Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae.
The derepressible Pho84 high-affinity phosphate permease of Saccharomyces cerevisiae, encoded by the PHO84 gene belongs to a family of phosphate:proton symporters (PHS). The protein contains 12 native cysteine residues of which five are predicted to be located in putative transmembrane regions III, VI, VIII, IX, and X, and the remaining seven in the hydrophilic domains of the protein. Here we report on the construction of a Pho84 transporter devoid of cysteine residues (C-less) in which all 12 native residues were replaced with serines using PCR mutagenesis and the functional consequences of this. Our results clearly demonstrate that the C-less Pho84 variant is able to support growth of yeast cells to the same extent as the wild-type Pho84 and is stably expressed under derepressible conditions and is fully active in proton-coupled phosphate transport across the yeast plasma membrane.
Berhe
A
A
Department of Biochemistry and Biophysics, Wallenberg Laboratory, S-106 91 Stockholm, Sweden.
Zvyagilskaya
R
R
Lagerstedt
J O
JO
Pratt
J R
JR
Persson
B L
BL
D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016259:Genes, myc / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D017433:Protein Structure, Secondary; D028081:Proton-Phosphate Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; C105495:PHO84 protein, S cerevisiae; D010710:Phosphates; D028081:Proton-Phosphate Symporters; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine