title: 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 transport-deficient pcp1 mutants. Exogenous spermidine rapidly inactivated spermidine transport with a half-life of approximately 10-15 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 NH4(+)-rich media but did not prevent its down-regulation by spermidine. The defect in spermidine transport was more pronounced in NH4(+)- 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.
authors.affiliation: Department of Anatomy and Physiology, CHUL Research Center, Ste. Foy, Quebec, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Kaouass
authors.forename: I
authors.initials: I
authors.lastname: Gamache
authors.forename: D
authors.initials: D
authors.lastname: Ramotar
authors.forename: M
authors.initials: M
authors.lastname: Audette
authors.forename: R
authors.initials: R
authors.lastname: Poulin
mesh_terms: D001692:Biological Transport; D015815:Cell Adhesion Molecules / Q000378:metabolism; D015536:Down-Regulation; D004705:Endocytosis; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005656:Fungal Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D011073:Polyamines / Q000378:metabolism; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013095:Spermidine / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; C092275:NPR2 protein, S cerevisiae; D011073:Polyamines; D029701:Saccharomyces cerevisiae Proteins; C066958:NPR1 protein, S cerevisiae; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D013095:Spermidine