Genetic analysis reveals domain interactions of Arabidopsis Hsp100/ClpB and cooperation with the small heat shock protein chaperone system

Abstract

We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaperone (AtHsp101) in acquired thermotolerance by isolating recessive, loss-of-function mutations and a novel semidominant, gain-of-function allele [hot1-4 (A499T)]. The hot1-4 allele is unusual in that it not only fails to develop thermotolerance to 45 degrees C after acclimation at 38 degrees C, but also is sensitive to 38 degrees C, which is a permissive temperature for wild-type and loss-of-function mutants. hot1-4 lies between nucleotide binding domain 1 (NBD1) and NBD2 in a coiled-coil domain that is characteristic of the Hsp100/ClpB proteins. We then isolated two classes of intragenic suppressor mutations of hot1-4: loss-of-function mutations (Class 1) that eliminated the 38 degrees C sensitivity, but did not restore thermotolerance function to hot1-4, and Class 2 suppressors that restored acquired thermotolerance function to hot1-4. Location of the hot1-4 Class 2 suppressors supports a functional link between the coiled-coil domain and both NBD1 and the axial channel of the Hsp100/ClpB hexamer. In addition, the strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps) after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. These results also demonstrate that quantitative phenotypes can be used for in vivo genetic dissection of protein mechanism in Arabidopsis.

Figure 1.

Location and Phenotype of Mutations in AtHsp101 (See Also Supplemental Figure 1 Online). (A) Location of hot1 mutations with a thermotolerance phenotype on a schematic diagram of the AtHsp101 protein. The conserved AAA modules consist of two domains, a nucleotide binding domain (NBD1 or 2), containing conserved motifs [Walker A, Walker B, Sensor 1(S1), and Arg finger (R)], and a C-terminal small domain (gray boxes), containing Sensor 2 (S2) in AAA2. The coiled-coil domain contains two signature motifs (Lee et al., 2003), with hot1-4 located in signature motif II. (B) Location of missense mutations that are wild type for thermotolerance, which were obtained by Tilling analysis (see Methods). (C) Quantitative assessment of thermotolerance in hot1 mutant seedlings compared with their corresponding wild type (Columbia for hot1-1 and hot1-4, Columbia erecta for all others). After growth for 2.5 d in the dark at 22°C, seedlings were pretreated at 38°C for 90 min and returned to 22°C for 2.5 d (38°C samples), or pretreatment was followed by 2 h at 22°C then 2 or 3 h at 45°C before 2.5 d of recovery (38°C > 45°C samples). hot1-4 seedlings are more sensitive to 38°C treatment than wild-type or null alleles. Asterisks indicate values equal to zero. Mean and standard deviations were derived by measurement from three independent experiments performed with 60 or more seedlings per mutant and values plotted as a percentage of the 22°C value. (D) Protein gel blot analysis of AtHsp101 protein levels in wild-type and hot1 seedlings after treatment at 38°C.

Figure 2.

Unusual Sensitivity of hot1-4 to 38°C. Hypocotyl elongation of hot1 mutant seedlings treated for different times at 38°C, as measured after an additional 2.5 d of growth in the dark at 22°C. Replicated measurements performed as in Figure 1C.

Figure 3.

Expression of Mutant HOT1-4 Protein in Wild-Type Plants Recapitulates the 38°C Sensitivity Phenotype of hot1-4. The hot1-4 genomic DNA under its own promoter was expressed in transgenic wild-type plants. Two independent transgenic lines (T3:1 and T3:2) are shown compared with the wild type and hot1-4 (three seedlings each). The hypocotyl elongation assay was performed as in Figure 1C. Equal protein samples from heat-stressed seedlings (38°C for 90 min followed by 2 h at 22°C) were analyzed with the indicated antisera against AtHsp101, sHsps, or glyceraldehyde-3-phosphate dehydrogenase (GAPC).

Figure 4.

Location of hot1-4 Suppressor Mutations in AtHsp101. Class 1 loss-of-function suppressors are labeled in smaller gray font and indicated with closed circles. Restoration-of-function Class 2 suppressors are indicated in larger black font with open circles. The strongest Class 2 suppressors, as discussed in the text, are underlined (see also Supplemental Figure 1 online).

Figure 5.

Thermotolerance Phenotype of 10-d-Old Suppressor Mutants. After growth for 10 d in the light at 22°C, seedlings were treated either at 38°C for 3 h (A) or at 38°C for 90 min followed by 2 h at 22°C and then 2 h at 45°C (B). Seedlings were photographed 7 d after treatment.

Figure 6.

Accumulation of AtHsp101 Protein in the Suppressor Mutants. Protein samples from heat-stressed seedlings (38°C for 90 min followed by 2 h at 22°C) were analyzed with AtHsp101 antiserum. (A) Class 1 suppressor mutants. (B) Class 2 suppressor mutants.

Figure 7.

Reversal of sHsp Insolubility in the Suppressor Mutants. Ten-day-old seedlings were treated either at 38°C for 3 h (38) or at 38°C for 90 min followed by 2 h at 22°C and then 60 min at 45°C (45), and total protein (T) was isolated after further recovery for 3 h at 22°C. The insoluble protein fraction (P) was separated from the soluble fraction (S) by centrifugation and analyzed with AtHsp101, AtHsp17.6C-I (sHsp I), and AtHsp17.6C-II (sHsp II) antiserum.

Figure 8.

Location of the hot1 Mutations on the T. thermophilus ClpB Structure. (A) The available structure of the T. thermophilus ClpB monomer (PDB file 1QVR; chain in [A]) is shown with the coiled-coil domain and AAA modules indicated. Within each AAA module, the NBD domain is colored blue, with the Walker A and B motifs in orange, sensor 1 in pink, and the Arg finger in yellow (see also Supplemental Figure 1 online). The small, α-helical domain of each AAA module is shown in gray. Nucleotide (AMP-PNP) is space-filled in CPK coloring (hydrogen, white; oxygen, red; phosphorous, orange; sodium, blue). Motif 1 as defined by Schirmer et al. (1996) is colored purple in the coiled-coil domain. The residue corresponding to each of the hot1 mutation sites is space-filled and colored green. (B) Alternative view of the motif II region of the coiled-coil domain indicating the L2, L3, and L4 helices and the residue positions of the hot1-4 and hot1-6 mutations (in CPK coloring). (C) View of interactions of the hot1-5 position (in CPK coloring) with residues within NDB2 and NBD1 as discussed in the text. (D) Position of hot1-7 (CPK coloring) relative to the NBD2 ATP binding site. The sensor 2 Arg residue, which is part of the GAR motif, as well as sensor 1 are shown in pink. Residue numbering corresponds to AtHsp101. Figure was prepared with Swiss PDB viewer.

Figure 9.

Location of the hot1-4 Suppressor Mutations on the T. thermophilus ClpB Monomer Structure. The major domains and motifs of HCP100/ClpB are colored as indicated and described in Figure 8. The sites of Class 1 suppressor mutations are space-filled dark gray, and sites of the Class 2 suppressor mutations are space-filled in red. The three strongest Class 2 suppressors are indicated in bold and are underlined. For A297, G653, and G649, which are in unresolved segments of the structure, dotted lines are used to indicate the relative positions of those segments. The arrow indicates the general position of the axial channel of the hexameric form of the protein. Residue numbering corresponds to AtHsp101. Figure was prepared with Swiss PDB viewer.