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.

Stimulation of Yeast Telomerase Activity by the Ever Shorter Telomere 3 (Est3) Subunit Is Dependent on Direct Interaction with the Catalytic Protein Est2

“…In agreement with the effects on TLC1 association, Est3p ETN -HA 3 and Est3p DQ -HA 3 were reduced in their ability to co-immunoprecipitate with Myc 18 -Est2p (Fig. 2B) [13][14][15][16][17][18] in the identical immunoprecipitation. Results are representative of two independent biological replicates (see supplemental Fig.…”

“…Telomerase-We showed previously that Est1p stimulates the assembly of Est3p with telomerase (15). However, our failure to detect an Est1p/Est3p interaction by coimmunoprecipitation from cell extract in the absence of Est2p suggested that Est1p might not be the primary binding site for Est3p (15).…”

“…Extracts were normalized to 20 mg/ml and incubated with antibodies as described previously (15,22). 10 l of anti-Myc immunoprecipitated material (1/6 of total) or anti-HA immunoprecipitated material was separated by 10 -12% step-gradient SDS-PAGE.…”

“…For Northern blot analysis, RNA was isolated from immunoprecipitation beads and detected by Northern blotting as described previously (15,22). Whole cell RNA was prepared from 10 ml of mid-log phase cultures (26).…”

Telomerase and retrotransposons: Reverse transcriptases that shaped genomes

“…Intriguingly, mutations in CDC13 and EST1 that abolished telomere maintenance in vivo did not have an obvious effect on this physical interaction, suggesting that a step other than recruitment may be affected. Besides interacting with Cdc13, Est1 has also been implicated in promoting the assembly of Est3 into the telomerase complex (44,45). Using a similar approach, Zakian and colleagues also demonstrated a direct and specific physical interaction between Est1 and Est3, thus providing a plausible mechanism for how this assembly function is achieved (46).…”