The Spermidine Transport System Is Regulated by Ligand Inactivation, Endocytosis, and by the Npr1p Ser/Thr Protein Kinase in Saccharomyces cerevisiae

Abstract

We have characterized the regulation of spermidine transport in yeast and identified some of the genes involved in its control. Disruption of the SPE2 gene encoding S-adenosylmethionine decarboxylase, which catalyzes an essential step in polyamine biosynthesis, upregulated the initial velocity of spermidine uptake in wild-type cells as well as in the polyamine transportdeficient pcp1 mutants. Exogenous spermidine rapidly inactivated spermidine transport with a half-life of Ϸ10 -15 min via a process that did not require de novo protein synthesis but was accelerated by cycloheximide addition. Conversely, reactivation of spermidine influx upon polyamine deprivation required active protein synthesis. The stability of polyamine carrier activity was increased 2-fold in polyamine-depleted spe2 deletion mutants, indicating that endogenous polyamines also contribute to the down-regulation of spermidine transport. Ligand-mediated repression of spermidine transport was delayed in end3 and end4 mutants that are deficient in the initial steps of the endocytic pathway, and spermidine uptake activity was increased 4-to 5-fold in end3 mutants relative to parental cells, although the stability of the transport system was similar in both strains. Disruption of the NPR1 gene, which encodes a putative Ser/Thr protein kinase essential for the reactivation of several nitrogen permeases, resulted in a 3-fold decrease in spermidine transport in NH 4 ؉ -rich media but did not prevent its down-regulation by spermidine. The defect in spermidine transport was more pronounced in NH 4 ؉ -than proline-grown npr1 cells, suggesting that NPR1 protects against nitrogen catabolite repression of polyamine uptake activity. These results suggest that (a) the polyamine carrier is an unstable protein subject to down-regulation by spermidine via a process involving ligand inactivation followed by endocytosis and that (b) NPR1 expression fully prevents nitrogen catabolite repression of polyamine transport, unlike the role predicted for that gene by the inactivation/reactivation model proposed for other nitrogen permeases.

AGP2 Encodes the Major Permease for High Affinity Polyamine Import in Saccharomyces cerevisiae

“…As expected from our previous observation that END3 deletion up-regulates polyamine transport activity (37), end3⌬ mutants exhibited a marked increase to the toxic effects of high concentrations of putrescine, spermidine, and spermine, confirming that the assay accurately reflected changes in polyamine transport activity. These data suggest that, like Ptk2p and Sky1p, the L-carnitine transporter Agp2p, as well as Brp1p and Fes1p, may also play a role in polyamine transport.…”

“…Cells growing exponentially in YPD were harvested, and total RNA was extracted and processed as described under "Experimental Procedures." C, effect of END3 disruption on the time course of [ Agp2p was previously shown to localize mainly at the plasma membrane by expression of a HA epitope-tagged construct of the gene, using either immunogold electron microscopy or subcellular fractionation (37). A minor fraction of the Agp2p-HA is also recovered in the endoplasmic reticulum and vacuole, although the significance of this subcellular localization is uncer- FIG.…”

“…6A). Therefore, cells devoid of endogenous polyamine synthesis do not significantly derepress its transport pathway, although this would be expected, since polyamines accumulated from the rich YPD medium probably exert feedback regulation on the expression of the polyamine transport system (37).…”

“…Agp2p-We have previously shown that defects in an early step of the endocytic pathway enhance the maximal velocity of spermidine uptake, which we postulated to be the result of a decreased turnover of the plasma membrane polyamine transporter (37). We therefore examined whether basal Agp2p abundance is increased in end3⌬ mutants, which lack a cytoskeletal protein that plays a key role in the early steps of internalization of plasma membrane proteins, and are thus defective in both fluid phase and clathrin-dependent endocytosis (45,46).…”

Histatin 5 Uptake by Candida albicans Utilizes Polyamine Transporters Dur3 and Dur31 Proteins

“…In S. cerevisiae, addition of spermidine can alter gene expression involved in various metabolic pathways and activate other transport systems in the absence of the preferred transporter (54,55); thus it is possible that the presence of Hst 5 modulates C. albicans metabolism or carrier affinities. Also, intracellular Hst 5 may be recognized as contributing to high levels of cellular polyamines, and then be excreted from the cell via polyamine TPO permeases.…”