title: 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 approximately 83% 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 Vmax 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.
authors.affiliation: CHUL Research Center, Ste. Foy, Quebec, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Kaouass
authors.forename: M
authors.initials: M
authors.lastname: Audette
authors.forename: D
authors.initials: D
authors.lastname: Ramotar
authors.forename: S
authors.initials: S
authors.lastname: Verma
authors.forename: D
authors.initials: D
authors.lastname: De Montigny
authors.forename: I
authors.initials: I
authors.lastname: Gamache
authors.forename: K
authors.initials: K
authors.lastname: Torossian
authors.forename: R
authors.initials: R
authors.lastname: Poulin
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active / Q000235:genetics; D015152:Blotting, Northern; D018844:Cyclin-Dependent Kinases; D007700:Kinetics; D008094:Lithium / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011073:Polyamines / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011700:Putrescine / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D013095:Spermidine / Q000378:metabolism*; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D011073:Polyamines; D029701:Saccharomyces cerevisiae Proteins; D008094:Lithium; D012964:Sodium; C522753:PTK2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D013095:Spermidine; D011700:Putrescine; D000095043:Cyclin-Dependent Kinase-Activating Kinase