Document Detail

Cysteine residues and the structure of the rat renal proximal tubular type II sodium phosphate cotransporter (rat NaPi IIa).
MedLine Citation:
PMID:  10926678     Owner:  NLM     Status:  MEDLINE    
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.
G Lambert; I C Forster; J Biber; H Murer
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of membrane biology     Volume:  176     ISSN:  0022-2631     ISO Abbreviation:  J. Membr. Biol.     Publication Date:  2000 Jul 
Date Detail:
Created Date:  2000-09-27     Completed Date:  2000-09-27     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0211301     Medline TA:  J Membr Biol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  133-41     Citation Subset:  IM    
Physiologisches Institut, Universität Zürich Irchel, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
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MeSH Terms
Amino Acid Sequence
Amino Acid Substitution
Carrier Proteins / chemistry*,  genetics
Cysteine / chemistry*,  genetics
Ethyl Methanesulfonate / analogs & derivatives,  pharmacology
Kidney Tubules, Proximal / chemistry*
Mesylates / pharmacology
Methyl Methanesulfonate / analogs & derivatives,  pharmacology
Molecular Sequence Data
Mutagenesis, Site-Directed
Oocytes / drug effects
Protein Structure, Tertiary
Reducing Agents / pharmacology
Serine / genetics
Sodium-Phosphate Cotransporter Proteins
Sodium-Phosphate Cotransporter Proteins, Type II
Sodium-Phosphate Cotransporter Proteins, Type IIa
Xenopus laevis
Reg. No./Substance:
0/Carrier Proteins; 0/Disulfides; 0/Mesylates; 0/Reducing Agents; 0/Slc34a1 protein, rat; 0/Sodium-Phosphate Cotransporter Proteins; 0/Sodium-Phosphate Cotransporter Proteins, Type II; 0/Sodium-Phosphate Cotransporter Proteins, Type IIa; 0/Symporters; 0/methanethiosulfonate ethylammonium; 155450-07-0/(2-sulfonatoethyl)methanethiosulfonate; 155450-08-1/(2-(trimethylammonium)ethyl)methanethiosulfonate; 2949-92-0/methyl methanethiosulfonate; 52-90-4/Cysteine; 56-45-1/Serine; 62-50-0/Ethyl Methanesulfonate; 66-27-3/Methyl Methanesulfonate

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