Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation.
The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.
Uemura
Takeshi
T
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
Tachihara
Ken
K
Tomitori
Hideyuki
H
Kashiwagi
Keiko
K
Igarashi
Kazuei
K
D000595:Amino Acid Sequence; D017920:Antiporters; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005838:Genotype; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D027701:Organic Cation Transport Proteins; D010766:Phosphorylation; D011073:Polyamines / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D013095:Spermidine / Q000378:metabolism; D013096:Spermine / Q000378:metabolism
D017920:Antiporters; D026901:Membrane Transport Proteins; D027701:Organic Cation Transport Proteins; D011073:Polyamines; D029701:Saccharomyces cerevisiae Proteins; C475058:TPO1 protein, S cerevisiae; D013096:Spermine; D011494:Protein Kinases; D013095:Spermidine