Tail-anchored (TA) proteins are a class of proteins that possess a single transmembrane domain at their C-terminal end and perform essential functions on the cytosolic face of organellar membranes. The Arabidopsis genome encodes over 500 TA proteins, which are involved in diverse functions including redox reactions, vesicular trafficking and programmed cell death (PCD). We have previously reported that Arabidopsis Triphosphate Tunnel Metalloenzyme proteins TTM1 and TTM2 are mitochondrial outer membrane-localized TA proteins that show pyrophosphatase activity. Interestingly, both TTM1 and TTM2 are involved in different types of PCD, senescence and immunity, respectively. The knockout mutant of TTM1 (ttm1) displays delayed senescence not only during natural aging but also upon prolonged dark or ABA treatment, indicating its universal positive role in senescence-related PCD. Here, we show that TTM1 undergoes multiple phosphorylation steps at three major phosphorylation sites upon a perception of senescence cues (ABA treatment or dark treatment). One of the sites, S437, is strongly phosphorylated upon ABA or dark treatment in planta. Since a phospho-mimetic, but not a phospho-dead mutant version of TTM1 could complement the delayed senescence phenotype of the ttm1 mutant, the phosphorylation at this residue is essential for TTM1 function to positively regulate senescence. We conducted extensive in vitro kinase assays and identified a set of MAP kinases that phosphorylate TTM1 at S437, connecting these kinases to senescence via TTM1. Furthermore, we also found MAP kinases that phosphorylate two additional sites. Interestingly, current data suggest that phosphorylation of these sites regulates TTM1 protein turnover. Taken together, we found that senescence induces multiple phosphorylation events in TTM1 that regulate TTM1 protein function in senescence. This work provides the first mechanistic evidence connecting the mitochondrial outer membrane-localized protein TTM1 with ABA and dark-induced leaf senescence.
Coauthors: Wolfgang Moeder – University of Toronto;Keiko Yoshioka – University of Toronto