Protein repair L-isoaspartyl methyltransferase in plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley seeds (original) (raw)
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Plant Physiology, 2013
PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2′) differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct en...
Protein Repair L-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor in Arabidopsis, 2008
The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein L-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from L-isoaspartyl formation by catalyzing the conversion of abnormal L-isoaspartyl residues to their normal L-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of L-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of L-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal L-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments.
The New phytologist, 2016
PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of...
A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana
Plant Molecular Biology, 1996
Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5'-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.
Journal of Biological Chemistry, 2010
The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by onblot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An overrepresentation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.
Physiologia Plantarum, 2006
Protein L-isoaspartyl (D-aspartyl) O-methyltransferases (EC 2.1.1.77; PIMT or PCMT) are enzymes that initiate the full or partial repair of damaged L-aspartyl and L-asparaginyl residues, respectively. These enzymes are found in most organisms and maintain a high degree of sequence conservation. Arabidopsis thaliana (Arabidopsis L. Heynh.) is unique among eukaryotes in that it contains two genes, rather than one, that encode PIMT isozymes. We describe a novel Arabidopsis PIMT isozyme, designated AtPIMT2αω, encoded by the PIMT2 gene (At5g50240). We characterized the enzymatic activity of the recombinant AtPIMT2αω in comparison to the other AtPIMT2 isozymes, AtPIMT1, and to the human PCMT ortholog, to better understand its role in Arabidopsis. All Arabidopsis PIMT isozymes are active over a relatively wide pH range. For AtPIMT2αω maximal activity is observed at 50 °C (a lethal temperature for Arabidopsis); this activity is almost ten times greater than the activity at the growth temperature of 25 °C. Interestingly, enzyme activity decreases after preincubation at temperatures above 30°C. A similar situation is found for the recombinant AtPIMT2ψ and the AtPIMT2ω isozymes, as well as for the AtPIMT1 and human PCMT1 enzymes. These results suggest that the short-term ability of these methyltransferases to initiate repair under extreme temperature conditions may be a common feature of both the plant and animal species.
PLANT PHYSIOLOGY, 2004
The spontaneous and deleterious conversion of l-asparaginyl and l-aspartyl protein residues to l-iso-Asp or d-Asp occurs as proteins age and is accelerated under stressful conditions. Arabidopsis (Arabidopsis L. Heynh.) contains two genes (At3g48330 and At5g50240) encoding protein-l-isoaspartate methyltransferase (EC 2.1.1.77; PIMT), an enzyme capable of correcting this damage. The gene located on chromosome 5 (PIMT2) produces two proteins differing by three amino acids through alternative 3′ splice site selection in the first intron. Recombinant protein from both splicing variants has PIMT activity. Subcellular localization using cell fractionation followed by immunoblot detection, as well as confocal visualization of PIMT:GFP fusions, demonstrated that PIMT1 is cytosolic while a canonical nuclear localization signal, present in PIMT2ψ and the shorter PIMT2ω, is functional. Multiplex reverse transcription-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, ...
2000
The spontaneous and deleterious conversion of L-asparaginyl and L-aspartyl protein residues to L-iso-Asp or D-Asp occurs as proteins age and is accelerated under stressful conditions. Arabidopsis (Arabidopsis L. Heynh.) contains two genes (At3g48330 and At5g50240) encoding protein-L-isoaspartate methyltransferase (EC 2.1.1.77; PIMT), an enzyme capable of correcting this damage. The gene located on chromosome 5 (PIMT2) produces two proteins differing by three amino acids through alternative 3# splice site selection in the first intron. Recombinant protein from both splicing variants has PIMT activity. Subcellular localization using cell fractionation followed by immunoblot detection, as well as confocal visualization of PIMT:GFP fusions, demonstrated that PIMT1 is cytosolic while a canonical nuclear localization signal, present in PIMT2c and the shorter PIMT2v, is functional. Multiplex reverse transcription-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to b-TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous abscisic acid (ABA). Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA and applied stress presumably through an ABA-dependent pathway. Furthermore, cleaved amplified polymorphic sequences from PIMT2 amplicons determined that ABA preferentially enhances the production of PIMT2v transcript in leaves and possibly in tissues other than germinating seeds.
Plant Science, 2006
Proteome analysis of mature barley (Hordeum vulgare subsp. vulgare) seeds has led to the identification of proteins in about 450 spots on 2Dgels. To shed light on the role of some of these proteins, their temporal appearance was monitored over 5 weeks during grain-filling and maturation of field-grown barley. Appearance profiles are described for 105 proteins identified in 185 2D-gel spots in the overlapping pI ranges 4-7 and 6-11. Grouping of proteins according to appearance across functional categories revealed instances of differential regulation of protein forms. Thus, a single 1-cys-peroxiredoxin isoform was identified in three spots, one present throughout grain filling, one appearing during desiccation and one observed only in mature seeds. This suggested post-translational modification of the protein to different degrees during seed maturation. Distinct isoforms of several proteins were identified in spots with individual appearance profiles, indicating differential expression of isoforms. Three isoforms of b-1,3 endoglucanase, including one not previously observed, each had a different temporal appearance pattern probably reflecting involvement in diverse processes such as cell wall modification or defence against fungal pathogens. Comparison of two cultivars, Barke and Morex, led to identification of protein spots appearing earlier in Morex than Barke, reflecting the faster maturation of Morex seeds. #