Pun1p is a metal ion-inducible, calcineurin/Crz1p-regulated plasma membrane protein required for cell wall integrity

Abstract

Under conditions of environmental stress, the plasma membrane is involved in several regulatory processes to promote cell survival, like maintenance of signaling pathways, cell wall organization and intracellular ion homeostasis. PUN1 encodes a plasma membrane protein localizing to the ergosterol-rich membrane compartment occupied also by the arginine permease Can1. We found that the PUN1 (YLR414c) gene is transcriptionally induced upon metal ion stress. Northern blot analysis of the transcriptional regulation of PUN1 showed that the calcium dependent transcription factor Crz1p is required for PUN1 induction upon heavy metal stress. Here we report that mutants deleted for PUN1 exhibit increased metal ion sensitivity and morphological abnormalities. Microscopical and ultrastructural observations revealed a severe cell wall defect of pun1∆ mutants. By using chemical cross-linking, Blue native electrophoresis, and co-immunoprecipitation we found that Pun1p forms homo-oligomeric protein complexes. We propose that Pun1p is a stress-regulated factor required for cell wall integrity, thereby expanding the functional significance of lateral plasma membrane compartments.

Fig. 1

Metal ion stress-dependent expression of PUN1. Induction of PUN1 by metal ion stress. Logarithmically grown BY4741 wild-type cells were induced by different concentrations of metal ions for 30 min as indicated in the figure. RNA samples were separated on a formaldehyde gel, blotted and incubated with radio labeled probes against PUN1 and ACT1, used as a loading control. Intensities of Northern blot signals were quantified by ImageQuant 5.1 software and normalized to untreated cells and the actin control.

Fig. 2

Complementation and phenotyping of the pun1 deletion. A. Strains FY1679 wild-type and MApun1∆ were grown in YPD and YPD supplemented with 1 mM MnCl₂, 1.5 mM AsCl₃, 1 mM NiSO₄, and 25 mM CaCl₂ at 28 °C for 24 h under shaking. Growth was followed by measuring OD₆₀₀. Growth curves shown represent an average of three individual measurements. MApun1∆ cells exhibited increased metal ion sensitivity compared to their isogenic wild-type. B. Complementation of the pun1∆ growth phenotype. Serial 10-fold dilutions of strains FY1679, carrying the empty plasmid YCplac22 and MApun1∆ carrying the empty plasmid or expressing PUN1 under the control of its endogenous promoter (YCp-PUN1), were spotted on YPD plates with and without metal ions (concentrations as indicated in the figure), and incubated at 28 °C for 48 h. Plasmid-based expression of PUN1 restored metal ion sensitivity of pun1∆ mutant cells. C. Measurement of intracellular AsCl₃ concentration. Strains FY1679 and MApun1∆ carrying the empty plasmid YEpM351HA and MApun1∆ overexpressing C-terminally HA-tagged PUN1 from plasmid YEpM-PUN1-HA (MApun1∆(PUN1)ⁿ) were grown in SD medium containing 250 μM AsCl₃ and SD medium (w/o AsCl₃) for 6 h at 28 °C. Cells were washed, OD₆₀₀ and dry weight were defined and the total intracellular As³⁺ concentration of whole cells (μg As³⁺/g cells) was determined by ICP-MS (ARC Seibersdorf research GmbH, Austria). Values shown represent an average of three individual measurements. Absence of PUN1 caused a highly increased intracellular As³⁺ concentration.

Fig. 3

Subcellular localization of Pun1p. Plasma membrane ergosterol patches colocalize with Pun1p-GFP. Logarithmically grown FY1679 cells expressing C-terminally GFP-tagged Pun1p from plasmid pUG35-PUN1-GFP were treated with Filipin, which stains plasma membrane ergosterol (false colored red). The merged images demonstrate the high degree of overlap of the ergosterol patches and Pun1p-GFP. DIC, differential interference contrast.

Fig. 4

Induction of PUN1 upon metal stress. A. Yeast strain BY4741, bearing a chromosomal PUN1-GFP fusion, was stressed with various metal ions as indicated in liquid YDP for 1 h (a–e) and 2 h (f–j) at 28 °C. Pun1p-GFP was highly induced upon exposure to 200 mM CaCl₂ and 2 mM MnCl₂. GFP-fluorescence and corresponding DIC images were obtained from living cells without fixation. B. The identical strain as shown in Fig. 4A was grown in YPD or treated with various metal ions as indicated for 2 h at 28 °C. Cells were collected and washed and total protein of whole cells was precipitated with TCA. Proteins were separated by SDS-PAGE and immunoblotted against GFP and Hexokinase-1 (Hxk1), used as a loading control. Western blot results clearly confirmed metal ion-induced expression of Pun1p-GFP. C. Influence of upstream regulatory elements on metal ion-induced transcription. Wild-type cells and mutants deleted for HOG1, CRZ1, RLM1, STE12, SKO1, DIG1, and MSN2/4 were analyzed by Northern blot for MnCl₂ induced expression of PUN1. Red boxes highlight absent induction of PUN1 in strain BYcrz1∆ compared to the wild-type.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Modified cell wall composition of pun1∆ mutant cells. A. Staining of cell wall components. FY1679 wild-type and MApun1∆ cells were treated as indicated with Aniline Blue, concanavalin A–FITC, and Calcofluor White, staining cell wall 1,3-β-d-glucan, mannoproteins, and chitin, respectively. Fluorescence microscopy revealed reduced fluorescence of pun1∆ cells. B. Treatment with zymolyase. FY1679 wild-type and MApun1∆ cells were grown logarithmically in YPD medium, diluted to an equal OD₆₀₀ value and treated with 0, 30, or 100 μg/ml zymolyase 20T. At time points indicated in the figure, OD₆₀₀ was determined (upper panel). Equal amounts of FY1679 wild-type and MApun1∆ cells were treated for 30 min with identical zymolyase concentrations as used before. After centrifugation, equal amounts of the supernatant were immunodetected for cytoplasmic hexokinase 1 (Hxk1p), representing Hxk1p release caused by cell lysis (middle panel). Intracellular Hxk1p levels of both strains were compared by immunodetection after TCA precipitation of equal cell amounts (lower panel). C. Electron microscopic images of strains FY1679 wild-type (a-b), MApun1∆ (c and d), and MApun1∆ expressing PUN1 from plasmid YCp-PUN1. Principal cell wall constituents (GL and MP) and the plasma membrane (PM) are indicated. Images b, d, and f correspond to white frames in images a, c, and e, respectively. Plasmid-based expression of PUN1 restored the defective cell wall composition of MApun1∆ cells. PM, plasma membrane; GL, β-glucan layer; MP, mannoprotein layer. Black bars, 100 nm.

Fig. 5

Modified cell wall composition of pun1∆ mutant cells. A. Staining of cell wall components. FY1679 wild-type and MApun1∆ cells were treated as indicated with Aniline Blue, concanavalin A–FITC, and Calcofluor White, staining cell wall 1,3-β-d-glucan, mannoproteins, and chitin, respectively. Fluorescence microscopy revealed reduced fluorescence of pun1∆ cells. B. Treatment with zymolyase. FY1679 wild-type and MApun1∆ cells were grown logarithmically in YPD medium, diluted to an equal OD₆₀₀ value and treated with 0, 30, or 100 μg/ml zymolyase 20T. At time points indicated in the figure, OD₆₀₀ was determined (upper panel). Equal amounts of FY1679 wild-type and MApun1∆ cells were treated for 30 min with identical zymolyase concentrations as used before. After centrifugation, equal amounts of the supernatant were immunodetected for cytoplasmic hexokinase 1 (Hxk1p), representing Hxk1p release caused by cell lysis (middle panel). Intracellular Hxk1p levels of both strains were compared by immunodetection after TCA precipitation of equal cell amounts (lower panel). C. Electron microscopic images of strains FY1679 wild-type (a-b), MApun1∆ (c and d), and MApun1∆ expressing PUN1 from plasmid YCp-PUN1. Principal cell wall constituents (GL and MP) and the plasma membrane (PM) are indicated. Images b, d, and f correspond to white frames in images a, c, and e, respectively. Plasmid-based expression of PUN1 restored the defective cell wall composition of MApun1∆ cells. PM, plasma membrane; GL, β-glucan layer; MP, mannoprotein layer. Black bars, 100 nm.

Fig. 6

Oligomerization of Pun1p. A. Chemical cross-linking of Pun1p-HA. Membranes of FY1679 cells expressing plasmid YEpM-PUN1-HA were separated by ultracentrifugation on a discontinuous sucrose gradient (see Materials and methods). Equal amounts of the membrane extract were treated with increasing concentrations (0–300 μM) of the cross-linking reagent o-PDM. Proteins were separated by SDS-PAGE and immunoblotted against the HA epitope. Black arrows point to the Pun1p-HA monomer and higher-order oligomers (dimer to hexamer from bottom to top). o-PDM, o-phenylenedimaleimide. B. Blue native electrophoresis of Pun1p-HA. Equal amounts of the same membrane fraction used for chemical cross-linking were solubilized with the indicated detergents, applied to a native gradient gel and immunoblotted against the HA epitope. Arrowheads indicate putative oligomeric states of Pun1p-HA (dimer to dodecamer from bottom to top). Solid asterisk indicates an undefined protein complex containing Pun1p-HA. D, digitonin; TX, Triton X-100; DM, n-Dodecyl-β-d-maltoside; M, protein ladder. C. Co-immunoprecipitation (Co-IP) of Pun1p. Membrane fractions of strain BY4741 expressing chromosomally GFP-tagged Pun1p, either coexpressing HA-tagged Pun1p from plasmid YCp-PUN1-HA (lanes in blot area 1 and 3) or bearing the empty plasmid YCplac33 (lanes in blot area 2 and 4), were solubilized using 0.1% TX and incubated with anti-HA serum-coated (HA Co-IP) or anti-GFP serum-coated (GFP Co-IP) beads for 1 h at 4 °C. Unbound (supernatant, SN) and bound (elution, E) fractions were separated by SDS-PAGE and analyzed by immunoblotting with antibodies directed against HA (upper panel) or GFP (lower panel).

Fig. 7

Comprehensive model of transcriptional induction of PUN1 upon different stress conditions.