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. 2008 Oct 3;283(40):27100-9.
doi: 10.1074/jbc.M803923200. Epub 2008 Aug 6.

Functional divergence between co-chaperones of Hsc70

Stefan Tzankov  1 , Michael J H Wong, Kun Shi, Christina Nassif, Jason C Young
Affiliations

Functional divergence between co-chaperones of Hsc70

Stefan Tzankov et al. J Biol Chem. .

Abstract

The ATPase cycle of the chaperone Hsc70 is regulated by co-chaperones; Hsp40/DnaJ-related proteins stimulate ATP hydrolysis by Hsc70 and can bind unfolded polypeptides themselves. Conversely, various nucleotide exchange factors (NEFs) stimulate ADP-ATP exchange by Hsc70. We analyzed the purified Hsp40-related co-chaperones DJA1 (Hdj2) and DJA2 (Hdj3) and found that they had a distinct pattern of binding to a range of polypeptides. DJA2 alone could stimulate Hsc70-mediated refolding of luciferase in the absence of NEF, whereas DJA1 was much less active. The addition of the Bag1 NEF increased refolding by Hsc70 and DJA2, as did the newly characterized NEF Hsp110, but each NEF had a different optimal concentration ratio to Hsc70. Notably, the NEF HspBP1 could not increase refolding by Hsc70 and DJA2 at any concentration, and none of the NEFs improved the refolding activity with DJA1. Instead, DJA1 was inhibitory of refolding with DJA2 and Hsc70. All combinations of DJA1 or DJA2 with the three NEFs stimulated the Hsc70 ATPase rate, although Hsp110 became less effective with increasing concentrations. A chimeric DJA2 having its Hsc70-stimulatory J domain replaced with that of DJA1 was functional for polypeptide binding and ATPase stimulation of Hsc70. However, it could not support efficient Hsc70-mediated refolding and also inhibited refolding with DJA2 and Hsc70. These results suggest a more complex model of Hsc70 mechanism than has been previously thought, with notable functional divergence between Hsc70 co-chaperones.

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Figures

FIGURE 1
FIGURE 1. Polypeptide binding by DJAs
The indicated polypeptides were radiolabeled by cell-free translation and co-precipitated either with nickel-Sepharose alone or with purified His-tagged DJA1, DJA2, or DJA4 and nickel-Sepharose. Bound polypeptide was quantified by SDS-PAGE and phosphorimaging analysis. In these and all experiments, the error bars represent the standard deviations from the mean of at least three independent trials. The amount of polypeptide bound by negative control beads or by DJA1, DJA2, or DJA4 was plotted as a percentage of input translated material. cytb5, cytochrome b5; syb2, synaptobrevin 2.
FIGURE 2
FIGURE 2. Polypeptide refolding by Hsc70, DJA2, and NEFs
Luciferase was denatured in 6 M guanidine and diluted 1:100 into refolding reactions containing RL or the indicated combinations of Hsc70 and co-chaperones. Refolding at 30 °C was monitored by luciferase activity. A, activity upon refolding in RL or with 4 μM Hsc70 alone or with 4 μM DJA1 or DJA2, was monitored over time and plotted as a percentage of the activity at 60 min of the RL positive control. B–D, refolding activity after 60 min with 4 μM Hsc70, 4 μM DJA2, and the indicated amounts of C-Bag, HspBP1, and Hsp110 was plotted relative to that with Hsc70 and DJA2 alone. The y axis scales are identical. In this and all subsequent experiments, refolding after 60 min with 4 μM Hsc70 and 4 μM DJA2 was used as control reactions, with the amount of refolded luciferase activity set to 1. E, refolding activity over time with 4 μM Hsc70, 4 μM DJA2, and either 4 μM C-Bag or 1 μM Hsp110 was plotted relative to Hsc70 and DJA2 alone at 60 min. For comparison, the data for Hsc70 and DJA2 alone from A is replotted here.
FIGURE 3
FIGURE 3. DJA1 inhibits polypeptide refolding
Luciferase refolding was monitored as in Fig. 2, with the indicated combinations of Hsc70 and co-chaperones. A, refolding activity after 60 min with 4 μM Hsc70, 4 μM DJA1, and the indicated amounts of C-Bag, HspBP1, and Hsp110 was plotted relative to the Hsc70 and DJA2 control reactions. B, refolding activity after 60 min with 4 μM Hsc70, the indicated amounts of DJA2, and either no addition or 4 μM C-Bag or 1 μM Hsp110 was plotted relative to the Hsc70 and DJA2 control reactions. C, refolding activity after 60 min with 4 μM Hsc70, the indicated amounts of DJA2 and DJA1, and either 4 μM C-Bag or 1 μM Hsp110 was plotted relative to the Hsc70 and DJA2 control reactions.
FIGURE 4
FIGURE 4. NEFs diverge in Hsc70 ATPase stimulation
ADP production in 30 °C reactions containing the indicated combinations of Hsc70 and co-chaperones was monitored by thin layer chromatography separation of radiolabeled ADP from ATP and phosphorimaging analysis. A, examples of ADP production over time in reactions with 4 μM Hsc70, 4 μM DJA2, and either 4 μM C-Bag, 4 μM HspBP1, or 1 μM Hsp110. Steady-state (linear) ATPase rates in Table 1 are based on similar experiments. B, Hsc70 ATPase rates were measured for reactions with the indicated combinations of 4 μM Hsc70, 4 μM DJA1 or DJA2, 4 μM C-Bag, 4 μM HspBP1, or 0.5 μM Hsp110. C and D, ATPase rates were measured with 4 μM Hsc70, 4 μM of either DJA1 or DJA2, and the indicated amounts of C-Bag, HspBP1, and Hsp110.
FIGURE 5
FIGURE 5. Activity of a DJA1-DJA2 chimera
A, upper panel, schematic of the domain architecture of DJA1, DJA2 and the DJA1–2 mutant. The positions of the J domain, linker, middle domain with zinc fingers, and C-terminal domain are marked. DJA1–2 contains residues 1–95 of DJA1 and residues 96 – 412 of DJA2. Lower panel, CLUSTALW2 alignment of the N-terminal regions of DJA1 and DJA2. The linker sequences are in italics, and the sequence of DJA1–2 is underlined. B, binding of radiolabeled polypeptides to DJA2 and DJA1–2 was tested as in Fig. 1, and the amount bound by DJA1–2 was plotted relative to the amount bound by DJA2. C, Hsc70 ATPase rates were measured as in Fig. 4 for reactions with 4 μM Hsc70, 4 μM DJA1–2, and the indicated amounts of C-Bag, HspBP1, and Hsp110. D and E, luciferase refolding was monitored as in Fig. 2, with the indicated combinations of Hsc70 and co-chaperones. D, refolding activity after 60 min with 4 μM Hsc70, 4 μM DJA1–2, and the indicated amounts of C-Bag, HspBP1, and Hsp110, was plotted relative to the Hsc70 and DJA2 control reactions. E, refolding activity after 60 min with 4 μM Hsc70 and the indicated amounts of DJA2 and DJA1–2 was plotted relative to the Hsc70 and DJA2 control reactions.
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