.
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U2 - 10.1006/prep.1997.0809
DO - 10.1006/prep.1997.0809
M3 - Article
C2 - 9473459
AN - SCOPUS:0038929229
SN - 1046-5928
VL - 12
SP - 67
EP - 74
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -