Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae

Jennifer L. Osterhage; Jennell M. Talley; Katherine L. Friedman

doi:10.1038/nsmb1125

Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae

Jennifer L. Osterhage, Jennell M. Talley, Katherine L. Friedman

Biology

Research output: Contribution to journal › Article › peer-review

74 Scopus citations

Abstract

Telomerase counteracts loss of terminal sequences incurred during DNA replication. In S. cerevisiae, telomerase contains an RNA template (TLC1), a reverse transcriptase (Est2p) and at least two regulatory proteins (Est1p and Est3p). Whereas Est2p is constitutively telomere bound, Est1p associates in late S phase, coincident with telomere lengthening. Here we directly demonstrate by coimmunoprecipitation that the composition of telomerase varies during the cell cycle. The absence of Est1p and Est3p from the complex during G1 phase can be attributed to proteasome-dependent degradation of Est1p. Stabilization of Est1p during G1 phase promotes telomerase assembly, revealing a previously uncharacterized role for Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, complexes assembled during G1 cannot lengthen telomeres. We conclude that telomerase assembly during G1 phase is regulated by Est1p stability, but assembly is insufficient to activate telomerase at telomeres.

Original language	English
Pages (from-to)	720-728
Number of pages	9
Journal	Nature Structural and Molecular Biology
Volume	13
Issue number	8
DOIs	https://doi.org/10.1038/nsmb1125
State	Published - Aug 2006

Bibliographical note

Funding Information:
We thank J. Banchant, C. Bartholomew and T. Higginbotham for advice and technical assistance with flow-cytometry experiments; M. Platts, H. Ji and C. Brown for critical reading of the manuscript; K. Pulliam, G. Todd and J. Ferguson for strain construction; C. Hardy and T. Graham (Vanderbilt University) for the antibodies to actin and Arf1, respectively; and J. Brown and M. Platts for technical assistance. E. Schneble contributed to the Est1p overexpression experiments. This work was supported by Research Scholar Grant RSG-04-048-01-GMC from the American Cancer Society to K.L.F. and grant 52003905 of the Howard Hughes Medical Institute Professors Program. J.L.O. and J.M.T. were supported by graduate training grant NIH5T32 GM08554 from the US National Institutes of Health. The VUMC Institution Flow Cytometry core was supported by the Vanderbilt Ingram Cancer Center (P30 CAC8485).

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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title = "Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae",

abstract = "Telomerase counteracts loss of terminal sequences incurred during DNA replication. In S. cerevisiae, telomerase contains an RNA template (TLC1), a reverse transcriptase (Est2p) and at least two regulatory proteins (Est1p and Est3p). Whereas Est2p is constitutively telomere bound, Est1p associates in late S phase, coincident with telomere lengthening. Here we directly demonstrate by coimmunoprecipitation that the composition of telomerase varies during the cell cycle. The absence of Est1p and Est3p from the complex during G1 phase can be attributed to proteasome-dependent degradation of Est1p. Stabilization of Est1p during G1 phase promotes telomerase assembly, revealing a previously uncharacterized role for Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, complexes assembled during G1 cannot lengthen telomeres. We conclude that telomerase assembly during G1 phase is regulated by Est1p stability, but assembly is insufficient to activate telomerase at telomeres.",

author = "Osterhage, {Jennifer L.} and Talley, {Jennell M.} and Friedman, {Katherine L.}",

note = "Funding Information: We thank J. Banchant, C. Bartholomew and T. Higginbotham for advice and technical assistance with flow-cytometry experiments; M. Platts, H. Ji and C. Brown for critical reading of the manuscript; K. Pulliam, G. Todd and J. Ferguson for strain construction; C. Hardy and T. Graham (Vanderbilt University) for the antibodies to actin and Arf1, respectively; and J. Brown and M. Platts for technical assistance. E. Schneble contributed to the Est1p overexpression experiments. This work was supported by Research Scholar Grant RSG-04-048-01-GMC from the American Cancer Society to K.L.F. and grant 52003905 of the Howard Hughes Medical Institute Professors Program. J.L.O. and J.M.T. were supported by graduate training grant NIH5T32 GM08554 from the US National Institutes of Health. The VUMC Institution Flow Cytometry core was supported by the Vanderbilt Ingram Cancer Center (P30 CAC8485).",

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N1 - Funding Information: We thank J. Banchant, C. Bartholomew and T. Higginbotham for advice and technical assistance with flow-cytometry experiments; M. Platts, H. Ji and C. Brown for critical reading of the manuscript; K. Pulliam, G. Todd and J. Ferguson for strain construction; C. Hardy and T. Graham (Vanderbilt University) for the antibodies to actin and Arf1, respectively; and J. Brown and M. Platts for technical assistance. E. Schneble contributed to the Est1p overexpression experiments. This work was supported by Research Scholar Grant RSG-04-048-01-GMC from the American Cancer Society to K.L.F. and grant 52003905 of the Howard Hughes Medical Institute Professors Program. J.L.O. and J.M.T. were supported by graduate training grant NIH5T32 GM08554 from the US National Institutes of Health. The VUMC Institution Flow Cytometry core was supported by the Vanderbilt Ingram Cancer Center (P30 CAC8485).

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N2 - Telomerase counteracts loss of terminal sequences incurred during DNA replication. In S. cerevisiae, telomerase contains an RNA template (TLC1), a reverse transcriptase (Est2p) and at least two regulatory proteins (Est1p and Est3p). Whereas Est2p is constitutively telomere bound, Est1p associates in late S phase, coincident with telomere lengthening. Here we directly demonstrate by coimmunoprecipitation that the composition of telomerase varies during the cell cycle. The absence of Est1p and Est3p from the complex during G1 phase can be attributed to proteasome-dependent degradation of Est1p. Stabilization of Est1p during G1 phase promotes telomerase assembly, revealing a previously uncharacterized role for Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, complexes assembled during G1 cannot lengthen telomeres. We conclude that telomerase assembly during G1 phase is regulated by Est1p stability, but assembly is insufficient to activate telomerase at telomeres.

AB - Telomerase counteracts loss of terminal sequences incurred during DNA replication. In S. cerevisiae, telomerase contains an RNA template (TLC1), a reverse transcriptase (Est2p) and at least two regulatory proteins (Est1p and Est3p). Whereas Est2p is constitutively telomere bound, Est1p associates in late S phase, coincident with telomere lengthening. Here we directly demonstrate by coimmunoprecipitation that the composition of telomerase varies during the cell cycle. The absence of Est1p and Est3p from the complex during G1 phase can be attributed to proteasome-dependent degradation of Est1p. Stabilization of Est1p during G1 phase promotes telomerase assembly, revealing a previously uncharacterized role for Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, complexes assembled during G1 cannot lengthen telomeres. We conclude that telomerase assembly during G1 phase is regulated by Est1p stability, but assembly is insufficient to activate telomerase at telomeres.

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Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae

Abstract

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