| Abstract
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Polyamines are small organic molecules involved in a variety of cellular functions. However, their exact mechanisms of action are largely unknown. Cellular polyamine levels are tightly regulated, since low levels lead to inhibited growth and high levels are cytotoxic. Toxic polyamine analogues are being examined as anti-cancer drugs, however as some cells are resistant to them their efficacy is reduced. In search of genes involved in mechanisms leading to polyamine resistance, our lab previously identified SKY1, the SR protein kinase of the budding yeast as a regulator of polyamine transport and resistance. Since the known substrates of Sky1p were not associated with the polyamine tolerance phenotype, we have investigated additional potential substrates. HRB1, an mRNA shuttling protein was identified as a potential substrate both in a biochemical and in a bioinformatic screen and was therefore further examined. Hrb1p was phosphorylated by Sky1p in vitro, requiring the RS domain but not its serine residues. However, Sky1p did not affect the phosophorylation or cellular localization of Hrb1p in intact yeast cells. The segment of amino acids 25-50 (located within the RS domain) was essential for the proper intracellular localization, possibly by interacting with the export factor Mtr10p. Additional search for potential Sky1p substrates yielded several other proteins. However, although several of them were phosphorylated by Sky1p in vitro, no correlation with the polyamine tolerant phenotype was observed. It is possible that other substrates not detected in our screen are involved in regulating polyamine uptake, or that the polyamine resistant phenotype is the result of concerted action of more than one substrate, a situation that could not be detected through manipulation of a single substrate. In order to further understand the cellular mechanisms for tolerating excessive polyamines, we searched for additional genes involved in regulating this process. We show here that Yeast cells deleted for the carnitine transporter AGP2 or the HSP70 nucleotide exchange factor FES1 can resist high polyamine levels, and show decreased polyamine uptake. Interestingly, the polyamine tolerant phenotype is not correlated with the cellular polyamine profile, as the polyamine resistant cells accumulate significant amounts of polyamines. In addition, these cells have higher putrescine levels, a result of increased ODC activity. In contrast to the salt resistance observed in cells deleted for SKY1, cells deleted for AGP2 or FES1 are actually sensitive to salt ions. In addition, we found additional genes that their deletion confers intermediate polyamine tolerance. These include the nitrogen permease regulator NPR2 and the transcription factors STP1 and STP2, involved in amino acid sensing. These results suggest that polyamine tolerance mechanisms are complex and involve other factors besides uptake. It was shown that mammalian antizyme (mAz) is involved in mediating polyamine uptake, in addition to its role in degradation and inhibition of ODC activity. The recent identification of an antizyme orthologue in yeast (yAz) enabled us to determine whether as demonstrated in mammalian cells yAz inhibits polyamine uptake in yeast cells. Our analysis have demonstrated that in contrast to the effect of Az in mammalian cells, overexpression of yAz in yeast cells actually slightly increased polyamine uptake, while its genomic deletion resulted in slightly reduced uptake. Nevertheless, overexpression of yAz resulted in a reduction of cellular polyamine levels and growth inhibition. Interestingly, yAz failed to promote yODC degradation in a mammalian system, either in vitro or in vivo. Appending the mODC C-terminal degradation signal, known to destabilaize other proteins did not enhance yODC degradation. Since only the addition of purified yeast 26S proteasomes enabled efficient yAz-dependent yODC degradation, we concluded that the degradation signal of the yAz/yODC complex is not recognized by the mammalian proteasome. We further ident fied a region of yODC that is essential for yAz binding, as its deletion mutant failed to bind yAz and did not exhibit yAz-induced degradation. Further analysis of the yAz protein using a series of deletions revealed that most of the protein is essential for ODC activity inhibition and degradation, while deletion of amino acids 101-140 and to some extent 1-20 and retains Az function. Our findings thus add valuable information and shed more light on the complex mechanisms of polyamine tolerance and regulation.
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