“…The results suggest that both AIP1 and AIP3 regulatory subunits localize to the chloroplast as well as the peroxisome. Given the presence of a cryptic PTS1 motif, together with C-terminal peroxisomal targeting following masking of the plastid transit peptide, the structural and experimental results support the suggestion of a dual subcellular localization of these two proteins that in itself may be an object of regulation during development [19]. Fig.…”
Section: Resultsmentioning
confidence: 63%
“…These findings suggest that both AIP1 and AIP3 regulatory subunits have acquired functional attributes independent of the ALS holoenzyme. Interestingly, a similar phenomenon has been described for several other proteins in Arabidopsis and other plant species [19]. While BCAA synthesis has been localized chiefly to plastids, the degradation of BCAAs have been hypothesized to occur mainly in mitochondria and peroxisomes [24].…”
BackgroundBranched-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.ResultsTo 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.ConclusionsTaken 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 materialThe online version of this article (doi:10.1186/s12870-017-1022-6) contains supplementary material, which is available to authorized users.
“…The results suggest that both AIP1 and AIP3 regulatory subunits localize to the chloroplast as well as the peroxisome. Given the presence of a cryptic PTS1 motif, together with C-terminal peroxisomal targeting following masking of the plastid transit peptide, the structural and experimental results support the suggestion of a dual subcellular localization of these two proteins that in itself may be an object of regulation during development [19]. Fig.…”
Section: Resultsmentioning
confidence: 63%
“…These findings suggest that both AIP1 and AIP3 regulatory subunits have acquired functional attributes independent of the ALS holoenzyme. Interestingly, a similar phenomenon has been described for several other proteins in Arabidopsis and other plant species [19]. While BCAA synthesis has been localized chiefly to plastids, the degradation of BCAAs have been hypothesized to occur mainly in mitochondria and peroxisomes [24].…”
BackgroundBranched-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.ResultsTo 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.ConclusionsTaken 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 materialThe online version of this article (doi:10.1186/s12870-017-1022-6) contains supplementary material, which is available to authorized users.
“…First, it cannot be excluded that overexpression under the strong 35S promoter results in mistargeting of the GFP fusion protein into chloroplasts. Second, it has already been seen with dually targeted proteins that one location can be favored versus the other (Carrie and Whelan, 2013). That seems to be the case for Arabidopsis RECA2, which is dually targeted but appeared to affect only the maintenance of the mtDNA (Shedge et al, 2007;Miller-Messmer et al, 2012).…”
Section: Roles Of Recg1 In Controlling Proper Homologous Recombinatiomentioning
The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift.
“…Since the first report of a plant protein targeted to two different subcompartments [3], the record of dual-targeted proteins, mostly soluble proteins, has increased to approximately 160 in Arabidopsis and more than 90 in other plants [4, 5]. The majority of dual-localized proteins, roughly 110 in Arabidopsis , are targeted to mitochondria and chloroplasts, where most of them are associated with prokaryote-type processes, for example DNA replication, recombination and repair, nucleotide metabolism, tRNA biogenesis and translation.…”
The reports of dual-targeted proteins in plants have steadily increased over the past years. The vast majority of these proteins are soluble proteins distributed between compartments of the non-secretory pathway, predominantly chloroplasts and mitochondria. In contrast, dual-targeted transmembrane proteins, especially of the secretory pathway, are rare and the mechanisms leading to their differential targeting remain largely unknown. Here, we report dual-targeting of the Arabidopsis DUF679 Membrane Protein 1 (DMP1) to the tonoplast (TP) and the plasma membrane (PM). In Arabidopsis and tobacco two equally abundant DMP1 isoforms are synthesized by alternative translation initiation: a full length protein, DMP1.1, and a truncated one, DMP1.2, which lacks the N-terminal 19 amino acids including a TP-targeting dileucine motif. Accumulation of DMP1.1 and DMP1.2 in the TP and the PM, respectively, is Brefeldin A-sensitive, indicating transit via the Golgi. However, DMP1.2 interacts with DMP1.1, leading to extensive rerouting of DMP1.2 to the TP and “eclipsed” localization of DMP1.2 in the PM where it is barely visible by confocal laser scanning microscopy but clearly detectable by membrane fractionation. It is demonstrated that eGFP fusion to either DMP1 terminus can cause mistargeting artifacts: C-terminal fusion to DMP1.1 or DMP1.2 results in altered ER export and N-terminal fusion to DMP1.1 causes mistargeting to the PM, presumably by masking of the TP targeting signal. These results illustrate how the interplay of alternative translation initiation, presence or absence of targeting information and rerouting due to protein-protein interaction determines the ultimate distribution of a transmembrane protein between two membranes.
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