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. 1999 Apr 1;13(7):817-26.
doi: 10.1101/gad.13.7.817.

Functional requirement of p23 and Hsp90 in telomerase complexes

S E Holt  1 , D L Aisner, J Baur, V M Tesmer, M Dy, M Ouellette, J B Trager, G B Morin, D O Toft, J W Shay, W E Wright, M A White
Affiliations

Functional requirement of p23 and Hsp90 in telomerase complexes

S E Holt et al. Genes Dev. .

Abstract

Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity.

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Figures

Figure 1
Figure 1
In vitro assembly of active telomerase from hTERT and hTR requires additional components present in reticulocyte lysates. In vitro-transcribed and -translated hTERT was mixed with purified hTR in the presence of increasing concentrations of rabbit reticulocyte lysate (0, 12.5%, 25%, 37.5%, and 50% of reaction volume). Following incubation for 90 min at 30°C, samples were assayed for telomerase activity by TRAP analysis. The ratio of signal from extended products vs. the 36-bp internal TRAP assay standard (ITAS) was determined by densitometetry analysis. Values shown represent these ratios normalized to that observed in the absence of additional RRLs, which was set at 1. This experiment has been repeated numerous times with identical results.
Figure 2
Figure 2
p23 and Hsp90 bind hTERT. (A) Transformants of the yeast two-hybrid reporter strain L40 selected to express LexA–hTERT (1–195) and VP16–p23, alone or together, were tested for the ability to grow on medium lacking histidine. Growth on the selective plate indicates a positive two-hybrid interaction. (B) The indicated antibodies were used to immunoprecipitate proteins from RRLs containing HA-tagged hTERT synthesized with [35S]methionine in the absence of the template RNA. Following extensive washing, the immunoprecipitates were separated by SDS-PAGE, and the presence of coprecipitated hTERT was determined by PhosphorImager analysis. Ten percent of the total translation reaction was loaded to reveal the amount of labeled hTERT present in the reticulocyte lysates. Repeated experiments gave similar results. (C) The indicated antibodies were used to immunoprecipitate proteins from lysates of untransfected HT1080 cells. The immunoprecipitates were washed extensively and assayed for telomerase activity by TRAP. The input lane corresponds to activity present in lysate from 1000 cells. TRAP assays were performed on the pellets (P) and supernatents (S) of immunoprecipitates from lysates of 2000 cells each. Antibodies to p23 and Hsp90 were precoated with saturating levels of purified p23 (+p23) or Hsp90 (+hsp90) to demonstrate specificity of the immunoprecipitation.
Figure 2
Figure 2
p23 and Hsp90 bind hTERT. (A) Transformants of the yeast two-hybrid reporter strain L40 selected to express LexA–hTERT (1–195) and VP16–p23, alone or together, were tested for the ability to grow on medium lacking histidine. Growth on the selective plate indicates a positive two-hybrid interaction. (B) The indicated antibodies were used to immunoprecipitate proteins from RRLs containing HA-tagged hTERT synthesized with [35S]methionine in the absence of the template RNA. Following extensive washing, the immunoprecipitates were separated by SDS-PAGE, and the presence of coprecipitated hTERT was determined by PhosphorImager analysis. Ten percent of the total translation reaction was loaded to reveal the amount of labeled hTERT present in the reticulocyte lysates. Repeated experiments gave similar results. (C) The indicated antibodies were used to immunoprecipitate proteins from lysates of untransfected HT1080 cells. The immunoprecipitates were washed extensively and assayed for telomerase activity by TRAP. The input lane corresponds to activity present in lysate from 1000 cells. TRAP assays were performed on the pellets (P) and supernatents (S) of immunoprecipitates from lysates of 2000 cells each. Antibodies to p23 and Hsp90 were precoated with saturating levels of purified p23 (+p23) or Hsp90 (+hsp90) to demonstrate specificity of the immunoprecipitation.
Figure 2
Figure 2
p23 and Hsp90 bind hTERT. (A) Transformants of the yeast two-hybrid reporter strain L40 selected to express LexA–hTERT (1–195) and VP16–p23, alone or together, were tested for the ability to grow on medium lacking histidine. Growth on the selective plate indicates a positive two-hybrid interaction. (B) The indicated antibodies were used to immunoprecipitate proteins from RRLs containing HA-tagged hTERT synthesized with [35S]methionine in the absence of the template RNA. Following extensive washing, the immunoprecipitates were separated by SDS-PAGE, and the presence of coprecipitated hTERT was determined by PhosphorImager analysis. Ten percent of the total translation reaction was loaded to reveal the amount of labeled hTERT present in the reticulocyte lysates. Repeated experiments gave similar results. (C) The indicated antibodies were used to immunoprecipitate proteins from lysates of untransfected HT1080 cells. The immunoprecipitates were washed extensively and assayed for telomerase activity by TRAP. The input lane corresponds to activity present in lysate from 1000 cells. TRAP assays were performed on the pellets (P) and supernatents (S) of immunoprecipitates from lysates of 2000 cells each. Antibodies to p23 and Hsp90 were precoated with saturating levels of purified p23 (+p23) or Hsp90 (+hsp90) to demonstrate specificity of the immunoprecipitation.
Figure 3
Figure 3
p23 and Hsp90 are required to assemble active telomerase in vitro. (A) In vitro-transcribed and -translated hTERT was diluted 1/20 into buffer (−RRL), fresh RRLs (+RRL), p23-depleted RRLs (p23 dep RRL), or p23-depleted RRLs supplemented with recombinant p23 at the indicated concentrations, and mixed with 0.5 μg of in vitro-transcribed hTR. The mixtures were incubated at 30°C for 90 min to allow reconstitution of hTERT and its hTR template. Aliquots were then removed and assayed for activity by TRAP. Depletion of p23 from RRLs was verified by Western analysis using the anti-p23 antibody (right). The results shown are representative of two independent experiments. Quantitation was performed by densitometry analysis as described in Fig. 1. Among many possibilities, failure to fully reconstitute activity with purified p23 may be due to codepletion of p23-associated factors. (B) Telomerase was reconstituted as described in A except that translated hTERT was diluted in RRLs that had been incubated in the presence of geldanamycin (+GA-pre) (100 μg/ml) or DMSO carrier for 30 min. +GA-Post indicates addition of geldanamycin (100 μg/ml) to the TRAP assay after the reconstitution of hTERT and hTR in normal RRLs. Repeated experiments gave identical results. (C) 35S-labeled HA-tagged hTERT synthesized in the presence of DMSO carrier (hTERT) or 50 μg/ml geldanamycin (hTERT/GA) was assayed for association with p23 and Hsp90 as described in Fig 1. An antibody to TCP-1, an abundant chaperone, was included as an additional control for specificity.
Figure 3
Figure 3
p23 and Hsp90 are required to assemble active telomerase in vitro. (A) In vitro-transcribed and -translated hTERT was diluted 1/20 into buffer (−RRL), fresh RRLs (+RRL), p23-depleted RRLs (p23 dep RRL), or p23-depleted RRLs supplemented with recombinant p23 at the indicated concentrations, and mixed with 0.5 μg of in vitro-transcribed hTR. The mixtures were incubated at 30°C for 90 min to allow reconstitution of hTERT and its hTR template. Aliquots were then removed and assayed for activity by TRAP. Depletion of p23 from RRLs was verified by Western analysis using the anti-p23 antibody (right). The results shown are representative of two independent experiments. Quantitation was performed by densitometry analysis as described in Fig. 1. Among many possibilities, failure to fully reconstitute activity with purified p23 may be due to codepletion of p23-associated factors. (B) Telomerase was reconstituted as described in A except that translated hTERT was diluted in RRLs that had been incubated in the presence of geldanamycin (+GA-pre) (100 μg/ml) or DMSO carrier for 30 min. +GA-Post indicates addition of geldanamycin (100 μg/ml) to the TRAP assay after the reconstitution of hTERT and hTR in normal RRLs. Repeated experiments gave identical results. (C) 35S-labeled HA-tagged hTERT synthesized in the presence of DMSO carrier (hTERT) or 50 μg/ml geldanamycin (hTERT/GA) was assayed for association with p23 and Hsp90 as described in Fig 1. An antibody to TCP-1, an abundant chaperone, was included as an additional control for specificity.
Figure 3
Figure 3
p23 and Hsp90 are required to assemble active telomerase in vitro. (A) In vitro-transcribed and -translated hTERT was diluted 1/20 into buffer (−RRL), fresh RRLs (+RRL), p23-depleted RRLs (p23 dep RRL), or p23-depleted RRLs supplemented with recombinant p23 at the indicated concentrations, and mixed with 0.5 μg of in vitro-transcribed hTR. The mixtures were incubated at 30°C for 90 min to allow reconstitution of hTERT and its hTR template. Aliquots were then removed and assayed for activity by TRAP. Depletion of p23 from RRLs was verified by Western analysis using the anti-p23 antibody (right). The results shown are representative of two independent experiments. Quantitation was performed by densitometry analysis as described in Fig. 1. Among many possibilities, failure to fully reconstitute activity with purified p23 may be due to codepletion of p23-associated factors. (B) Telomerase was reconstituted as described in A except that translated hTERT was diluted in RRLs that had been incubated in the presence of geldanamycin (+GA-pre) (100 μg/ml) or DMSO carrier for 30 min. +GA-Post indicates addition of geldanamycin (100 μg/ml) to the TRAP assay after the reconstitution of hTERT and hTR in normal RRLs. Repeated experiments gave identical results. (C) 35S-labeled HA-tagged hTERT synthesized in the presence of DMSO carrier (hTERT) or 50 μg/ml geldanamycin (hTERT/GA) was assayed for association with p23 and Hsp90 as described in Fig 1. An antibody to TCP-1, an abundant chaperone, was included as an additional control for specificity.
Figure 4
Figure 4
Exposure of cells to an inhibitor of Hsp90 blocks induction of telomerase activity. HT1080 cells were serum starved for 14 days, followed by addition of 10% serum, together with the indicated concentrations of geldanamycin, DMSO carrier, FK506, or cyclosporin A (CsA). (A) Treated cells were lysed and assayed for telomerase activity by TRAP. Data shown are representative of three independent experiments. (B) Parallel plates were assayed for [3H]thymidine incorporation. Effects on plating efficiency were assayed by cell counts before and after stimulation followed by replating. All samples had similar plating efficiencies of 70%–80% except for treatment with 40 μg/ml of CsA, which reduced plating efficiencies to 60% (data not shown). (C) Telomerase negative primary human fibroblasts (BJ cells) were infected with retrovirus expressing HA-tagged hTERT. Four hours postinfection, cells were incubated with 100 ng/ml geldanamycin; 24 hr postinfection, cells were harvested and assayed for telomerase activity by TRAP analysis and expression of the HA-tagged hTERT by Western analysis using the anti-HA (12CA5) antibody. Lysates from a selected population of BJ cells expressing HA-tagged hTERT were used as a positive control for the Western analysis. (D) Cells were treated as described in C with a range of concentrations of geldanamycin. TRAP activity following treatment was quantitated as in Fig 1. Values plotted are the average of two experiments.
Figure 4
Figure 4
Exposure of cells to an inhibitor of Hsp90 blocks induction of telomerase activity. HT1080 cells were serum starved for 14 days, followed by addition of 10% serum, together with the indicated concentrations of geldanamycin, DMSO carrier, FK506, or cyclosporin A (CsA). (A) Treated cells were lysed and assayed for telomerase activity by TRAP. Data shown are representative of three independent experiments. (B) Parallel plates were assayed for [3H]thymidine incorporation. Effects on plating efficiency were assayed by cell counts before and after stimulation followed by replating. All samples had similar plating efficiencies of 70%–80% except for treatment with 40 μg/ml of CsA, which reduced plating efficiencies to 60% (data not shown). (C) Telomerase negative primary human fibroblasts (BJ cells) were infected with retrovirus expressing HA-tagged hTERT. Four hours postinfection, cells were incubated with 100 ng/ml geldanamycin; 24 hr postinfection, cells were harvested and assayed for telomerase activity by TRAP analysis and expression of the HA-tagged hTERT by Western analysis using the anti-HA (12CA5) antibody. Lysates from a selected population of BJ cells expressing HA-tagged hTERT were used as a positive control for the Western analysis. (D) Cells were treated as described in C with a range of concentrations of geldanamycin. TRAP activity following treatment was quantitated as in Fig 1. Values plotted are the average of two experiments.
Figure 4
Figure 4
Exposure of cells to an inhibitor of Hsp90 blocks induction of telomerase activity. HT1080 cells were serum starved for 14 days, followed by addition of 10% serum, together with the indicated concentrations of geldanamycin, DMSO carrier, FK506, or cyclosporin A (CsA). (A) Treated cells were lysed and assayed for telomerase activity by TRAP. Data shown are representative of three independent experiments. (B) Parallel plates were assayed for [3H]thymidine incorporation. Effects on plating efficiency were assayed by cell counts before and after stimulation followed by replating. All samples had similar plating efficiencies of 70%–80% except for treatment with 40 μg/ml of CsA, which reduced plating efficiencies to 60% (data not shown). (C) Telomerase negative primary human fibroblasts (BJ cells) were infected with retrovirus expressing HA-tagged hTERT. Four hours postinfection, cells were incubated with 100 ng/ml geldanamycin; 24 hr postinfection, cells were harvested and assayed for telomerase activity by TRAP analysis and expression of the HA-tagged hTERT by Western analysis using the anti-HA (12CA5) antibody. Lysates from a selected population of BJ cells expressing HA-tagged hTERT were used as a positive control for the Western analysis. (D) Cells were treated as described in C with a range of concentrations of geldanamycin. TRAP activity following treatment was quantitated as in Fig 1. Values plotted are the average of two experiments.
Figure 4
Figure 4
Exposure of cells to an inhibitor of Hsp90 blocks induction of telomerase activity. HT1080 cells were serum starved for 14 days, followed by addition of 10% serum, together with the indicated concentrations of geldanamycin, DMSO carrier, FK506, or cyclosporin A (CsA). (A) Treated cells were lysed and assayed for telomerase activity by TRAP. Data shown are representative of three independent experiments. (B) Parallel plates were assayed for [3H]thymidine incorporation. Effects on plating efficiency were assayed by cell counts before and after stimulation followed by replating. All samples had similar plating efficiencies of 70%–80% except for treatment with 40 μg/ml of CsA, which reduced plating efficiencies to 60% (data not shown). (C) Telomerase negative primary human fibroblasts (BJ cells) were infected with retrovirus expressing HA-tagged hTERT. Four hours postinfection, cells were incubated with 100 ng/ml geldanamycin; 24 hr postinfection, cells were harvested and assayed for telomerase activity by TRAP analysis and expression of the HA-tagged hTERT by Western analysis using the anti-HA (12CA5) antibody. Lysates from a selected population of BJ cells expressing HA-tagged hTERT were used as a positive control for the Western analysis. (D) Cells were treated as described in C with a range of concentrations of geldanamycin. TRAP activity following treatment was quantitated as in Fig 1. Values plotted are the average of two experiments.
Figure 5
Figure 5
Purified foldosome components substitute for RRLs to assemble active telomerase in vitro. In vitro-transcribed and -translated hTERT was diluted 1/20 into an assembly reaction containing purified hTR and buffer in the absence (−) or presence (+ATP alone) of 5 mm ATP, or in the presence of 5 mmATP and purified foldosome components: p23, Hsp90, Hop, Hsp70, YDJ1 (+ATP+cofactors). Identical assembly reactions contained increasing concentrations of RRLs. All reactions were incubated for 90 min at 30°C followed by TRAP analysis. The experiment shown is representative of three independent experiments.
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