Characterization of orchardgrass p23, a flowering plant Hsp90 cohort protein

Abstract

p23 is a heat shock protein 90 (Hsp90) co-chaperone and stabilizes the Hsp90 heterocomplex in mammals and yeast. In this study, we isolated a complementary DNA (cDNA) encoding p23 from orchardgrass (Dgp23) and characterized its functional roles under conditions of thermal stress. Dgp23 is a 911 bp cDNA with an open reading frame predicted to encode a 180 amino acid protein. Northern analysis showed that expression of Dgp23 transcripts was heat inducible. Dgp23 has a well-conserved p23 domain and interacted with an orchardgrass Hsp90 homolog in vivo, like mammalian and yeast p23 homologs. Recombinant Dgp23 is a small acidic protein with a molecular mass of approximately 27 kDa and pI 4.3. Dgp23 was also shown to function as a chaperone protein by suppression of malate dehydrogenase thermal aggregation. Differential scanning calorimetry thermograms indicated that Dgp23 is a heat-stable protein, capable of increasing the T (m) of lysozyme. Moreover, overexpression of Dgp23 in a yeast p23 homolog deletion strain, Deltasba1, increased cell viability. These results suggest that Dgp23 plays a role in thermal stress-tolerance and functions as a co-chaperone of Hsp90 and as a chaperone.

Fig. 1

Comparison of orchardgrass p23 amino acid sequence with homologs from other organisms. a Alignment of orchardgrass p23 protein (accession no. DQ172836) with homologs from corn (ACF82936), rice (NP_001050241), rape (AAG41763), grape (CAO46199), Arabidopsis (CAC16575), japanese cedar (BAE92292), moss (XP_001768873), budding yeast (P28707), fission yeast (Q11118), human (Q15185), mouse (AAD39543), and chicken (B56211). Numbers at the right indicate amino acid residue position. Fully identical residues are shaded in black; six or seven identities in gray; and five identities in light gray. The conserved p23 domain is represented as a box; a solvent-accessible surface as a bar below the sequence; and conserved identical residues as an asterisk above the sequence. b Phylogenetic relationships between Dgp23 and other p23 homologs. Phylogenetic tree shows a graphical representation of evolutionary relationships and was constructed by using EBI-ClustalW algorithm

Fig. 2

Northern blot analyses of Dgp23.a Accumulation of Dgp23 transcripts after heat treatment at various temperatures for 1 h. b Time-course accumulation of Dgp23 transcripts during heat treatment at 35°C and subsequent recovery after heat treatment. Ethidium bromide staining of the RNA gel was used to show equal loading

Fig. 3

In vivo interaction analysis of Dgp23 and DgHsp90 by yeast two-hybrid system. +His Transformants selected on SD medium lacking tryptophan and leucine; −His transformants selected on SD medium lacking tryptophan, leucine, and histidine; X-Gal β-galactosidase assay for the protein–protein interaction

Fig. 4

SDS-PAGE and IEF analysis of recombinant Dgp23. a SDS-PAGE (12%) of protein samples from the purification steps of Dgp23. Lane 1 Molecular weight markers; lane 2 crude extracts after IPTG induction; lane 3 eluate from affinity chromatography; lane 4 purified Dgp23 after thrombin digestion. b IEF of purified Dgp23. Carbonic anhydrase (pI 6.0), bovine serum albumin (5.6), ovalbumin (4.6), and soybean calmodulin (4.1) as IEF standards (M), and 10 μg of purified Dgp23 was subjected to IEF gel. All protein bands were stained with Coomassie brilliant blue

Fig. 5

Chaperone assay of recombinant Dgp23. a Chaperone activity of Dgp23. MDH (0.3 μM) was incubated in the absence (open circle) or presence of 0.3 μM (closed square), 0.6 μM (closed diamond), 1.5 μM (closed triangle) Dgp23 or 0.3 μM EcTrx (open diamond). Dgp23 (0.3 μM) was incubated without MDH (open square). b p23 protection against MDH thermal aggregation. Two micromolar MDH, p23 or MDH-p23 mixture, was heat-treated at the indicated temperatures for 15 min. After centrifugation, supernatants were analyzed by 18% SDS-PAGE. The blots of each gel were hybridized with anti-His antibody for MDH and anti-Dgp23 antibody for Dgp23

Fig. 6

DSC. Thermal unfolding of lysozyme (closed square) and lysozyme–Dgp23 mixture (closed circle) were measured in DSC. The inset shows the melting temperature

Fig. 7

Thermotolerance of yeast cells overexpressing Dgp23. Yeast spot assays (a and b) and survival rate (c) were carried out to determine thermotolerance of yeast transformants. a Wild-type (BY4741, WT), Δsba1 (pYES alone), and Δsba1 + Dgp23 were incubated at 30°C or 37°C for 3 days. b Serially diluted yeast cells were exposed to 25°C or 50°C for 30 min before spotting and incubated at 30°C for 3 days. c Survival rate after heat treatment at 50°C for the indicated times. After 2 days incubation at 30°C, visualized colonies were counted to calculate the percentage survival relative to yeast cells treated at 25°C