Polyamine Transport in Yeast

Polyamine uptake in yeast is energy-dependent and the Km value for putrescine, spermidine, and spermine are 770, 8.3, and 18 |iM, respectively. A distinctive feature of the polyamine transport system in Saccharomyces cerevisiae is that it is strongly inhibited by Mg2+ (42). Thus in a Mg2+-limited medium, polyamines, especially sper-mine, overaccumulate in cells and become toxic for growth. We isolated a mutant (YTM22-8) whose growth was tolerant to spermine in a Mg2+-limited medium. This mutant was defective in polyamine uptake and did not accumulate spermine. We obtained clones of two yeast genes that restored spermine sensitivity to YTM22-8. Those genes were PTK1 and PTK2, which encode serine/threonine kinases (43,44).

Polyamine Transport
Fig. 6. Polyamine transporters in yeast. Polyamine-specific uptake proteins are not yet identified.

This suggests that spermine uptake in yeast is regulated by phosphorylation and dephosphorylation. Poulin and his coworkers also reported that another putative serine/threonine protein kinases, NPR1, essential for the reactivation of several nitrogen permeases, is involved in the activation of spermidine uptake in addition to PTK1 (= STK1) and PTK2 (= STK2) (45,46). However, a gene for a polyamine-specific transporter on the plasma membrane of Saccharomyces cerevisiae has not yet been isolated. Recently, we found that UGA4, which is a transporter of 4-aminobutyric acid on the vacuolar membrane can take up putrescine (47), and that GAP1, which is a general transporter of amino acids on the plasma membrane, can take up putrescine and spermidine (48).

It has been reported that excretion of spermidine can be catalyzed by the Bacillus subtilis multidrug transporter Blt (49). Thus we reasoned that the polyamine transporter in yeast may have some sequence similarity to Blt, and we searched for such amino acid sequence and proteins encoded by the yeast genome (50). We could identify four genes that encode polyamine transport proteins TPO1 through TPO4 (51,52). When expressed from chromosomes, these proteins were located on the plasma membrane (53). When expressed from a multicopy vector, these proteins were mainly located on the plasma membrane, but with some localization on the vacuolar membrane (51,52,54). Polyamine transport by TPO1 was dependent on pH. Uptake of polyamines occurred at alkaline pH (8.0), whereas excretion of polyamines occurred at acidic pH (5.0). Thus the function of TPO1 to 4 was similar to PotE and CadB. Because yeast cells usually grow at acidic pH, TPO1 to 4 function as excretion proteins for polyamines (Fig. 6). 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 sper-mine (52). TPO1 consists of 586 amino acid residues (50) and has 12 putative transmembrane segments. Three glutamic acid residues (Glu207, Glu324, and Glu574), which may intracellular extracellular

Orn intracellular extracellular


Fig. 7. Functions of antizyme. Polyamine transporters involved in uptake and excretion in mammalian cells are not yet identified.

Poly amines Acetyl poly a mines


Fig. 7. Functions of antizyme. Polyamine transporters involved in uptake and excretion in mammalian cells are not yet identified.

interact with polyamines, are located in positions similar to those of the key residues in PotE (Glu77, Glu207, and Glu433) (52). When the amino acid sequence of TPO1 and PotE were compared, TPO1 possessed a longer hydrophilic N^-terminal region in which many serine and threonine residues are included, suggesting that polyamine transport is positively regulated by protein kinases. In support of this, phosphorylation of Ser19 by protein kinase C and that of Thr52 by casein kinase enhanced the transport activity of TPO1 (54). Furthermore, the sorting of TPO1 protein to plasma membrane was enhanced by phosphorylation of Ser342 by cAMP-dependent protein kinases 1 and 2 (54). Thus both uptake and excretion of polyamines are regulated by phosphorylation and dephosphorylation.

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