The STK2 Gene, Which Encodes a Putative Ser/Thr Protein Kinase, Is Required for High-Affinity Spermidine Transport in Saccharomyces cerevisiae

Abstract

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify 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 polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued 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 comprising 11 of the 12 catalytic subdomains of Stk2p is Ϸ83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower V max than in the parental strain. 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 of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li ؉ and Na ؉ , suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

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

“…2 and data not shown). These data and the fact that ptk2⌬ and sky1⌬ mutants are defective in polyamine transport (17,19,36) suggested that the inability of the agp2⌬ mutant, in particular, to efficiently accumulate F-SPM in the vacuoles might result from a defect at the level of transport. Moreover, the molecular defect in the agp2⌬ mutant is likely to affect polyamine transport at the plasma membrane rather than vacuolar level, since deficient vacuolar accumulation would have translated into increased sensitivity to polyamines rather than the marked resistance observed here.…”

“…On the other hand, neither Brp1 (i.e. YGL007W, an open reading frame of unknown function) or Fes1p, a protein that binds to a complex involved in the regulation of protein translation, has any documented role in transport function (17,22,24,36). Thus, the most likely candidate gene having a role in polyamine transport among the mutants screened was clearly AGP2.…”

“…Agp2p Is a Major Effector of High Affinity Polyamine Transport-To address the hypothesis that Agp2p is a polyamine transporter, we monitored the uptake of 14 C-labeled spermidine in the agp2⌬ mutant and its parental strain as described (17). As shown in Fig.…”

“…In yeast, spermidine and spermine are thought to be imported across the plasma membrane via both high and low affinity transport systems (17), whereas putrescine transport occurs only via an apparently nonsaturable, low affinity pathway (17,18). An important determinant of polyamine transport is the Ser/Thr protein kinase Ptk2p, which pleiotropically acts as an activator of plasma membrane permeability to various cations, including polyamines (17,19).…”

“…An important determinant of polyamine transport is the Ser/Thr protein kinase Ptk2p, which pleiotropically acts as an activator of plasma membrane permeability to various cations, including polyamines (17,19). Via the glucose-dependent activation of the Pma1p H ϩ -ATPase, Ptk2p activity increases the proton motive force that energizes the transport of a variety of cationic compounds (20).…”