Lambert, G; Forster, I C; Biber, J; Murer, H (2000). Cysteine residues and the structure of the rat renal proximal tubular type II sodium phosphate cotransporter (rat NaPi IIa). Journal of Membrane Biology, 176(2):133-141.
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The rat renal Na/P(i) cotransporter type IIa (rat NaP(i) IIa) is a 637 amino acid protein containing 12 cysteine residues. We examined the effect of different cysteine modifying methanethiosulfonate (MTS)-reagents and the disulfide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transport activity of wild-type and 12 single cysteine substitution mutants of rat NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethiosulfonate (MMTS) and TCEP inhibited the transport activity of both the wild-type, as well as all the single mutant proteins. This indicated the existence of more than one functionally important cysteine residue, not accessible extracellularly, and at least 2 disulfide bridges. To identify the disulfide bridges, three double mutants lacking 2 of the 3 cysteine residues predicted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; reconsideration of the topological model predictions suggested a second disulfide bridge between C225 and C520. Evaluation of a fourth double mutant indicated that at least one of two disulfide bridges (C306 and C334; C225 and C520) has to be formed to allow the surface expression of a functional cotransporter. A revised secondary structure is proposed which includes two partially repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520.
|Item Type:||Journal Article, refereed|
|Communities & Collections:||04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
|DDC:||570 Life sciences; biology|
|Date:||15 July 2000|
|Deposited On:||11 Feb 2008 12:22|
|Last Modified:||27 Nov 2013 23:20|
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