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Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development

View Article: PubMed Central - PubMed

ABSTRACT

Background: Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking.

Results: To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised.

Conclusions: Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-017-1022-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

ALS and AIP interaction analysis using yeast two-hybrid. Assessment of ALS-AIP and AIP protein interaction using Y2H assays. Colonies expressing the designated constructs and grown on –Leu –Trp + His plates (top panel) and test plates containing the indicated concentrations of 3-AT in the absence of Leu/Trp/His (bottom panels) were imaged. Strong homo-oligomers are observed for both ALS and AIPs. Heterodimers are observed between ALS and AIPs but not between AIP1 and AIP3. GUS CDS was used as a negative control
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Fig1: ALS and AIP interaction analysis using yeast two-hybrid. Assessment of ALS-AIP and AIP protein interaction using Y2H assays. Colonies expressing the designated constructs and grown on –Leu –Trp + His plates (top panel) and test plates containing the indicated concentrations of 3-AT in the absence of Leu/Trp/His (bottom panels) were imaged. Strong homo-oligomers are observed for both ALS and AIPs. Heterodimers are observed between ALS and AIPs but not between AIP1 and AIP3. GUS CDS was used as a negative control

Mentions: To gain a better understanding of the ALS protein interactome, we conducted a yeast two-hybrid (Y2H) screen using the ALS catalytic subunit as bait, leading to the identification of several proteins. Following the sequencing of the corresponding fusion cDNA sequences involved, the deduced protein sequence of the identified ALS interactors fell into eight classes (Table 1) and were denoted as ALS-interacting proteins (AIPs). All interacting pairs were again independently verified using Y2H (data not shown). Two out of the eight classes, AIP1 (AT2G31810) and AIP3/VAT1 (AT5G16290), showed high deduced protein similarity to prokaryotic and eukaryotic regulatory ALS subunits (Additional file 1: Figure S1), suggesting that both these putative regulatory subunits associate with the ALS catalytic subunit in planta. In the absence of information pertaining to the binding affinities of the regulatory subunits with the catalytic subunit, we assessed the qualitative binding stability of both proteins using the indirect measure of Y2H signal intensity in yeast. No detectable difference in subunit-binding stability was observed among the catalytic subunit and the two regulatory proteins (Fig. 1).Table 1


Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development
ALS and AIP interaction analysis using yeast two-hybrid. Assessment of ALS-AIP and AIP protein interaction using Y2H assays. Colonies expressing the designated constructs and grown on –Leu –Trp + His plates (top panel) and test plates containing the indicated concentrations of 3-AT in the absence of Leu/Trp/His (bottom panels) were imaged. Strong homo-oligomers are observed for both ALS and AIPs. Heterodimers are observed between ALS and AIPs but not between AIP1 and AIP3. GUS CDS was used as a negative control
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384131&req=5

Fig1: ALS and AIP interaction analysis using yeast two-hybrid. Assessment of ALS-AIP and AIP protein interaction using Y2H assays. Colonies expressing the designated constructs and grown on –Leu –Trp + His plates (top panel) and test plates containing the indicated concentrations of 3-AT in the absence of Leu/Trp/His (bottom panels) were imaged. Strong homo-oligomers are observed for both ALS and AIPs. Heterodimers are observed between ALS and AIPs but not between AIP1 and AIP3. GUS CDS was used as a negative control
Mentions: To gain a better understanding of the ALS protein interactome, we conducted a yeast two-hybrid (Y2H) screen using the ALS catalytic subunit as bait, leading to the identification of several proteins. Following the sequencing of the corresponding fusion cDNA sequences involved, the deduced protein sequence of the identified ALS interactors fell into eight classes (Table 1) and were denoted as ALS-interacting proteins (AIPs). All interacting pairs were again independently verified using Y2H (data not shown). Two out of the eight classes, AIP1 (AT2G31810) and AIP3/VAT1 (AT5G16290), showed high deduced protein similarity to prokaryotic and eukaryotic regulatory ALS subunits (Additional file 1: Figure S1), suggesting that both these putative regulatory subunits associate with the ALS catalytic subunit in planta. In the absence of information pertaining to the binding affinities of the regulatory subunits with the catalytic subunit, we assessed the qualitative binding stability of both proteins using the indirect measure of Y2H signal intensity in yeast. No detectable difference in subunit-binding stability was observed among the catalytic subunit and the two regulatory proteins (Fig. 1).Table 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking.

Results: To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised.

Conclusions: Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-017-1022-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus