Human telomerase exists in two distinct active complexes in vivo.
Telomerase, a stable complex of telomerase reverse transcriptase (TERT) and template RNA (TERC), is responsible for telomere maintenance. During purification trials of recombinant human telomerase of the two components reconstituted in insect cells, we identified two complexes of human telomerase of molecular masses 680 and 380 kDa, both of which retain telomerase activity in vitro. We show here that the former complex does not include Hsp90 (heat shock protein 90) and its telomerase activity is resistant to Hsp90 inhibitors, whereas the latter contains Hsp90 and its telomerase activity is sensitive to Hsp90 inhibitors. N-terminal of FLAG-hTERT in the former is exposed, as this complex was efficiently purified with anti-FLAG M2 affinity resin. We also identified two different telomerase complexes in HeLa cells, in addition to ectopically expressed hTERT. Most of endogenous hTERT and FLAG-hTERT was detected around 680 kDa. These two complexes in HeLa cells have the same properties as their respective reconstituted telomerases. The unstable property of the telomerase complex with Hsp90, especially in the presence of Hsp90 inhibitors, was due to proteasome-mediated degradation of hTERT, since proteasome inhibitors prevented hTERT degradation in vivo. To our knowledge, this is the first demonstration of two distinct active complexes of human telomerase ectopically expressed in insect and mammalian cells.
Mizuno
Hideki
H
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Japan.
Khurts
Shilagardi
S
Seki
Takahiko
T
Hirota
Yasuhide
Y
Kaneko
Shuichi
S
Murakami
Seishi
S
D000818:Animals; D002850:Chromatography, Gel; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera; D019098:Telomerase / Q000737:chemistry* / Q000302:isolation & purification
D018841:HSP90 Heat-Shock Proteins; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; D019098:Telomerase