Fig. 1. Effect of the penton base on co-chaperone activity of BAG3. (A). Recombinant
proteins used in the assay were obtained as described in Material and Methods. 1µl-portions
of the final preparation of GST-BAG3 (BAG), GSTBAG3C (C) and Ad2 base (Base) were
resolved on SDS/PAGE and stained with CBB. (B). Effect of J proteins on luciferase
refolding. (C). ATPase activity of Hsp70/Hdj1 system was measured in the presence of
BAG3 as described in Materials and Methods. (D). Effect of Ad2 penton base protein (Base)
and Ad3 penton base protein (Dd) on ATPase activity of Hsp70/Hdj1 system under a
stimulatory concentration of BAG3. BAG3 was preincubated with viral proteins for 1 hour at
room temperature. The molar concentration of BSA was equal to that of the other proteins.
(E). Effect of BAG3 on the Hsp70/Hdj1 system measured with the luciferase refolding assay
as described in Materials and Methods. (F). Effect of Ad2 penton base protein (Base) and
Ad3 penton base protein (Dd) on luciferase refolding activity of the Hsp70/Hdj1 system under
a stimulatory concentration of BAG3. The effect of base proteins was analysed after
preincubation with BAG3. The starting level of assay is shown in black in C, D, E, F.
Material and Methods
Proteins. BAG1S was overexpressed using pBAD24 vector in E.coli RIL BL21 strain. Protein
was purified as described by Höhfeld and Jentsch (1997). Hsp70 (HSPA1A) and Hdj1 were
overexpressed from pET11b and pET21d vectors respectively, in E.coli RIL BL21 strain and
purified according to King et al (2001). Bacterially expressed Tpr2 was a kind gift of Dr W.
Obermann.
ATPase assay. Hsp70 (4M) was incubated for 2h at 37C in 25l of 40mM Hepes buffer,
pH 7.5, containing 150mM KCl, 5mM MgCl2, 400M ATP and 1.25 Ci [32P]-ATP (spec.
activity 6000 Ci/mmol, 10.0 mCi/ml), together with proteins. Samples of 1l of the incubation
mixture were spotted on PEI-cellulose plates. Plates were resolved in 1M LiCl:1M HCOOH
1:1, dried and the radioactivity of spots corresponding to free phosphate was measured with
Phosphoimager (Amersham Pharmacia). When needed 0.25M Hdj1 protein was added.
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GST-BAG3, GST-BAG3∆C, and GST concentrations varied between 0.4M and 4.0M.
Dodecahedra concentrations varied between 0.32M and 1.6M. Viral proteins were
preincubated with BAG3 for 1h at room temperature. ATPase activity was defined as the
percent of total ATP added to each sample that underwent hydrolysis. All results were
corrected for spontaneous ATP hydrolysis.
Luciferase refolding assay. Luciferase at final concentration 0.5M (14,6 mg/ml, Promega)
and 10M Hsp90 were incubated in 25mM Tris buffer, pH 7.8, containing 8mM MgSO4, 1%
BSA, 10% glycerol, 0.25 % Triton X-100, for 5min at 50C. After cooling the incubation
mixture was diluted 6-fold in the renaturation buffer: 10 mM Tris, pH 7.5, containing 3mM
MgCl2, 50 mM KCl, 2mM DTT, 8mM creatine phosphate, 0.02 units/l creatine kinase, 2mM
ATP, 4mM Hsp70. Concentration of Hdj1, Tpr2 and base proteins varied between 0.08 and
3.2µM (Fig. 3A). In other experiments the concentration of Hdj1 was set at 0.25µM
(stimulatory for Hsp70) and concentrations of GST-BAG3, GST-BAG3∆C, GST, BAG1S and
BSA varied between 0.4 to 4µM (Fig. 3E). In the experiment presented in Fig. 3F the
concentration of BAG3 was 0.4µM (stimulatory for Hsp70), and that of penton base and
dodecahedron varied between 0.32 to 3.2 µM. Viral proteins were preincubated with BAG3
for 1h at room temperature. Renaturation was carried out at room temperature. After 2h, 5l
samples were withdrawn, the substrate (Promega) was added and the activity of renatured
luciferase was measured in luminometer (BMG Labtechnologies). Chaperoning activity was
expressed as percent of refolded luciferase in comparison to heat-nondenatured luciferase.
The assay is described in more detail by Walerych et al (2004).
Acknowledgements
We are indebted to Aleksandra Helwak for bacterially expressed Hdj1 and BAG1S
proteins and to Wolfgang Obermann for bacterially expressed Tpr2 protein.
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