Conserved Ser/Arg-rich motif in PPZ orthologs from fungi is important for its role in cation tolerance

Abstract

PPZ1 orthologs, novel members of a phosphoprotein phosphatase family of phosphatases, are found only in fungi. They regulate diverse physiological processes in fungi e.g. ion homeostasis, cell size, cell integrity, etc. Although they are an important determinant of salt tolerance in fungi, their physiological role remained unexplored in any halotolerant species. In this context we report here molecular and functional characterization of DhPPZ1 from Debaryomyces hansenii, which is one of the most halotolerant and osmotolerant species of yeast. Our results showed that DhPPZ1 knock-out strain displayed higher tolerance to toxic cations, and unlike in Saccharomyces cerevisiae, Na(+)/H(+) antiporter appeared to have an important role in this process. Besides salt tolerance, DhPPZ1 also had role in cell wall integrity and growth in D. hansenii. We have also identified a short, serine-arginine-rich sequence motif in DhPpz1p that is essential for its role in salt tolerance but not in other physiological processes. Taken together, these results underscore a distinct role of DhPpz1p in D. hansenii and illustrate an example of how organisms utilize the same molecular tool box differently to garner adaptive fitness for their respective ecological niches.

FIGURE 1.

Growth of dhppz1 mutant in the presence of toxic cations. A, 5 μl of 10-fold serial dilutions of logarithmic phase culture of D. hansenii strains DBH9 (wild type) and DBH91 (dhppz1) were spotted on YPD plates supplemented with different concentrations of LiCl and NaCl. Growth on plates was observed after 2 days of incubation at 28 °C. B, saturated cultures of DBH9 and DBH91 grown in YPD medium were re-inoculated (at initial A₆₀₀ of 0.025) into 50 ml of YPD medium at different pH values containing different concentrations of NaCl. After 24 h of growth at 28 °C, the A₆₀₀ of these cultures was measured. Relative growth of the culture at each concentration of salt was expressed as the percentage of the growth measured in the absence of added salt at same pH. Data presented are the results of three independent experiments (mean ± S.D.). C, shown is dilution spotting of DBH9 and DBH91 on YPD plates containing different amounts of hygromycin and spermine. Results are representative of three independent experiments.

FIGURE 2.

Effect of DhPPZ1 knock out on cation ion transport in D. hansenii. A and B, shown is intracellular concentration of Li⁺ and K⁺ in wild type D. hansenii (DBH93) and dhppz1 mutant (DBH936) at different pH values. D. hansenii cells at early log phase were exposed to 200 mm LiCl in incubation buffer at different pH values. Cells were collected at different time points by filtration, and the total intracellular concentration (ppm per A₆₀₀ of the cells) of Li⁺ and K⁺ ion in these cells were measured by atomic absorption spectrophotometer as described under “Materials and Methods.” Data (mean ± S.D.) from duplicate biological samples with three replicates each are shown. C–E, shown is a comparison of the levels of DhENA1, DhTRK1, and DhNHA1 transcript in wild type and dhppz1 strains as analyzed by RT-qPCR. Total RNA was isolated from logarithmic phase cultures of wild type (DBH9) and dhppz1 mutant (DBH91) strain grown in YPD medium or after exposing them to 200 mm LiCl for 20 min. The RT-qPCR was performed with one step SYBR-I Green reaction kit (Invitrogen) using gene specific primers. Relative expression levels were normalized using DhGPDH as an internal control. The average relative expression levels for each gene in wild type and dhppz1 mutant was calculated using the ΔΔCt method and expressed as -fold difference (mean ± S.D.).

FIGURE 3.

DhPPZ1 knock-out affects growth of D. hansenii. A, growth curve of dhppz1 mutant (DBH936) and control parental (DBH93) D. hansenii strains are shown. Saturated cultures DBH936 and DBH93 were re-inoculated 100 ml of YPD medium at initial A₆₀₀ of 0.05. Growth of these cultures was monitored by measuring A₆₀₀ at regular intervals. B, shown is reversal of slow growth phenotype of dhppz1 mutant by expressing DhPPZ1 from plasmid. Saturated cultures of DBH936 strains transformed with indicated plasmids were re-inoculated into 25 ml of SD medium (at initial A₆₀₀of 0.025). After 24 h of growth at 28 °C, A₆₀₀ of these cultures was measured. DBH93 strain transformed with plasmids pDA1 and pDH4 was used as control. Data were expressed as their relative growth with respect to the control. C, relative growth of dhppz1 mutant at different pH values is shown. Saturated cultures of DBH936 and DBH93 (control) were re-inoculated (initial A₆₀₀ 0.025) into 50 ml of YPD medium buffered to different pH values. After 24 h of growth at 28 ºC, A₆₀₀ of the culture was measured. The growth at pH 6.0 for the respective culture was taken 100%. Data presented are the results of three independent experiments (mean ± S.D.).

FIGURE 4.

Overexpression of DhPpz1p suppresses phenotypic defects of dhmpk1 deletion in D. hansenii. A, suppression of the toxic cation and caffeine sensitivity of dhmpk1 mutant is shown. DBH932 harboring plasmids pAN5, pAN6, or pDA1 (vector control) were grown in SD medium to logarithmic phase (A₆₀₀ ∼ 1.0). 10-Fold serial dilutions of these cultures were on SD plates containing LiCl or caffeine as indicated. B, shown is relative growth of dhmpk1 mutant harboring different plasmids as indicated. A₆₀₀ after 24 h of growth for control (DBH93 harboring pDA1 and pDH4) was taken 100%. Data presented are results of three independent experiments (mean ± S.D.).

FIGURE 5.

Structure-function analysis of N-terminal domain of DhPpz1p. Schematics depicting wild type and mutant DhPpz1p are shown. C-terminal catalytic domain (light box), N-terminal non-catalytic domain (dark box), and the position of different deletions are shown. The right panel shows phenotypic complementation of dhppz1 mutation in DBH936 by different plasmid constructs as determined by dilution spotting on SD plates containing LiCl, hygromycin (Hyg), and caffeine (Caf). + indicates complementation, and − indicates no complementation. Experiments were repeated three times with similar results.

FIGURE 6.

Mutational analysis serine/arginine-rich motif in DhPpz1p and Ppz1p. A, sequence alignment of the serine/arginine-rich motif present in different Ppz1p orthologs from different species is shown: YL, Yarrowia lipolytica XP_504610.2; CD, Candida dubliniensis XP_002422157.1); Pzl1, Neurospora crassa AF071752; CL, Clavispora lusitaniae XP_002619471; PG, Pichia guilliermondii EDK41476.2; Ppz1, S. cerevisiae NP_013696.1; Ppz2, S. cerevisiae NP_010724.1; DhPpz1, D. hansenii XP_459586.2. Conserved residues are shown by asterisks. B, phenotypic complementation of wild type DhPpz1p and mutants carrying point mutations in serine/arginine-rich motif along with the sequence of respective mutated region are shown. + indicates complementation, and − indicates not complementation. C, dilution spotting of S. cerevisiae strains expressing wild type Ppz1p or its mutants on SD plate containing LiCl is shown. PPZ1 (parental strain BY4742/pRS423), ppz1 mutant (Y10557/pRS423), and Δ43–52 (Y10557/PPZ1-Δ43–52) were grown overnight on SD without histidine before serial dilution. Representative data of three independent experiments are shown. D, effect of mutation in individual serine and arginine residues of Ser/Arg motif on salt tolerance exhibited by dhppz1 mutant is shown. 10-Fold serial dilution of the DBH936 strain expressing different point mutations was spotted on SD plates containing 0.4 and 0.6 m LiCl. Plates were incubated at 28 ºC for 3–4 days before being photographed. A culture of DBH936 harboring empty vector pDA1 was used as the control. Representative data of two independent experiments are shown.

FIGURE 7.

Role of Ser/Arg-rich motif in the interaction of DhPpz1p with DhHal3p. A, two-hybrid assay for interactions between DhHal3p and DhPpz1p. Wild type DhPPZ1 or its mutant having a deletions in the Ser/Arg motif (Δ27–36) cloned in pEG202 vector was used as bait, whereas DhHAL3 cloned in plasmid pJG4-5 was used as prey. Bait and prey constructs (in pairs as indicated) or in combination with empty prey or bait (as control) were transformed into S. cerevisiae strain EGY48. Growth of the transformants on Gal-Raf minimal medium is shown after dilution spotting. Experiments were repeated twice with pool of four independent transformants. Representative data are shown. B, growth of EGY48 harboring DhPPZ1 and Δ27–36 bait along with empty prey or DhHal3 in prey vector were grown overnight in minimal media with 2% raffinose (without tryptophan and histidine). The cultures were re-inoculated in minimal media with 1% raffinose and 2% galactose (without tryptophan, histidine, and leucine) at A₆₀₀ ∼0.10 and grown further for 41 h. Data (mean ± S.D.) of two independent experiments, each performed in duplicate with a pool of four different transformants, is shown here.