Cysteine residues and the structure of the rat renal proximal tubular type II sodium phosphate cotransporter (rat NaPi IIa).
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.
Lambert
G
G
Physiologisches Institut, Universität Zürich Irchel, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
Forster
I C
IC
Biber
J
J
Murer
H
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000737:chemistry*; D008698:Mesylates / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D019163:Reducing Agents / Q000494:pharmacology; D012694:Serine / Q000235:genetics; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D004220:Disulfides; D008698:Mesylates; D019163:Reducing Agents; C493556:Slc34a1 protein, rat; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters; C093171:methanethiosulfonate ethylammonium; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C014674:methyl methanethiosulfonate; D012694:Serine; D005020:Ethyl Methanesulfonate; D008741:Methyl Methanesulfonate; D003545:Cysteine