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

Abstract

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.

The plant Pontin and Reptin homologues, RuvBL1 and RuvBL2a, colocalize with TERT and TRB proteins in vivo, and participate in telomerase biogenesis

“…We observed a clear nuclear interaction between AtRuvBL1 protein and AtTERT N‐terminal fragments covering AtTERT domains localized in positions 1‐233 and 1‐271 in the A. thaliana leaf protoplasts using BiFC (Figure b). These results supported the observation from tobacco BY2 culture protoplasts where N‐terminal fragments of AtTERT interact with AtRuvBL1 (Majerská et al ., ). As the central reverse transcriptase (RT) domain of hTERT is implicated in hRuvBL1 binding (Venteicher et al ., ), we expanded our interest to the other AtTERT fragments.…”

“…Additionally, we expanded our BiFC study (Majerská et al ., ) and tested heteromerization of AtRuvBL1 and AtRuvBL2a not only in Nicotiana tabacum BY‐2 protoplasts, but also in A. thaliana leaf protoplasts (Figure d). Analysis of subcellular localization of the AtRuvBL1‐AtRuvBL2a interactions further showed that one reciprocal interaction of nYFP/AtRuvBL1 and cYFP/AtRuvBL2a was negative, and cYFP/AtRuvBL1 and nYFP/AtRuvBL2a showed nuclear, but not nucleolar localization, maybe due to the presence of a tag that may induce conformational changes of the AtRuvBL proteins (Cheung et al ., ).…”

“…In mammals, hRuvBL1 and hRuvBL2 proteins, Pontin and Reptin, respectively, are present in early steps of telomerase RNP biogenesis. We characterized plant homologues of RuvBL proteins: AtRuvBL1 and AtRuvBL2a, previously co‐purified together with telomerase protein subunit AtTERT from A. thaliana suspension cultures (Majerská et al ., ). Here we show that AtRuvBL1 protein colocalizes with N‐terminal part of AtTERT subunit of plant telomerase also in vivo .…”

“…The Gateway‐compatible donor vectors carrying AtRuvBL1, AtRuvBL2a, AtPOT1a, AtPOT1b and AtGAUT10 were described in Majerská et al . (). The AtTRB1, 2 and 3 constructs have described previously (Schrumpfová et al ., ).…”

“…Moreover, they observed that T‐DNA insertion mutation in AtRuvBL1 gene was lethal. In our laboratory, AtRuvBL1 protein and also one of two AtRuvBL2 homologues, named AtRuvBL2a (AT5G67630), were purified together with AtTERT using Tandem Affinity Purification (TAP) from A. thaliana suspension cultures (Majerská et al ., ).…”