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. 2003 Jul;23(14):4948-58.
doi: 10.1128/MCB.23.14.4948-4958.2003.

Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells

Harm H Kampinga  1 , Bart Kanon, Florian A Salomons, Alexander E Kabakov, Cam Patterson
Affiliations

Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells

Harm H Kampinga et al. Mol Cell Biol. 2003 Jul.

Abstract

CHIP is a cochaperone of Hsp70 that inhibits Hsp70-dependent refolding in vitro. However, the effect of altered expression of CHIP on the fate of unfolded proteins in mammalian cells has not been determined. Surprisingly, we found that overexpression of CHIP in fibroblasts increased the refolding of proteins after thermal denaturation. This effect was insensitive to geldanamycin, an Hsp90 inhibitor, and required the tetratricopeptide repeat motifs but not the U-box domain of CHIP. Inhibition of Hsp70 chaperone activity abolished the effects of CHIP on protein folding, indicating that the CHIP-mediated events were Hsp70 dependent. Hsp40 competitively inhibited the CHIP-dependent refolding, which is consistent with in vitro data indicating that these cofactors act on Hsp70 in the ATP-bound state and have opposing effects on Hsp70 ATPase activity. Consistent with these observations, CHIP overexpression did not alter protein folding in the setting of ATP depletion, when Hsp70 is in the ADP-bound state. Concomitant with its effects on refolding heat-denatured substrates, CHIP increased the fraction of nascent chains coimmunoprecipitating with Hsc70, but only when sufficient ATP was present to allow Hsp70 to cycle rapidly. Our data suggest that, consistent with in vitro studies, CHIP attenuates the Hsp70 cycle in living cells. The impact of this effect on the fate of unfolded proteins in cells, however, is different from what might be expected from the in vitro data. Rather than resulting in inhibited refolding, CHIP increases the folding capacity of Hsp70 in eukaryotic cells.

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Figures

FIG. 1.
FIG. 1.
Luciferase degradation and CHIP overexpression. (A) O23 cells were transfected with Hsp70 (solid squares), CHIP (solid triangles) CHIP and Hsp70 (open squares), or a control plasmid (solid circles) together with pRSVLL/V, encoding firefly luciferase targeted to the cytoplasm. At 48 h after transfection, the cells were treated with cycloheximide (CHX) to inhibit new luciferase synthesis, and luciferase activity was measured and plotted relative to the activity prior to cycloheximide treatment (=100%). Data are means ± standard errors of the means from three to four independent experiments performed in quadruplicate. (B) O23 cells were transfected with Hsp70 (lanes 4 to 6), CHIP (lanes 7 to 9), CHIP and Hsp70 (lanes 10 to 12), or a control plasmid (lanes 1 to 3) together with pRSVLL/V, encoding firefly luciferase targeted to the cytoplasm. At 48 h after transfection, the cells were lysed either immediately (C), directly after a 30-min heat shock at 45°C (H), or 180 min at 37°C after the heat shock (H+R), and luciferase expression was measured by Western blotting.
FIG. 2.
FIG. 2.
Overexpressed CHIP shows partial colocalization with cytosolic luciferase before and after heat shock. O23 cells were transfected with CHIP and cytoplasmic firefly luciferase-encoding constructs. At 48 h after transfection, the cells were processed for immunohistochemistry and subsequent confocal microscopic analysis to detect the localization of ectopically expressed CHIP (A and E) and luciferase (B and F) under control (A to D) and heat shock (E to H) conditions. The cells were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (C and G). Merged images are shown in panels D and H.
FIG. 3.
FIG. 3.
Enhanced refolding of heat-denatured luciferase by CHIP is independent of Hsp90 function but dependent on interaction with and functional activity of Hsp70. O23 cells were transiently transfected with various chaperone-encoding constructs together with pRSVLL/V, encoding firefly luciferase targeted to the cytoplasm. At 48 h after transfection, the cells were treated with cycloheximide to inhibit new luciferase synthesis, heated for 30 min at 45°C, and reincubated for 0 to 60 min at 37°C to allow luciferase refolding. Samples were taken at the indicated time points, and luciferase activity was measured and plotted relative to the activity prior to treatment (=100%). Data are means ± standard errors of the means from 3 to 10 independent experiments performed in quadruplicate. (A) Expression levels of full-length (lane 1, arrow) and mutant CHIP (lane 2, CHIPΔTPR, arrow; lane 3, CHIPΔUbox, arrow) after transient transfection. Endogenous CHIP expression is marked with an asterisk; (B) Luciferase refolding in cells expressing CHIP alone (solid triangles), Hsp70 alone (solid squares), or Hsp70 and CHIP (open squares) compared to cells transfected with an empty vector (control, solid circles). (C) Luciferase refolding in cells overexpressing full-length CHIP (solid triangles) compared to CHIP lacking the U-box domain (CHIPΔUbox; open diamonds), CHIP lacking its TPR domain (CHPΔTPR; open triangles), and cells transfected with an empty vector (control; solid circles). (D) Luciferase refolding in cells overexpressing CHIP (triangles) compared to cells transfected with an empty vector (control; circles) in the absence (solid symbols) or presence of the Hsp90-disrupting agent geldanamycin (GA; 1 μg/ml added 10 min prior to the heat shock) (open symbols). (E) Luciferase refolding in cells overexpressing CHIP (triangles) or Hsp70 (squares) either alone (open symbols) or when cotransfected with the J-domain of Hsp40 (solid symbols). Cells were (co)transfected with 1:1 ratios of the plasmids encoding the respective (co)chaperones.
FIG. 4.
FIG. 4.
Thermal inactivation and insolubilization of luciferase is retarded in CHIP-overexpressing cells. O23 cells were transiently transfected with constructs expressing Hsp70 (solid squares), CHIP (solid triangles), CHIP lacking the TPR domain (CHIPΔTPR; open triangles), or an empty vector (control; solid circles) together with constructs encoding (EGFP-tagged) firefly luciferase targeted to the cytoplasm. At 48 h after transfection, the cells were heated at either 42°C (A) or 45°C (B). Samples were taken at the indicated time points during heating, and luciferase activity was measured and plotted relative to the activity prior to treatment (=100%). Data are from a typical experiment performed in quadruplicate. In panel C, unheated and heated (30 min at 42°C) samples from control cells (lanes 1 to 3 and 10 to 12, respectively), CHIP-overexpressing cells (lanes 4 to 6 and 13 to 15, respectively), and Hsp70-overexpressing cells (lanes 7 to 9 and 16 to 18, respectively) were fractionated, and total cell lysates (T), the Triton X-100-soluble (S), and insoluble (P) fractions were loaded on SDS-polyacrylamide gels. Luciferase-EGFP was detected with EGFP antibodies. Data are from a typical experiment.
FIG. 5.
FIG. 5.
CHIP acts competitively with Hsp40 but not Bag-1 in affecting luciferase denaturation and refolding. O23 cells were transiently transfected with different ratios of plasmids encoding Hsp40, CHIP, and an empty vector (A, C, and E) or at a 1:1 ratio with a Bag-1-expressing plasmid (B, D, and F) together with 1 μg of pRSVLL/V, encoding firefly luciferase targeted to the cytoplasm. At 48 h after transfection, the cells were heated for 30 min at 45°C, and samples were taken immediately after heating (A and B) or 1 h (C and D) or 3 h (E and F) after heating for measurement of luciferase activity. The activity data are plotted relative to the activity prior to treatment (=100%). Data are means from three to four independent experiments performed in quadruplicate. Error bars were omitted for reasons of clarity but never exceeded 10% of the average value.
FIG. 6.
FIG. 6.
Rate of inactivation of luciferase due to ATP depletion is not retarded by CHIP. CHO cells were transiently transfected with constructs expressing Hsp70 (solid squares), CHIP (solid triangles), CHIP lacking the TPR domain (CHIPΔTPR; open triangles), or an empty vector (control; solid circles) together with pRSVLL/V, encoding firefly luciferase targeted to the cytoplasm. At 48 h after transfection, the cells were treated with CCCP in medium containing 2-deoxyglucose, which reduces cellular ATP levels to below 2% within 1 h. Samples were taken at the indicated time points during heating, and luciferase activity was measured and plotted relative to the activity prior to treatment (=100%). Data are means ± standard error of the mean from three independent experiments performed in quadruplicate.
FIG. 7.
FIG. 7.
CHIP enhances the amount of nascent polypeptide chains that coimmunoprecipitate with Hsp70 in the presence of an ATP-regenerating system. CHO cells were transfected with Myc-tagged Hsc70 with (+) or without (−) CHIP and pulse-labeled with [35S]methionine at 48 h after transfection. Lysates were prepared in the presence of apyrase (lanes 1 to 3) or an ATP-regenerating (ATP) system (lanes 4 to 6). Immunoprecipitation was performed with Myc-Hsp70, and the precipitated material was run on polyacrylamide-SDS gels. Arrows indicate the positions of Hsc70 and CHIP. Data are from a typical experiment (see Table 1 for quantitative data).
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