Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster

Abstract

The Drosophila melanogaster family of small heat shock proteins (sHsps) is composed of 4 main members (Hsp22, Hsp23, Hsp26, and Hsp27) that display distinct intracellular localization and specific developmental patterns of expression in the absence of stress. In an attempt to determine their function, we have examined whether these 4 proteins have chaperone-like activity using various chaperone assays. Heat-induced aggregation of citrate synthase was decreased from 100 to 17 arbitrary units in the presence of Hsp22 and Hsp27 at a 1:1 molar ratio of sHsp to citrate synthase. A 5 M excess of Hsp23 and Hsp26 was required to obtain the same efficiency with either citrate synthase or luciferase as substrate. In an in vitro refolding assay with reticulocyte lysate, more than 50% of luciferase activity was recovered when heat denaturation was performed in the presence of Hsp22, 40% with Hsp27, and 30% with Hsp23 or Hsp26. These differences in luciferase reactivation efficiency seemed related to the ability of sHsps to bind their substrate at 42 degrees C, as revealed by sedimentation analysis of sHsp and luciferase on sucrose gradients. Therefore, the 4 main sHsps of Drosophila share the ability to prevent heat-induced protein aggregation and are able to maintain proteins in a refoldable state, although with different efficiencies. The functional reasons for their distinctive cell-specific pattern of expression could reflect the existence of defined substrates for each sHsp within the different intracellular compartments.

Fig 1.

Blocking the N-terminus of Hsp22 with a histidine tag reduces its efficiency to prevent citrate synthase (CS) heat-induced aggregation. CS (0.1 μM) was incubated at 42°C for 90 minutes either alone (open lozenge) or in the presence of 0.1 μM of His-Hsp22 purified under native conditions (A and B, open triangle) or purified with urea (A, open circle) or of untagged-Hsp22 purified under native conditions (B, closed triangle). Protein aggregation was determined by the light-scattering assay at 320 nm. Data are representative of 6 trials and are expressed as the mean ± standard deviation

Fig 2.

D melanogaster sHsps inhibits heat-induced aggregation of citrate synthase (CS). CS (0.1 μM) was incubated at 42°C for 90 min either alone (opened lozenge) or in presence of BSA (0.5 μM, opened square), Hsp22 (A), Hsp23 (B), Hsp26 (C) or Hsp27 (D) at 2 different concentrations (0.05 μM: closed square, 0.1 μM: closed circle or 0.5 μM: closed triangle). Protein aggregation was determined by the light-scattering assay at 320 nm. Data are representative of 6 trials and are expressed as the mean ± standard deviation

Fig 3.

D melanogaster sHsps inhibit heat-induced aggregation of luciferase. Luciferase (0.1 μM) was incubated at 42°C for 30 minutes either alone (opened lozenge) or in the presence of BSA (0.5 μM, open square), Hsp22 (A), Hsp23 (B), Hsp26 (C), or Hsp27 (D) at 2 different concentrations (0.1 μM: closed circle, 0.5 μM: closed triangle). Protein aggregation was determined by the light-scattering assay at 320 nm. Data are representative of 6 trials and are expressed as the mean ± standard deviation

Fig 4.

The sHsps of D melanogaster maintain heat-denatured luciferase in a refoldable state in vitro. (A) Luciferase (0.2 μM) was incubated at 42°C for 15 minutes either alone (open lozenge) or with 1 μM BSA (open square), Hsp22 (closed triangle), Hsp23 (closed lozenge), Hsp26 (closed square), or Hsp27 (closed circle). The refolding step was performed at 30°C for 90 minutes in reticulocyte lysate supplemented with ATP, and luciferase activity was determined at different time points. Data are representative of 3–5 trials and are presented as a percentage of luciferase activity after 15 minutes of incubation at 22°C. Data are expressed as mean ± standard deviation. (B) Luciferase was incubated with Hsp22 or Hsp23 for 15 minutes at 4°C, 15 minutes at 42°C, or 15 minutes at 42°C and 90 minutes at 30°C in the presence of reticulocyte lysate and ATP. Samples were applied on 10–40% sucrose gradients and were analyzed by SDS-PAGE. Small Hsps and luciferase were localized by Western blotting. 1–25: sucrose gradients fractions (1 being at the bottom of the gradient and 25 at the top); bold boxes: fractions in which protein precipitation was likely disturbed by the hemoglobin present in the reticulocyte lysate

Fig 5.

Summary of chaperone activity of D melanogaster sHsps. (A) Prevention of citrate synthase (CS) heat-induced aggregation at a 1:1 (sHsp:CS) molar ratio after 90 minutes at 42°C. See the legend of Figure 2 for details. (B) Reactivation of luciferase at a 5:1 (sHsp: luciferase) molar ratio after a 15-minute heat shock (42°C) and 90 minutes of recovery at 30°C in the presence of reticulocyte lysate. See the legend of Figure 4A for details