PhD Exit Seminar – Purva Karia (Yoshioka Lab)

Tail-anchored Triphosphate Tunnel Metalloenzymes in Arabidopsis thaliana: An Investigation into the Molecular Mechanisms underlying their Biological Functions

Abstract

The Triphosphate Tunnel Metalloenzymes (TTMs) superfamily is a group of proteins that contain a tunnel-shaped β-barrel structure, called a TTM domain. They exist in all domains of life where they act on a range of triphosphate substrates. However, the biological function of most TTM proteins is not well understood. The Arabidopsis genome encodes three TTM proteins named TTM1, 2, and 3. Our research group previously found that both TTM1 and TTM2 are involved in different types of programmed cell death (PCD), dark-induced senescence and immunity, respectively. It was concluded that this functional differentiation was governed by their transcriptional regulation. In this thesis, I show that the knockout mutant of TTM1 (ttm1) also displays delayed natural and ABA-induced senescence, indicating its universal role as a positive regulator of senescence-related PCD. I further found that TTM1 undergoes multiple phosphorylation events at three major sites upon perception of senescence cues (i.e. ABA or dark treatment). One of the sites, S437, shows a strong increase in phosphorylation after dark treatment and I have shown that phosphorylation at S437 is crucial for TTM1’s function in senescence. Our extensive in vitro kinase assays identified two MAP kinases that phosphorylate TTM1 at S437, connecting these kinases to senescence via TTM1. I also identified a set of MAP kinases that phosphorylate TTM1 at two additional sites, S10 and S490. My data suggest that phosphorylation of these additional sites regulates TTM1 protein turnover via the 26S proteasome. These data provided the first mechanistic evidence connecting the mitochondrial outer membrane-localized protein TTM1 with ABA and leaf senescence. Further, to understand the transcriptional regulation which determines the biological functions of TTM1 and TTM2, a combination of a yeast one-hybrid assay and various in silico analyses were performed. Based on my analyses, I propose five transcription factors that potentially regulate TTM1 expression during senescence and two transcription factors that likely contribute to the transcriptional down-regulation of TTM2 upon pathogen infection. In summary, my thesis work describes a mechanism for the post-translational regulation of plant TTM1 and also identifies possible transcriptional regulators of TTM1 and TTM2 for future study.