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Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

Majerská, Jana, Schrumpfová, PetraProcházková, Dokládal, Ladislav, Schořová, Šárka, Stejskal, Karel, Obořil, Michal, Honys, David, Kozáková, Lucie, Polanská, PavlaSováková, Sýkorová, Eva
Protoplasma 2017 v.254 no.4 pp. 1547-1562
Arabidopsis thaliana, biogenesis, bioinformatics, cell cycle, dimerization, mass spectrometry, protein subunits, protein-protein interactions, proteins, telomerase, telomeres
The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.