The ACT domain in chloroplast precursor–phosphorylating STY kinases binds metabolites and allosterically regulates kinase activity

Abstract

Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase–chorismate mutase–tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT domain is a widely distributed structural motif known to be important for allosteric regulation of many enzymes. In this work, using biochemical and biophysical techniques in vitro and in planta, including kinase assays, microscale thermophoresis, size exclusion chromatography, as well as site-directed mutagenesis approaches, we show that the ACT domain regulates autophosphorylation and substrate phosphorylation of the STY kinases. We found that isoleucine and S-adenosylmethionine bind to the ACT domain, negatively influencing its autophosphorylation ability. Moreover, we investigated the role of the ACT domain in planta and confirmed its involvement in chloroplast differentiation in vivo. Our results provide detailed insights into the regulation of enzyme activity by ACT domains and establish that it has a role in binding amino acid ligands during chloroplast biogenesis.

Acclimation in plants – the Green Hub consortium

“…(6) Transport processes across the inner membrane of the chloroplast envelope are adjusted in response to low temperatures (Guan et al ., 2019; Trentmann et al ., 2020). (7) Fine‐tuning of chloroplast protein import contributes to effective plant development and acclimation (Lamberti et al ., 2011; Eisa et al ., 2019a; Eisa et al ., 2019b).…”

“…The efficiency of protein uptake is influenced by phosphorylation of the precursor proteins, mediated by the cytosolic protein kinases STY8, 17 and 46 (Lamberti et al ., 2011). Our consortium recently showed that S ‐adenosyl methionine and isoleucine – both of which are synthesised in the chloroplast – bind to the highly conserved STY‐specific aspartate kinase–chorismate mutase–tyrA domain, which leads to the inhibition of kinase activity (Eisa et al ., 2019a). In addition, rates of precursor protein import are reduced under high light conditions owing to a decrease in the expression of the STY kinase genes (Eisa et al ., 2019b).…”

Coordination of Chloroplast Activity with Plant Growth: Clues Point to TOR

“…Finally, when a stress stimulus arrives, there is only a short window of time to modify and import nuclear-encoded proteins into the chloroplast before the gate closes. Protein phosphorylation is a very fast reaction occurring on a time scale of seconds to minutes, which has been observed in the regulation of TOC import capacity and of SSU import into the chloroplast [ 77 , 78 , 79 , 80 , 81 ]. Indeed, Serine/threonine/tyrosine (STY) protein kinases have been proposed to phosphorylate the SSU CTP to regulate the import of the related pre-protein [ 67 ].…”

High Light Acclimation Induces Chloroplast Precursor Phosphorylation and Reduces Import Efficiency

“…The coding regions for STY8, STY46, and STY17 were cloned in the expression vector pET21a + with a C-terminal His-tag (Novagen) and purified as described previously [8]. pOE23, pHCF101, pNdhM, and pFNRL1 were cloned in the expression vector pET21a + with the following primers introducing appropriate restriction sites: pOE23_SacI_fr 5 -AGTCGAGCTCATGGCGTACAGTG CGTGT-3 , pOE23_NotI_rev 5 -CCCACCGCGGCCGCAGCAACACTGAAAGAAGT-3 , pHCF101_ SacI_fr 5 -ACTCGAGCTCATGCCGCTTCTTCATCCACA-3 , pHCF101_NotI_rev 5 -CCCACCGCG GCCGCGACTTCGACTGGAGACAA-3 , pNdhM_SacI_fr 5 -AGTGAGCTCATGGTTGCAGCATTC-3 , pNdhM_NotI_rev 5 -TGCTTAGCGGCCGCAGCGTCCTCTTGAGG-3 , pFNRL1_SacI_fr 5 -AGTC GAGCTCATGGCTGCTGCTATA-3 , pFNRL1_SacI_rev 5 -CCCACCGCGGCCGCGTAGACTTCAA CATT-3 .…”

“…Identification of the responsible dual specificity kinases STY8, STY17, and STY46 provided a tool to investigate the impact of preprotein phosphorylation in vivo, revealing that their impairment results in delayed chloroplast biogenesis [6,7]. Moreover, we recently showed that the kinases are regulated metabolically via an ACT domain [8].…”