Screening for modulators of spermine tolerance identifies Sky1, the SR protein kinase of Saccharomyces cerevisiae, as a regulator of polyamine transport and ion homeostasis

Abstract

Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants of Saccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Delta cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity of PPZ1 but not of ENA1.

FIG. 1

Isolation and demonstration of the involvement of SKY1 in spermine tolerance. (A) Structure of the genomic clone selected for restoring spermine sensitivity to the spermine-tolerant sptC mutant. The four ORFs are shown as boxes and their names are indicated. (B) Deletion of SKY1 confers spermine tolerance. Yeast strains containing a disruption of three of the four indicated ORFs were tested for spermine tolerance by streaking on YPD agar plates containing 1.5 mM spermine (Spm). ptk2Δ cells, which were previously demonstrated to confer spermine tolerance, served as a positive control.

FIG. 2

Effect of SKY1 disruption and Sky1p overexpression on growth tolerance to spermine. (A) Basal growth rate of wild-type (WT), sky1Δ, and ptk2Δ cells. (B) Effect of 1.5 mM spermine on the growth of wild-type, sky1Δ, and ptk2Δ cells. (C) Effect of spermine concentration on the growth of wild-type, sky1Δ, ptk1Δ, and ptk2Δ cells as determined after 16 h of incubation. (D) Sky1p as well as Ptk1p and Ptk2p were overexpressed in wild-type cells from the pAD54 expression vector. Fivefold dilutions of the resulting transformants were spotted on MLSC plates with and without 0.1 mM spermine (SPM).

FIG. 3

Kinase activity of Sky1p is required for reversing the spermine-tolerant phenotype of sky1Δ cells. The growth of sky1Δ cells and of the sptC cells that were transformed with empty vector (pAD54) or with constructs encoding wild-type SKY1 or its catalytically inactive variant K187A (both with an amino-terminal HA tag) was determined on solid MLSC plates containing 1.5 mM spermine (Spm). The expression of wild-type Sky1 protein and of the catalytically inactive K187A mutant protein was determined by Western blot analysis using anti-HA antibodies as described in the text. Sizes are shown in kilodaltons.

FIG. 4

Effect of SKY1 disruption on the time course of putrescine, spermidine, and spermine uptake. The uptake of [¹⁴C]spermine, [³H]spermidine (both at 20 μM), and [¹⁴C]putrescine (10 μM) by wild-type (WT) and sky1Δ cells was determined at the indicated times as described in Materials and Methods. The results presented are averages of three determinations ± standard deviation.

FIG. 5

Initial velocity of [³H]spermidine uptake in wild-type (WT) and sky1Δ cells. The uptake of [³H]spermidine (Spd) at the indicated concentrations was determined after 1.5 min of incubation as described in Materials and Methods. The results presented are averages of three determinations ± standard deviation. The insert presents a double reciprocal plot of the initial rate of [³H]spermidine uptake in wild-type and sky1Δ cells.

FIG. 6

sky1Δ cells are tolerant to LiCl and NaCl and sensitive to osmotic shock. (A) Fivefold dilutions of wild-type (WT), sky1Δ, ptk1Δ, and ptk2Δ cells were spotted on YPD plates containing 0.2 M LiCl or 1.2 M NaCl. (B) Fivefold dilutions of wild-type cells transformed with the expression vector pAD54, encoding Sky1p, Ptk1p, and Ptk2p. The resulting transformants were spotted on MLSC plates with similar additives as in A. (C) Fivefold dilutions of wild-type, sky1Δ, ptk1Δ, and ptk2Δ cells were spotted on YPD plates containing 1.5 M KCl or 1.5 M sorbitol.

FIG. 7

Complementation analysis reveals that SKY1 and PTK2 act in two parallel signaling pathways. (A) sky1Δ and ptk2Δ cells were transformed with empty vector (pAD54) or with constructs that overexpress Sky1p or Ptk2p. Spermine tolerance of the resulting transformants was determined by drop tests in MLSC plates containing 1.5 mM spermine and SC plates containing 0.2 M LiCl. The growth of wild-type (WT), sky1Δ, ptk2Δ, and sky1Δ ptk2Δ double mutant cells was tested in the presence of the indicated concentrations of spermine (B) and LiCl (C).

FIG. 8

H⁺-ATPase activity in plasma membranes of sky1Δ and ptk2Δ cells and in wild-type cells overproducing Sky1p or Ptk2p. Plasma membranes were prepared from wild-type (WT) cells transformed with empty vector (WT) or SKY1 (WT+SKY1) or PTK2 (WT+PTK2)-overexpressing vectors and from sky1Δ or ptk2Δ cells transformed with empty vector (sky1Δ and ptk2Δ, respectively). ATPase activity was assayed (A) and the Pma1p protein was determined (B) as described under Materials and Methods.

FIG. 9

PPZ1 but not PPZ2 or ENA1 is required for the effect of SKY1, PTK1, and PTK2 on lithium tolerance phenotype. (A) ppz1Δ and ppz2Δ cells were transformed with the expression vector pAD54, encoding Sky1p, Ptk1p, and Ptk2p. The resulting transformants were spotted in fivefold dilutions on SC plates containing 0.2 M LiCl or MLSC plates containing 1.5 mM spermine. (B) The PPZ1 gene was deleted from cnb1Δ cells. The growth of the resulting double mutant cells was compared to that of wild-type (WT), cnb1Δ, and ppz1Δ cells. (C) Effect of LiCl concentration on the growth of ppz1Δ and sky1Δ ptk2Δ double mutant cells. (D) ena1-4Δ cells were transformed with an empty pAD54 vector or with this vector encoding Sky1p, Ptk1p, and Ptk2p, and the growth of the resulting transformants was tested on plates contains 17 mM LiCl as in panel A.