Abstract
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.
Highlights
Polyamines, which are essential for cell growth, are regulated by biosynthesis, degradation, and transport [1,2,3]
Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized putrescine, spermidine, and spermine
To exclude the possibility that the TPO1-HA3 found in the vacuolar membrane fraction reflects contamination from the plasma membrane fraction, refractionation of the vacuolar membrane fractions was performed by sucrose density gradient centrifugation
Summary
Plasmids—Plasmid YEpTPO1 in vector YEp351 (LEU2ϩ) [7] was constructed previously [4, 5]. For construction of the plasmid encoding HA-tagged TPO1 (YEpTPO1-HA3), the gene for the TPO1 open reading frame lacking the termination codon was amplified by PCR from genomic DNA of X2180-1A (MATa SUC2 mal0 gal CUP1) as a template using primer sets of HindIII-TPO1F (5Ј-TAGCCCAAGCTTGATCGTAGGAATTCCCTAAAG-3Ј) and SalI-TPO1R (5Ј-TAACGCGTCGACAGCGGCGTAAGCATACTT-3Ј). The resulting DNA fragment was digested with HindIII and SalI and inserted into the same restriction sites of YEp352 (URA3ϩ) [7]. The resulting DNA fragment was digested with SalI and EcoRI and inserted into the same restriction sites of the plasmid constructed as described above. The single-copy plasmid encoding HA-tagged TPO1 (YCpTPO1-HA3) was constructed by inserting the DNA fragment of the HA-tagged TPO1 gene of YEpTPO1-HA3.
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