ATP requirement for chloroplast protein import is set by the Km for ATP hydrolysis of stromal Hsp70 in Physcomitrella patens

Abstract

The 70-kD family of heat shock proteins (Hsp70s) is involved in a number of seemingly disparate cellular functions, including folding of nascent proteins, breakup of misfolded protein aggregates, and translocation of proteins across membranes. They act through the binding and release of substrate proteins, accompanied by hydrolysis of ATP. Chloroplast stromal Hsp70 plays a crucial role in the import of proteins into plastids. Mutations of an ATP binding domain Thr were previously reported to result in an increase in the Km for ATP and a decrease in the enzyme's kcat. To ask which chloroplast stromal chaperone, Hsp70 or Hsp93, both of which are ATPases, dominates the energetics of the motor responsible for protein import, we made transgenic moss (Physcomitrella patens) harboring the Km-altering mutation in the essential stromal Hsp70-2 and measured the effect on the amount of ATP required for protein import into chloroplasts. Here, we report that increasing the Km for ATP hydrolysis of Hsp70 translated into an increased Km for ATP usage by chloroplasts for protein import. This thus directly demonstrates that the ATP-derived energy long known to be required for chloroplast protein import is delivered via the Hsp70 chaperones and that the chaperone's ATPase activity dominates the energetics of the reaction.

Figure 1.

Hsp70-2^wt, Hsp70-2^T271E, and Hsp70-2^T271V cDNA Rescues the Hsp70-2 KO in P. patens Plants. (A) ClustalW alignment of a portion of the nucleotide binding sites of Pp-Hsp70-2 (P. patens), Ec-DnaK (E. coli), and Bt-Hsc70 (bovine). The position of T271 in the moss gene is identified. (B) Genotyping of moss strains generated from Hsp70-2^wt, Hsp70-2^T271E, and Hsp70-2^T271V cDNA-rescued KO plants. Primer pairs are indicated above the gel; G1, G2, R1, and R2 are forward and reverse primers in the genomic DNA and the resistance gene used for the KO, respectively. gDNA, PCR product amplified from the genomic locus (lane 1); cDNA, PCR product amplified from the cDNA construct rescuing the plant from the lethal Hsp70-2^wt KO (lanes 4, 7, and 10).

Figure 2.

Growth Rate Measurement for Hsp70-2^wt, Hsp70-2^T271E, and Hsp70-2^T27V cDNA-Rescued KO Transgenic Plants. (A) Three representative micrographs showing chlorophyll fluorescence (red) of 1-week-old plants. Hsp70-2, Hsp70-2^wt; T271E, Hsp70-2^T271E; T271V, Hsp70-2^T271V. Bar = 100 μm. (B) Growth rate of the indicated plants on BCD (phototrophic) media. Colony area was measured every 2 d for 16 d and normalized to the initial area on day 0. Data represent the means and standard errors from three colonies for each moss strain.

Figure 3.

Effect of Cochaperones on the in Vitro ATPase Activity of Hsp70-2^wt, Hsp70-2^T271E, and Hsp70-2^T27V. (A) Comparison of ATPase activities of bacterially produced moss Hsp70-2^wt, Hsp70-2^T271E, and Hsp70-2^T271V and E. coli DnaK with cochaperones from E. coli and moss. ATPase activity of these proteins was assayed colorimetrically with malachite green in a medium containing HEPES-KOH (40 mM, pH 7.0), KCl (75 mM), Mg(CH₃COO)₂, denatured NR substrate peptide (NRLLLTG) (200 nM), ATP (1 mM), and when added, DnaK or Hsp70 (0.3 μM), E. coli or P. patens DnaJ (1 μM), and GrpE or CGE (1 μM). Shown are means and standard errors from three replicate experiments. (B) Recombinant moss proteins used in the ATPase assays. Proteins were purified after overexpression in bacteria; the figure shows 1 μg of each on a Coomassie blue–stained 12.5% SDS-PAGE gel.

Figure 4.

Thr Hsp70 Mutants Display Lower V_max and Increased K_m for ATP Hydrolysis Compared with the Wild Type. Moss proteins were purified after overexpression in bacteria. ATPase activity was assayed with moss cochaperones as in Figure 3 using prSSU as the substrate. Data were fitted to the Michaelis-Menten equation with the indicated parameters using KaleidaGraph. Circles, squares, and triangles correspond to Hsp70-2^wt, Hsp70-2^T271V, and Hsp70-2^T271E, respectively. The data represent means and standard errors from three replicates.

Figure 5.

ATP-Dependent Import of tp22GFP and prL11 into 7-d-Old Wild-Type Moss Chloroplasts. L, import assay in the light; D, import assay in the dark with 3 mM added ATP; D+Th, import assay in the dark followed by thermolysin treatments to digest the unimported proteins; Pr, precursor; M, mature protein. ATP was removed from the precursors by gel filtration.

Figure 6.

Import of tp22GFP into Wild-Type and Transgenic Moss Chloroplasts as a Function of Added ATP. (A) ATP dependence of tp22GFP import in the dark into chloroplasts isolated from 7-d-old wild-type plants and from transgenic KO moss rescued with Hsp70-2^wt, Hsp70-2^T271E, or Hsp70-2^T271V cDNA. Pr, precursor; M, mature protein. (B) Quantification of (A). Data from three replicates (±se) were fitted to the Michaelis-Menten equation using KaleidaGraph; resulting values for K_m and V_max are indicated.

Figure 7.

Import of prL11 into Transgenic Moss Chloroplasts as a Function of Added ATP. (A) Import of prL11 to chloroplasts isolated from 7-d-old transgenic KO moss rescued with Hsp70-2^wt and Hsp70-2^T271E cDNA. Pr, precursor; M, mature protein. (B) Quantification of (A). Data from three replicates (±se) were fitted to the Michaelis-Menten equation using KaleidaGraph; resulting values for K_m and V_max are indicated.

Figure 8.

Immunoblot Showing Relative Protein Abundance of Hsp70 and Hsp93 in Chloroplasts. (A) Immunoblot using antibodies against Hsp70-2 and Hsp93. Chloroplasts were isolated from wild-type plants and KO plants rescued by Hsp70-2^wt, Hsp70-2^T271E, and Hsp70-2^T271V cDNA, respectively; CBB-LSU, loading control showing the large subunit of Rubisco stained with Coomassie blue. (B) Quantification of (A) showing means and standard errors from three independent experiments and normalized to the wild-type value.