Mutational analysis and localization of the inositol transporters of Saccharomyces cerevisiae.
Saccharomyces cerevisiae possesses two inositol transport proteins, Itr1p and Itr2p, encoded by the ITR1 and ITR2 genes, respectively. Itr1p and Itr2p are high and low affinity transporters, respectively. Eight out of nine cysteine residues in Itr1p, which are common in two transporters, were converted to serine residues by site directed mutagenesis. All mutant genes suppressed the growth defect caused by itr1 disruption, indicating that cysteine residues are not essential for its function. Chimeric genes that express Itr1p and Itr2p fused to the green fluorescent protein (GFP) under the control of the ADH1 promoter were constructed. Both genes were functional. Fluorescence microscopy analysis indicated that both GFP-fused Itr1p and Itr2p are localized to the plasma membrane. A multi-copy plasmid that expresses GFP-fused Itr1p under the control of the original ITR1 promoter was constructed. Under inositol-free culture conditions, GFP-fused Itr1p appeared and was localized to the plasma membrane. When the cells were cultured in the presence of inositol, GFP-fused Itr1p gradually disappeared from the plasma membrane, the fluorescence being redistributed within the cell. Prolonged culture of the cells also caused the relocalization of transporter proteins. These results clearly indicate that the cellular relocalization of transport proteins is responsible for a reduction of inositol transport activity, which is caused by the presence of inositol in the medium or culturing of cells to the stationary phase.
Miyashita
Miki
M
Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan.
Shugyo
Mie
M
Nikawa
Jun-Ichi
J