Membrane protein chaperone and sodium chloride modulate the kinetics and morphology of amyloid beta aggregation.

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins. Amyloid beta (Aß) peptides are derived from the cleavage of a larger membrane protein molecule and accumulate to form plaques extracellularly. According to the amyloid hypothesis, accumulation of Aß aggregates in the brain is primarily responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, the disassembly of Aß aggregates may provide opportunities for alleviating or treating AD. Here, we show that the novel protein targeting machinery from chloroplast, chloroplast signal recognition particle 43 (cpSRP43), is an ATP-independent membrane protein chaperone that can both prevent and reverse Aß aggregation effectively. Using of thioflavin T dye, we obtained the aggregation kinetics of Aß aggregation and determined that the chaperone prevents Aß aggregation in a concentration-dependent manner. Size exclusion chromatography and sedimentation assays showed that 10-fold excess of cpSRP43 can keep Aß in the soluble monomeric form. Electron microscopy showed that the fibril structure was disrupted in the presence of this chaperone. Importantly, cpSRP43 utilizes the binding energy to actively remodel the preformed Aß aggregates without assistance by a co-chaperone and ATP, emphasizing its unique function among protein chaperones. Moreover, when sodium chloride concentration is higher than 25 mm, the Aß aggregation rate increases drastically to form tightly associated aggregates and generate more oligomers. Our results demonstrate that the presence of cpSRP43 and low NaCl levels inhibit or retard Aß peptide aggregation, potentially opening new avenues to strategically develop an effective treatment for AD.

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12.

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

New Insights into the Chloroplast Outer Membrane Proteome and Associated Targeting Pathways.

Plastids are a dynamic class of organelle in plant cells that arose from an ancient cyanobacterial endosymbiont. Over the course of evolution, most genes encoding plastid proteins were transferred to the nuclear genome. In parallel, eukaryotic cells evolved a series of targeting pathways and complex proteinaceous machinery at the plastid surface to direct these proteins back to their target organelle. Chloroplasts are the most well-characterized plastids, responsible for photosynthesis and other important metabolic functions. The biogenesis and function of chloroplasts rely heavily on the fidelity of intracellular protein trafficking pathways. Therefore, understanding these pathways and their regulation is essential. Furthermore, the chloroplast outer membrane proteome remains relatively uncharted territory in our understanding of protein targeting. Many key players in the cytosol, receptors at the organelle surface, and insertases that facilitate insertion into the chloroplast outer membrane remain elusive for this group of proteins. In this review, we summarize recent advances in the understanding of well-characterized chloroplast outer membrane protein targeting pathways as well as provide new insights into novel targeting signals and pathways more recently identified using a bioinformatic approach. As a result of our analyses, we expand the known number of chloroplast outer membrane proteins from 117 to 138.

Plant LHC-like proteins show robust folding and static non-photochemical quenching.

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.

Protocol for evaluating protein relocalization from the plasma membrane to chloroplasts.

We present a protocol for analyzing and evaluating the relocalization of proteins from the plasma membrane to chloroplasts. Some plant membrane-bound proteins carry dual targeting signals, e.g., a membrane-anchoring N-myristoylation motif and a chloroplast transit peptide. These proteins are predominantly targeted to membranes; upon certain cues, however, they can undergo detachment from membranes and relocalization to chloroplasts. This protocol combines imaging and biochemical analyses to track in a reliable and quantitative manner the relocalization of proteins between subcellular organelles. For complete details on the use and execution of this protocol, please refer to Medina-Puche et al. (2020).

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair than for the C23-C31 pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

RsmD, a Chloroplast rRNA m2G Methyltransferase, Plays a Role in Cold Stress Tolerance by Possibly Affecting Chloroplast Translation in Arabidopsis.

Ribosomal RNA (rRNA) methylation is a pivotal process in the assembly and activity of ribosomes, which in turn play vital roles in the growth, development and stress responses of plants. Although few methyltransferases responsible for rRNA methylation have been identified in plant chloroplasts, the nature and function of these enzymes in chloroplasts remain largely unknown. In this study, we characterized ArabidopsisRsmD (At3g28460), an ortholog of the methyltransferase responsible for N2-methylguanosine (m2G) modification of 16S rRNA in Escherichia coli. Confocal microscopic analysis of an RsmD- green fluorescent protein fusion protein revealed that RsmD is localized to chloroplasts. Primer extension analysis indicated that RsmD is responsible for m2G methylation at position 915 in the 16S rRNA of Arabidopsis chloroplasts. Under cold stress, rsmd mutant plants exhibited retarded growth, i.e. had shorter roots, lower fresh weight and pale-green leaves, compared with wild-type (WT) plants. However, these phenotypes were not detected in response to drought or salt stress. Notably, the rsmd mutant was hypersensitive to erythromycin or lincomycin and accumulated fewer chloroplast proteins compared with the WT, suggesting that RsmD influences translation in chloroplasts. Complementation lines expressing RsmD in the rsmd mutant background recovered WT phenotypes. Importantly, RsmD harbored RNA methyltransferase activity. Collectively, the findings of this study indicate that RsmD is a chloroplast 16S rRNA methyltransferase responsible for m2G915 modification that plays a role in the adaptation of Arabidopsisto cold stress.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana.

BACKGROUND: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. METHODS: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. RESULTS: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. CONCLUSIONS: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract.

Recombinant expression, purification and SAXS analysis of Arabidopsis thaliana ClpC1.

Caseinolytic protease-associated chaperones (Clp chaperones) are HSP100 proteins belonging to the family of ATPases having diverse cellular functions, and they occur in various organisms ranging from bacteria to plants and mammals. Most Clp chaperones have a hexameric organization and associate with tetradecameric Clp proteases to recognize and unfold protein substrates that get degraded within the cellular milieu. Vascular plants have a diverse family of Clp chaperones compared to other organisms; wherein, the chloroplasts of Arabidopsis thaliana alone contain four distinct Clp chaperones, such as ClpC1, ClpC2, ClpD, and ClpB3. The paralogs AtClpC1 and AtClpC2 are more than 90% identical, though the extent of functional overlap between the two is not clear. Moreover, in vitro characterization reports are available only for AtClpC2, as AtClpC1 could not be expressed in recombinant form in the past. Herein, using a bacterial expression system, we have successfully expressed and purified AtClpC1 with a short N-terminal truncation, employing a three-step chromatographic purification strategy. We show that AtClpC1 exists as a hexamer in the presence of ATP and MgCl2, as known for other functional Clp chaperones. Further, our SAXS analyses provide a low-resolution envelope structure for the hexameric AtClpC1, which very well fits a ClpC hexamer model.

High-Resolution Crystal Structure of Chloroplastic Ribose-5-Phosphate Isomerase from Chlamydomonas reinhardtii-An Enzyme Involved in the Photosynthetic Calvin-Benson Cycle.

The Calvin-Benson cycle is the key metabolic pathway of photosynthesis responsible for carbon fixation and relies on eleven conserved enzymes. Ribose-5-phosphate isomerase (RPI) isomerizes ribose-5-phosphate into ribulose-5-phosphate and contributes to the regeneration of the Rubisco substrate. Plant RPI is the target of diverse post-translational modifications including phosphorylation and thiol-based modifications to presumably adjust its activity to the photosynthetic electron flow. Here, we describe the first experimental structure of a photosynthetic RPI at 1.4 Å resolution. Our structure confirms the composition of the catalytic pocket of the enzyme. We describe the homo-dimeric state of the protein that we observed in the crystal and in solution. We also map the positions of previously reported post-translational modifications and propose mechanisms by which they may impact the catalytic parameters. The structural data will inform the biochemical modeling of photosynthesis.

STARCH SYNTHASE5, a Noncanonical Starch Synthase-Like Protein, Promotes Starch Granule Initiation in Arabidopsis.

What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.

Arabidopsis BSD2 reveals a novel redox regulation of Rubisco physiology in vivo.

Plants need light energy to drive photosynthesis, but excess energy leads to the production of harmful reactive oxygen species (ROS), resulting in oxidative inactivation of target enzymes, including the photosynthetic CO2-fixing enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been demonstrated in vitro that oxidatively inactivated Rubisco can be reactivated by the addition of reducing agents. Busch et al. (in The Plant Journal, doi: 10.1111/tpj.14617, 2020) recently demonstrated that bundle-sheath defective 2 (BSD2), a stroma-targeted protein formerly known as a late-assembly chaperone for Rubisco biosynthesis, can be responsible for such reactivation in vivo. Here, we propose a working model of the novel redox regulation in Rubisco activity. Redox of Rubisco may be a new target for improving photosynthesis.

In Silico Analysis of ACE Inhibitory Peptides from Chloroplast Proteins of Red Alga Grateloupia asiatica.

Inhibition of angiotensin I-converting enzyme (ACE) is one of the key factors to repress high blood pressure. Although many studies have been reported that seaweed protein hydrolysates showed the ACE inhibitory activity, the comprehensive understanding of the relationship was still unclear. In this study, we employed chloroplast genome for in silico analysis and compared it with in vitro experiments. We first extracted water-soluble proteins (WSP) from red alga Grateloupia asiatica, which contained mainly PE, PC, APC, and Rbc, and prepared WSP hydrolysate by thermolysin, resulting that the hydrolysate showed ACE inhibitory activity. Then, we determined the complete chloroplast genome of G. asiatica (187,518 bp: 206 protein-coding genes, 29 tRNA, and 3 rRNA) and clarified the amino acid sequences of main WSP, i.e., phycobiliproteins and Rubisco, to perform in silico analysis. Consequently, 190 potential ACE inhibitory peptides existed in the main WSP sequences, and 21 peptides were obtained by in silico thermolysin digestion. By comparing in vitro and in silico analyses, in vitro ACE inhibitory activity was correlated to the IC50 value from in silico digestion. Therefore, in silico approach provides insight into the comprehensive understanding of the potential bioactive peptides from seaweed proteins.

Identification of a pigment cluster catalysing fast photoprotective quenching response in CP29.

Non-photochemical quenching is the photoprotective heat dissipation of chlorophyll-excited states. In higher plants, two quenching sites are located in trimeric LHCII and monomeric CP29 proteins. Catalysis of dissipative reactions requires interactions between chromophores, either carotenoid, chlorophyll or both. We identified CP29 protein domains involved in quenching by complementing an Arabidopsis deletion mutant with sequences deleted in pigment-binding or pH-sensitive sites. Acidic residues exposed to the thylakoid lumen were found not essential for activation of thermal dissipation in vivo. Chlorophylls a603 (a5) and a616 were identified as components of the catalytic pigment cluster responsible for quenching reaction(s), in addition to xanthophyll L2 and chlorophyll a609 (b5). We suggest that a conformational change induced by acidification in PsbS is transduced to CP29, thus bringing chlorophylls a603, a609 and a616 into close contact and activating a dissipative channel. Consistently, mutations on putative protonatable residues, exposed to the thylakoid lumen and previously suggested to regulate xanthophyll exchange at binding site L2, did not affect quenching efficiency.

Expression and Purification of DGD2, a Chloroplast Outer Membrane-Associated Glycosyltransferase for Galactolipid Synthesis.

Galactolipids are characteristic lipids of the photosynthetic membranes. They are highly enriched in the chloroplast and are present in photosystem structures. There are two major types of galactolipids, i.e., monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) in chloroplastic membranes, which amount to ∼50 and ∼20 mol % of the total chloroplast lipids, respectively. Under phosphate-limiting conditions, the amount of DGDG increases dramatically for rescuing phosphate from phospholipids. In Arabidopsis thaliana, the gene digalactosyldiacylglycerol synthase 2 (DGD2) encodes a membrane-associated glycosyltransferase. The gene expression is highly responsive to phosphate starvation and is significantly upregulated in this case. To understand the molecular mechanism of DGD2, we established a protocol for DGD2 expression and purification in an Escherichia coli-based system. The work involved optimization of the expression condition and the purification protocol and a careful selection of buffer additives. It was found that a removal of around 70 C-terminal residues was necessary to produce a homogeneous monomeric protein sample with high purity, which was highly active. The purified sample was characterized by an activity assay for enzyme kinetics in which a range of membrane mimetics with different lipid compositions were used. The results demonstrate that DGD2 activity is stimulated by the presence of negatively charged lipids, which highlight the importance of the membrane environment in modulating the enzyme's activity. The study also paves way for future biophysical and structural studies of the enzyme.

Tissue-Specific Regulation of Plastid Protein Import via Transit-Peptide Motifs.

Plastids differentiate into various functional types (chloroplasts, leucoplasts, chromoplasts, etc.) that have distinct proteomes depending on the specific tissue. Most plastid proteins are encoded by the nuclear genome, synthesized as higher molecular mass preproteins with an N-terminal transit peptide, and then posttranslationally imported from the cytosol. Evidence for tissue-specific regulation of import into plastids, and subsequent modulation of plastid proteomes, has been lacking. We quantified protein import into isolated pea (Pisum sativum) leaf chloroplasts and root leucoplasts and identified two transit-peptide motifs that specifically enhance preprotein import into root leucoplasts. Using a plastid preprotein expressed in both leaves and roots of stable transgenic plants, we showed that losing one of the leucoplast motifs interfered with its function in root leucoplasts but had no effect on its function in leaf chloroplasts. We assembled a list of all Arabidopsis (Arabid opsis thaliana) plastid preproteins encoded by recently duplicated genes and show that, within a duplicated preprotein pair, the isoform bearing the leucoplast motif usually has greater root protein abundance. Our findings represent a clear demonstration of tissue-specific regulation of organelle protein import and suggest that it operates by selective evolutionary retention of transit-peptide motifs, which enhances import into specific plastid types.

Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS.

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.

A novel 5'-hydroxyl dinucleotide hydrolase activity for the DXO/Rai1 family of enzymes.

Modifications at the 5'-end of RNAs play a pivotal role in determining their fate. In eukaryotes, the DXO/Rai1 family of enzymes removes numerous 5'-end RNA modifications, thereby regulating RNA turnover. Mouse DXO catalyzes the elimination of incomplete 5'-end caps (including pyrophosphate) and the non-canonical NAD+ cap on mRNAs, and possesses distributive 5'-3' exoribonuclease activity toward 5'-monophosphate (5'-PO4) RNA. Here, we demonstrate that DXO also catalyzes the hydrolysis of RNAs bearing a 5'-hydroxyl group (5'-OH RNA). The crystal structure of DXO in complex with a 5'-OH RNA substrate mimic at 2.0 Å resolution provides elegant insight into the molecular mechanism of this activity. More importantly, the structure predicts that DXO first removes a dinucleotide from 5'-OH RNA. Our nuclease assays confirm this prediction and demonstrate that this 5'-hydroxyl dinucleotide hydrolase (HDH) activity for DXO is higher than the subsequent 5'-3' exoribonuclease activity for selected substrates. Fission yeast Rai1 also has HDH activity although it does not have 5'-3' exonuclease activity, and the Rat1-Rai1 complex can completely degrade 5'-OH RNA. An Arabidopsis DXO1 variant is active toward 5'-OH RNA but prefers 5'-PO4 RNA. Collectively, these studies demonstrate the diverse activities of DXO/Rai1 and expands the collection of RNA substrates that can undergo 5'-3' mediated decay.

Calmodulin is involved in the dual subcellular location of two chloroplast proteins.

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

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

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.