Functional Characterization of an Arabidopsis Profilin Protein as a Molecular Chaperone under Heat Shock Stress

Abstract

Profilins (PFNs) are actin monomer-binding proteins that function as antimicrobial agents in plant phloem sap. Although the roles of Arabidopsis thaliana profilin protein isoforms (AtPFNs) in regulating actin polymerization have already been described, their biochemical and molecular functions remain to be elucidated. Interestingly, a previous study indicated that AtPFN2 with high molecular weight (HMW) complexes showed lower antifungal activity than AtPFN1 with low molecular weight (LMW). These were bacterially expressed and purified to characterize the unknown functions of AtPFNs with different structures. In this study, we found that AtPFN1 and AtPFN2 proteins have LMW and HMW structures, respectively, but only AtPFN2 has a potential function as a molecular chaperone, which has never been reported elsewhere. AtPFN2 has better protein stability than AtPFN1 due to its higher molecular weight under heat shock conditions. The function of AtPFN2 as a holdase chaperone predominated in the HMW complexes, whereas the chaperone function of AtPFN1 was not observed in the LMW forms. These results suggest that AtPFN2 plays a critical role in plant tolerance by increasing hydrophobicity due to external heat stress.

Keywords: AtPFN; heat shock; higher molecular weight; molecular chaperone; profilin.

Figure 1

Comparison of the hydrophobicity of AtPFN proteins and their purification from E. coli. (a) The Kyte-Doolittle analysis generated hydrophobicity plots of AtPFN1 (gray) and AtPFN2 (red). The y-axis indicates the hydrophobicity scores; positive scores on the y-axis indicate hydrophobic regions. (b) Recombinant AtPFN1 and AtPFN2 were isolated via E. coli expression, and purity was confirmed using 13% SDS-PAGE and Coomassie blue staining. Induction, IPTG induced total proteins; affinity resin, a soluble protein purified by an affinity column; SEC, pure protein fractionated from SEC.

Figure 2

Stability of AtPFN proteins under heat shock conditions. Heat stability analysis of AtPFN proteins and malate dehydrogenase (MDH; control). Approximately 5 μg each of AtPFN and MDH were incubated at 25 °C (left; Normal condition) or 43 °C (right; Heat condition) for 30 min and then centrifuged at 13,000× g for 15 min. Each protein’s supernatant (soluble fraction) and pellet (insoluble fraction) were fractionated and analyzed using 13% SDS-PAGE.

Figure 3

Structural analysis of recombinant AtPFN proteins in vitro. Recombinant AtPFN protein concentration-dependent aliquots were analyzed using a 10% native PAGE gel and silver staining. To confirm the native molecular mass of each AtPFN protein at normal condition, two recombinant proteins were analyzed via incubation at 25 °C for 30 min and centrifugation at 13,000× g for 15 min.

Figure 4

Comparison of holdase chaperone activity between AtPFN1 and AtPFN2. Thermal aggregation of 20 µg malate dehydrogenase (MDH) was examined at 43 °C for 20 min in the presence of AtPFN1 or AtPFN2 proteins. (a) Molar ratio of AtPFN2 to MDH: (▲) 3:1, (■) 5:1, and (●) 10:1. (◆) denotes the negative Control (MDH alone). (b) Molar ratios of AtPFN1 to MDH: (▲) 5:1, (■) 20:1, and (●) 40:1. (◆) denotes the negative control (MDH alone). Holdase chaperone activity by measuring the absorbance of the solutions at a wavelength of 340 nm.

Figure 5

Structural analysis of AtPFN2 via size-exclusion chromatography (SEC) and transmission electron microscopy (TEM). Purified recombinant AtPFN2 was separated using SEC based on MW. The HMW (a) and LMW (b) fractions of the AtPFN2 protein isolated through SEC were collected, and the structure of each fraction was determined. The HMW and LMW fractions were separated using 12% SDS-PAGE electrophoresis to confirm that they were indeed AtPFN2 proteins (inset). Oligomeric forms of AtPFN2 fractionated from SEC were observed under TEM (inset). The bar represents 50 nm.

Figure 6

Association of AtPFN2 holdase chaperone function with protein structure. After equalizing the AtPFN2 levels in the two SEC fractions (FI and FII) and an aliquot of the total protein, the specific chaperone activity of AtPFN2 was measured using MDH as a substrate at 340 nm (A340). (a) Comparison of chaperone activity between FI and FII fractions of AtPFN2. Thermal aggregation of 20 µg MDH was examined at 43 °C for 20 min in the presence of FI or FII fraction of AtPFN2 protein. The activities of the different protein fractions were compared as a titration manner. (b) The activities of the different protein fractions were compared to those of the total protein. Total protein activity was measured under our assay conditions and set to 1 (fold). Representative results represent the mean of at least three independent experiments.

Figure 7

Comparison of the chaperone activity and hydrophobicity of AtPFN1 and AtPFN2 under heat shock conditions. Comparison of the chaperone activity and hydrophobicity of AtPFN proteins at different temperatures. (a) Hydrophobicity analysis under normal and heat shock conditions upon incubation with bis-ANS for 30 min at 25 °C, 43 °C, and 60 °C, respectively. The fluorescence of bis-ANS was measured using a fluorometer with an excitation wavelength of 390 nm and emission wavelengths of 430–630 nm. (b) Relative chaperone activities of AtPFN1 and AtPFN2 at 25 °C (normal) and 43 °C (Heat shock). Representative results represent the mean of at least three independent experiments.

Figure 8

Antifungal activity of HMW and LMW fractions of AtPFN2 protein against four fungal strains. After 24 h incubation of fungal conidia ((a): Candida krusei, (b): C. tropicalis) and proteins, the solution was streaked on YPD agar, followed by additional 24 h incubation. c: control, 1: 0.5 mg/mL, 2: 0.25 mg/mL, 3: 0.125 mg/mL, 4: 0.0625 mg/mL, 5: 0.03125 mg/mL, 6: 0.0156 mg/mL, 7: 0.0078 mg/mL. After 24 h incubation of fungal conidia ((c): Fusarium graminearum, (d): Cryptococcus sp.) and proteins, the fungal growth was observed using a microscope.

Figure 9

A representative model of oligomeric status and function of AtPFN proteins. AtPFN2 has dual functions as a chaperone and antifungal agent in the HMW and LMW structures, respectively. However, AtPFN1 with a LMW structure acts as an antifungal protein.