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 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.
Kaouass
M
M
CHUL Research Center, Ste. Foy, Quebec, Canada.
Audette
M
M
Ramotar
D
D
Verma
S
S
De Montigny
D
D
Gamache
I
I
Torossian
K
K
Poulin
R
R
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
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