Essential cysteine residues of the type IIa Na+/Pi cotransporter.
The rat renal Na(+)/P(i) cotransporter (NaP(i)-IIa) contains 12 native cysteines. When individually replaced by a serine, none appears essential for proper expression and function. Nevertheless, the formation of one essential cysteine bridge (C5/C6), together with a postulated second bridge, is necessary. To determine the minimum cysteine residues required for functional NaP(i)-IIa, with the goal of generating a Cys-less backbone for structure-function studies, mutants were constructed in which multiple endogenous cysteines were replaced by serines in different combinations. In Xenopus oocytes, most mutants were functional, except those where cysteine pairs C4/C9, C4/C12 or C9/C12 were simultaneously deleted. This suggested that one of these pairs could form the second cysteine bridge essential for expression and/or protein function. Up to eight cysteines could therefore be removed to give a functional Cys-reduced NaP(i)-IIa with activity and kinetics comparable to the wild-type (WT). This construct, like all intermediate mutants and the WT, was insensitive to cysteine-modifying methanethiosulfonate (MTS) reagents. Moreover, by introducing a novel cysteine into the Cys-reduced NaP(i)-IIa at a site functionally important in the WT (Ser-460), the loss of transport function reported for mutant S460C, after exposure to MTS reagents, was recapitulated. This confirmed that the MTS reagent site of action was Cys-460 and that modification of native cysteines does not contribute to S460C behavior.
Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
Katja
K
Köhler
Ian C
IC
Forster
Gerti
G
Stange
Jürg
J
Biber
Heini
H
Murer
D000818:Animals; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D005260:Female; D009154:Mutation; D051381:Rats; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D014982:Xenopus laevis
C493556:Slc34a1 protein, rat; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters; D003545:Cysteine