THE STK2 GENE, WHICH ENCODES A PUTATIVE SER THR PROTEIN-KINASE, IS REQUIRED FOR HIGH-AFFINITY SPERMIDINE TRANSPORT IN SACCHAROMYCES-CEREVISIAE/

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identi fy genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polya mine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene re scued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprisi ng 11 of the 12 catalytic subdomains of Stk2p is approximate to 83% ho mologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recentl y described activator of low-affinity spermine uptake in yeast. Satura ble spermidine transport in stk2::lacZ mutants had an approximately fi vefold-lower affinity and twofold-lower V-max than in the parental str ain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild -type levels. Single mutants lacking the catalytic kinase subdomains o f STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accu mulation and a low-level resistance to toxic polyamine analogs. Spermi dine transport was repressed by prior incubation with exogenous spermi dine. Exogenous polyamine deprivation also derepressed residual spermi dine transport in stk2::lacZ mutants, but simultaneous disruption of S TK1 and STK2 virtually abolished high-affinity spermidine transport un der both repressed and derepressed conditions. On the other hand, putr escine uptake was also deficient in stk2::lacZ mutants but was not rep ressed by exogenous spermidine. Interestingly, stk2::lacZ mutants show ed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transp ort. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in y east whereas its close homolog STK1 mostly affects a lower-affinity, l ow-capacity polyamine transport activity.