Functional Studies on a Split Type II Na/Pi-Cotransporter

Functional Studies on a Split Type II Na/Pi-Cotransporter Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/Pi-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane distribution of this split NaPi-IIa transporter we followed two strategies. In one strategy we expressed the transporter as two complementary parts (p40 and p45) in Xenopus laevis oocytes and as another strategy we cleaved the WT protein with trypsin. Both strategies resulted in a split NaPi-IIa protein located in the plasma membrane. The two domains were tied together by a disulfide bridge, most likely involving the cysteines 306 and 334. Surface expression of the NaPi-IIa fragments was dependent on the presence of both domains. If both domains were coexpressed, the transporter was functional and transport characteristics were identical to those of the WT-NaPi-IIa protein. Corresponding to this, the transporter cleaved by trypsin also retains its transport capacity. These data indicate that cleavage of the type IIa Na/Pi-cotransporter at ECL-2 is compatible with its cotransport function. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Functional Studies on a Split Type II Na/Pi-Cotransporter

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Publisher
Springer-Verlag
Copyright
Copyright © 2002 by Springer-Verlag New York Inc.
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-001-0186-y
Publisher site
See Article on Publisher Site

Abstract

Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/Pi-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane distribution of this split NaPi-IIa transporter we followed two strategies. In one strategy we expressed the transporter as two complementary parts (p40 and p45) in Xenopus laevis oocytes and as another strategy we cleaved the WT protein with trypsin. Both strategies resulted in a split NaPi-IIa protein located in the plasma membrane. The two domains were tied together by a disulfide bridge, most likely involving the cysteines 306 and 334. Surface expression of the NaPi-IIa fragments was dependent on the presence of both domains. If both domains were coexpressed, the transporter was functional and transport characteristics were identical to those of the WT-NaPi-IIa protein. Corresponding to this, the transporter cleaved by trypsin also retains its transport capacity. These data indicate that cleavage of the type IIa Na/Pi-cotransporter at ECL-2 is compatible with its cotransport function.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Mar 18, 2014

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