THE SPERMIDINE TRANSPORT-SYSTEM IS REGULATED BY LIGAND INACTIVATION, ENDOCYTOSIS, AND BY THE NPR1P SER THR PROTEIN-KINASE IN SACCHAROMYCES-CEREVISIAE/

We have characterized the regulation of spermidine transport in yeast and identified some of the genes involved in its control, Disruption o f the SPE2 gene encoding S-adenosylmethionine decarboxylase, which cat alyzes an essential step in polyamine biosynthesis, up-regulated the i nitial velocity of spermidine uptake in wild-type cells as well as in the polyamine transport-deficient pcp1 mutants, Exogenous spermidine r apidly inactivated spermidine transport with a half-life of approximat e to 10-15 min via a process that did not require de nova protein synt hesis but was accelerated by cycloheximide addition, Conversely, react ivation of spermidine influx upon polyamine deprivation required activ e protein synthesis, The stability of polyamine carrier activity was i ncreased a-fold in polyamine-depleted spe2 deletion mutants, indicatin g that endogenous polyamines also contribute to the down-regulation of spermidine transport, Ligand-mediated repression of spermidine transp ort was delayed in end3 and end4 mutants that are deficient in the ini tial steps of the endocytic pathway, and spermidine uptake activity wa s increased 4- to 5-fold in end3 mutants relative to parental cells, a lthough the stability of the transport system was similar in both stra ins, Disruption of the NPR1 gene, which encodes a putative Ser/Thr pro tein kinase essential for the reactivation of several nitrogen permeas es, resulted in a 3-fold decrease in spermidine transport in NH4+-rich media but did not prevent its down-regulation by spermidine, The defe ct in spermidine transport was more pronounced in NH4+- than proline-g rown npr1 cells, suggesting that NPR1 protects against nitrogen catabo lite repression of polyamine uptake activity, These results suggest th at (a) the polyamine carrier is an unstable protein subject to down-re gulation by spermidine via a process involving Ligand inactivation fol lowed by endocytosis and that (b) NPR1 expression fully prevents nitro gen catabolite repression of polyamine transport, unlike the role pred icted for that gene by the inactivation/reactivation model proposed fo r other nitrogen permeases.