Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane - PubMed (original) (raw)
Comparative Study
Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane
C Navarre et al. EMBO J. 2000.
Abstract
Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide, Pmp3p, which has high sequence similarity to a novel family of plant polypeptides that are overexpressed under high salt concentration or low temperature treatment. The PMP3 gene is not essential under normal growth conditions. However, its deletion increases the plasma membrane potential and confers sensitivity to cytotoxic cations, such as Na(+) and hygromycin B. Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype of a mutant strain lacking the Pmr2p/Enap Na(+)-ATPases and the Nha1p Na(+)/H(+) antiporter, and suppresses the potassium dependency of a strain lacking the K(+) transporters, Trk1p and Trk2p. All these phenotypes could be reversed by the addition of high Ca(2+) concentration to the medium. These genetic interactions indicate that the major effect of the PMP3 deletion is a hyperpolarization of the plasma membrane potential that probably promotes a non-specific influx of monovalent cations. Expression of plant RCI2A in yeast could substitute for the loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue.
Figures
Fig. 1. Pmp3p is a novel 55 amino acid proteolipid of the yeast plasma membrane. (A) Yields (in pmol) of phenylthiohydantoin derivatives recovered after each cycle of Edman degradation for the proteolipid fraction extract derived from S.cerevisiae W303-1B plasma membranes. (B) Alignment of Pmp3p with RCI2A (A.thaliana), RCI2B (_A.thali_ana), ESI3 (L.elongatum) and BLT101 (Hordeum vulgare): amino acids identical in Pmp3p and one of the plant 54 amino acid polypeptides are boxed. (C) Silver-stained Tricine–SDS–polyacrylamide gels of purified plasma membrane preparations (50 µg/gel lane). Lane 1, molecular weight markers; lane 2, PMP3 wild-type strain W303-1B; lane 3, pmp3Δ mutant strain CN3.
Fig. 2. Effect of PMP3 deletion on NaCl tolerance. (A) Increased sensitivity to NaCl of a _pmp3_-deleted strain. Serial 10-fold dilutions of saturated cultures of wild-type W303-1B (PMP3) and CN3 (pmp3Δ) were spotted onto YD plates supplemented with the indicated concentrations of NaCl. The plates were photographed after a 2 day incubation at 30°C. (B) Effect of PMP3 deletion on the time course of intracellular Na+ accumulation during salt stress. Exponentially growing CN3 (pmp3Δ; open squares) and W303-1B (PMP3; closed squares) cells in YD medium were stressed by adding NaCl at time zero to a final concentration of 0.9 M. Aliquots were taken at the times indicated for determination of the intracellular Na+ concentration as described in Materials and methods.
Fig. 3. Deletion of PMP3 exacerbates the Na+ sensitivity phenotype of strains lacking Pmr2p/Enap and Nha1p. Strains W303-1B (PMR2 NHA1 PMP3; closed squares), CN3 (PMR2 NHA1 pmp3Δ; open squares), YR93 (pmr2Δ NHA1 PMP3; closed triangles), YR93-3 (pmr2Δ NHA1 pmp3Δ; open triangles), YR93-1 (pmr2Δ nha1Δ PMP3; closed circles) and YR93-31 (pmr2Δ nha1Δ pmp3Δ; open circles) were grown in YD medium to saturation. Then, 10 µl aliquots were transferred to 3 ml of YD medium supplemented with various NaCl concentrations and grown for 15 h at 30°C. Relative growth is the ratio of the OD600 of a given culture to the value obtained with control cells grown in YD. The experiment was repeated three times with similar results.
Fig. 4. Growth assays of trk1Δ trk2Δ and trk1Δ trk2Δ pmp3Δ. (A) Growth of CY162 (trk1Δ trk2Δ PMP3) (closed symbols) and CY162-3 (trk1Δ trk2Δ pmp3Δ) (open symbols) in YD medium without any potassium added supplemented with 1 mM EGTA (squares) or with 10 mM CaCl2 (triangles). (B) Serial 10-fold dilutions of overnight cultures of W303-1B (TRK1 TRK2 PMP3), CN3 (TRK1 TRK2 pmp3Δ), CY162 (trk1Δ trk2Δ PMP3) and CY162-3 (trk1Δ trk2Δ pmp3Δ) in YD medium containing 100 mM KCl were spotted onto YD plates without any KCl added. The plates were photographed after a 3 day incubation at 30°C. (C) Strains CY162 (trk1Δ trk2Δ PMP3; closed squares) and CY162-3 (trk1Δ trk2Δ pmp3Δ; open squares) were grown to saturation in YD medium containing 100 mM KCl. Then, 10 µl aliquots were transferred to 3 ml of YD containing 100 mM KCl at the indicated pH, and the OD600 was measured after 15 h at 30°C. Relative growth is expressed as the ratio of the OD600 of a given culture at the indicated pH to the OD600 of the control culture at pH 5.5. The experiment was repeated three times with similar results. (D) Serial 10-fold dilutions of overnight cultures of W303-1B (TRK1 TRK2 PMP3), CN3 (TRK1 TRK2 pmp3Δ), CY162 (trk1Δ trk2Δ PMP3) and CY162-3 (trk1Δ trk2Δ pmp3Δ) in YD medium containing 100 mM KCl were spotted onto YD plates containing 100 mM KCl at pH 3.0 and 10 mM CaCl2 when indicated. The plates were photographed after a 2 day incubation at 30°C.
Fig. 5. Effect of PMP3 deletion on membrane potential indicators. (A) Increased sensitivity to toxic cations of _pmp3_-deleted strains. Serial 10-fold dilutions of saturated cultures of wild-type W303-1B (PMP3) and CN3 (pmp3Δ) were spotted onto YD plates supplemented with the indicated concentrations of hygromycin B, TMA or H+. The plates were photographed after a 2 day incubation at 30°C. (B) Increased methylammonium uptake caused by deletion of PMP3. W303-1B (PMP3; closed squares) and CN3 (pmp3Δ; open squares) cells were grown to exponential phase in MM and washed. The uptake of 2 mM [14C]methylammonium was determined at the times indicated as described in Materials and methods. The experiment was repeated twice with similar results.
Fig. 6. Deletion of PMP3 has no effect on plasma membrane H+-ATPase activity. (A) ATP dependence of ATPase activity of plasma membranes from W303-1B (PMP1 PMP2 PMP3; closed squares), CN3 (PMP1 PMP2 pmp3Δ; open squares) and CN123 (pmp1Δ pmp2Δ pmp3Δ; open triangles). Each curve represents the average of three independent preprations. (B) ATP-dependent fluorescence quenching of ACMA versus ATP hydrolysis. The initial rate of fluorescence quenching in sealed plasma membranes from W303-1B (PMP3; closed squares) and CN3 (pmp3Δ; open squares) was determined in the presence of varying MgATP concentrations (from 0.05 to 4 mM) and plotted as a function of the ATP hydrolysis activity measured under the same conditions.
Fig. 7. Effect of divalent cations on the NaCl sensitivity due to the PMP3 deletion. (A) Strains YR93 (pmr2Δ PMP3; closed symbols) and YR93-3 (pmr2Δ pmp3Δ; open symbols) were grown in YD medium to saturation, then 10 µl aliquots were transferred to 3 ml of YD medium containing either 1 mM EGTA (circles) or 10 mM CaCl2 (triangles) supplemented with the indicated concentration of NaCl. After 15 h at 30°C, the OD600 of all cultures was measured. Relative growth is the ratio between the OD600 obtained for a given NaCl concentration and the OD600 of a control culture in YD. The experiment was repeated twice with similar results. (B) Strains YR93 (pmr2Δ PMP3; closed symbols) and YR93-3 (pmr2Δ pmp3Δ; open symbols) were grown in YD medium to saturation, then 10 µl aliquots of each culture were transferred to 3 ml of YD medium containing 0.2 M NaCl and either MgCl2 (squares) or CaCl2 (triangles) at the indicated concentration. Relative growth is the ratio between the OD600 obtained for a given divalent ion concentration and the OD600 of the corresponding culture containing 10 mM CaCl2. The experiment was repeated twice with similar results.
Fig. 8. The A.thaliana cDNA T20621 complements the PMP3 deletion with regard to NaCl and hygromycin B sensitivity. (A) Strains YR93-1 (pmr2Δ nha1Δ PMP3; closed circles), YR93-31 (pmr2Δ nha1Δ pmp3Δ; open circles) and YR93-31-RCI2A (pmr2Δ nha1Δ pmp3Δ RCI2A; closed triangles) were grown in YD medium to saturation, then 10 µl aliquots were transferred to 3 ml of YD supplemented with NaCl at the indicated concentration. After 15 h at 30°C, the OD600 of all cultures was measured. Relative growth is the ratio of the OD600 of a given culture to the OD600 of a control culture in YD. The experiment was repeated twice with similar results. (B) Serial 10-fold dilutions of saturated cultures of YR93-1 (pmr2Δ nha1Δ PMP3), YR93-31 (pmr2Δ nha1Δ pmp3Δ) and YR93-31-RCI2A (pmr2Δ nha1Δ pmp3Δ RCI2A) were spotted onto YD plates supplemented with 100 µg/ml hygromycin B. The plate was photographed after a 2 day incubation at 30°C.
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