Page 211 - PC2019 Program & Proceedings

PLANT CANADA 2019

S207. Arabidopsis CTP:phosphocholine cytidylyltransferase is phosphorylated and inactivated by
SnRK1
Caldo, K.; Y. Xu; L. Falarz; K. Jayawardana; J. Acedo; G. Chen
University of Alberta

De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic
biochemical reactions that must be fine-tuned with energy homeostasis. CTP:phosphocholine
cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway. In this study, an important
mechanism regulating plant CCT1 activity was identified. Comparative analysis showed that Arabidopsis
thaliana CCT1 (AtCCT1) exhibits homologous catalytic and membrane-binding domains to rat CCT1.
Homology modelling indicated that AtCCT1 has similar Rossmann fold and localization of active site
residues as rat CCT1. On the other hand, the C-terminal phosphorylation domain that is important for
stringent regulation in rat CCT1 is apparently missing in plant CCT. Instead, AtCCT1 contains a putative
consensus site (Ser187) of a sucrose-related non-fermenting kinase (SnRK1), a kinase involved in energy
homeostasis. Phos-tag SDS-PAGE coupled with MS analysis showed that SnRK1 phosphorylates
AtCCT1 primarily at this site. Phosphorylated AtCCT1 suffered a substantial reduction in enzyme
activity. Protein truncation and liposome binding studies indicated that SnRK1 phosphorylation of
AtCCT1 directly affects the catalytic domain instead of interfering with the phosphatidate-mediated
activation of the enzyme. Overexpression of AtCCT1 catalytic domain in Nicotiana benthamiana leaves
resulted in higher PC content, but its co-expression with SnRK1 reduced this effect. Taken together, our
results suggest that SnRK1 mediates the phosphorylation and inactivation of AtCCT1, revealing a new
mode of regulation for this key enzyme in plant PC biosynthesis.

Guanqun(Gavin) Chen (gc24@ualberta.ca)

S208. Identifying sequences required to piggyback AtADT5 into the nucleus
*
Clayton, E. ; S. Abolhassani Rad; M. Smith-Uffen; S. Kohalmi
The University of Western Ontario

In Arabidopsis thaliana the last step of phenylalanine (Phe) biosynthesis is catalyzed by a family of
AROGENATE DEHYDRATASES, and all six AtADTs localize to the chloroplast. AtADT5, however, is
the only AtADT that also localizes to the nucleus despite the lack of a nuclear localization signal (NLS).
We believe AtADT5 is a moonlighting protein, having a unique second function in the nucleus. Recently,
a unique interaction partner of AtADT5 was identified, and only heterodimers of AtADT5 and this
interactor localize to the nucleus. Compellingly, this interactor has two putative NLSs. We believe this
interactor is piggybacking AtADT5 into the nucleus. To identify the amino acids required for this
interaction, a series of domain-swapped constructs and targeted substitution constructs of AtADT4 and
AtADT5 were made as AtADT4 and AtADT5 share 90% sequence identity on the protein level. In
addition, deletion constructs were made removing the putative NLSs from the interactor to determine
whether one or both NLSs are required for nuclear import of the heterodimer. Heterodimer formation and
localization will be assessed using yeast-two-hybrid and bimolecular fluorescence complementation
assays. We will present and discuss our initial results that indicate that although specific amino acids are
involved, the story is more complex. This work will add to the growing body of research surrounding
moonlighting proteins, and the effect subtle sequence changes can have on protein function.

Emily Clayton (eclayto3@uwo.ca)

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