Intragenic suppressors of Hsp70 mutants: interplay between the ATPase- and peptide-binding domains

Abstract

ATP hydrolysis and polypeptide binding, the two key activities of Hsp70 molecular chaperones, are inherent properties of different domains of the protein. The coupling of these two activities is critical because the bound nucleotide determines, in part, the affinity of Hsp70s for protein substrate. In addition, cochaperones of the Hsp40 (DnaJ) class, which stimulate Hsp70 ATPase activity, have been proposed to play an important role in promoting efficient Hsp70 substrate binding. Because little is understood about this functional interaction between domains of Hsp70s, we investigated mutations in the region encoding the ATPase domain that acted as intragenic suppressors of a lethal mutation (I485N) mapping to the peptide-binding domain of the mitochondrial Hsp70 Ssc1. Analogous amino acid substitution in the ATPase domain of the Escherichia coli Hsp70 DnaK had a similar intragenic suppressive effect on the corresponding I462T temperature-sensitive peptide-binding domain mutation. I462T protein had a normal basal ATPase activity and was capable of nucleotide-dependent conformation changes. However, the reduced affinity of I462T for substrate peptide (and DnaJ) is likely responsible for the inability of I462T to function in vivo. The suppressor mutation (D79A) appears to partly alleviate the defect in DnaJ ATPase stimulation caused by I462T, suggesting that alteration in the interaction with DnaJ may alter the chaperone cycle to allow productive interaction with polypeptide substrates. Preservation of the intragenic suppression phenotypes between eukaryotic mitochondrial and bacterial Hsp70s suggests that the phenomenon studied here is a fundamental aspect of the function of Hsp70:Hsp40 chaperone machines.

Figure 1

Growth phenotype of yeast SSC1 mutants. pRS314-SSC1 carrying either a wt or mutant copy of SSC1 (as indicated) was transformed into strain DG252. Strains were grown in galactose minimal selective media overnight at 23°C, 1:10 serial dilutions were made, and equivalent numbers of cells were plated on rich glucose media and incubated at the indicated temperatures to test the ability of mutant SSC1 genes to support growth. (A) Growth of D107A, I485N, and D107A/I485N. (B) Growth of I485N in the presence of additional ATPase domain mutations.

Figure 2

Growth and precursor processing defect of SSC1-I485T is partly alleviated by D107A. (A) pRS314-SSC1 plasmids carrying either a wt or mutant copy of SSC1 (as indicated) were transformed into DG252 cells; transformants were streaked on plates containing 5-fluoroorotic acid to select for loss of the URA3-marked plasmid containing wt SSC1. 5-Fluoroorotic acid-resistant cells were grown in YPD overnight, 1:10 serial dilutions were made, and cells were plated on YPD and incubated at the indicated temperatures. (B and C) In vivo pulse–chase analysis of mitochondrial precursor processing. Strains containing wt or mutant copies of SSC1 were pulse labeled with [³⁵S]methionine and cysteine at 37°C for 2 min, at which time cold methionine and cysteine were added, and incubation continued for the indicated time in minutes before harvest and immunoprecipitation with antibodies to Hsp60 (B) or F₁β (C). Samples were analyzed by SDS/PAGE and visualized by using a PhosphorImager.

Figure 3

Location of residues on DnaK crystal structures (PDB entries 1DKG and 1DKX). ATPase domain (A) is rotated 180° relative to the traditional view; peptide-binding domain (B). Residues described in this study are blue: Ssc1 D107 (DnaK D79), E109 (E81), V110 (V82), Q116 (I88), N126 (N98), Q180 (Q152), F239 (F216), L260 (I237), I485 (I462). Peptide substrate is green. Residues identified in other studies (C) are yellow: DnaK R167, I169, T215 (24); DnaK Y145, N147, D148, E217, V218 (25).

Figure 4

Similar intragenic suppression phenotype is seen for analogous mutations in E. coli dnaK. pQE60 vector or pBB46 containing wt or mutant dnaK under the control of an isopropyl β-d-thiogalactoside (IPTG)-inducible promoter was transformed into strain BB205. Strains were grown overnight at 30°C, a permissive growth temperature for dnaK mutants. Serial dilutions (1:10) were made, equivalent numbers of cells were plated on media containing 15 μM IPTG, and cells were grown overnight at the indicated temperatures.

Figure 5

Peptide p5 stimulation of DnaK ATPase activity. Single-turnover ATPase assays were performed in the absence (□) or presence (♦) of p5. DnaK concentration is ≈0.35 μM; p5 is 5 μM. Fold stimulation of ATPase activity by p5 is shown in the box. (A) wt DnaK. (B) I462T. (C) D79A-I462T. (D) D79A.

Figure 6

Fluorescence anisotropy assay of peptide binding to wt and I462T DnaK. F-APPY (35 nM) fluorescein-labeled peptide was incubated with the indicated concentrations of wt (■) or I462T (⧫) DnaK overnight to achieve equilibrium. Fluorescence polarization was determined at 25°C with excitation at 490 nm and emission at 535 nm. Data were fitted to a quadratic single-site binding equation to determine the K_d of DnaK for peptide, which is shown in the box. Data is expressed as fraction of DnaK bound to F-APPY. At saturation, the maximum polarization for wt was approximately 240 polarization milliunits, whereas the maximum for I462T was approximately 205 polarization milliunits.

Figure 7

Surface plasmon resonance analysis of the interaction of immobilized DnaJ with wt and mutant DnaK proteins. DnaK (1 μM) was preincubated with 1 mM ATP for 5 min, and injection of DnaK over the sensor chip began at 0 seconds; buffer was injected at 210 seconds. A response in response units (RU) indicates binding of DnaK. Background binding to a flow cell containing immobilized biotin was subtracted from each sensorgram.

Figure 8

DnaJ stimulation of DnaK ATPase activity. Single-turnover ATPase assays were performed in the absence (□) or presence (♦) of DnaJ. DnaK concentration is ≈0.35 μM; DnaJ is 0.2 μM. Fold stimulation of ATPase activity by DnaJ is shown in the box. (A) wt DnaK. (B) I462T. (C) D79A-I462T. (D) D79A.

Figure 9

Intrinsic tryptophan fluorescence emission spectra of wt and mutant DnaK proteins. DnaK (0.65 μM) was preincubated with 1 mM ADP (♦) or ATP (□). To determine peak maxima (shown in box), data were fit by nonlinear regression analysis to an asymmetric curve described on the left by a Gaussian peak function and on the right by a mixed Gaussian/Laurentzian function by using Microsoft excel. (A) wt DnaK. (B) I462T. (C) D79A-I462T. (D) D79A.