A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana

Abstract

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.

Protein Repairl-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor inArabidopsis

“…In this work, we have isolated Arabidopsis mutant lines exhibiting varying levels of PIMT and investigated the consequences of such a genetic modulation on several seed characteristics, including isoAsp accumulation in proteins, seed longevity, and seedling establishment under salt or osmotic stressful conditions. The Arabidopsis genome contains two genes encoding PIMT enzymes (Mudgett and Clarke, 1996;Xu et al, 2004). Since PIMT2 is only weakly expressed in seeds compared with PIMT1 ( Figure 2D) (Xu et al, 2004), we have focused our study on PIMT1 using a pimt1-1 T-DNA insertion line affected in PIMT1 regulation and transgenic lines over-or underaccumulating the PIMT1 protein.…”

“…Sequence analyses of the pimt1-1 allele, which is in the Wassilewskija genetic background, identified a single T-DNA insertion in direct orientation located 187 bp upstream of the annotated translation start codon with exact left and right T-DNA borders and a deletion of 13 bp in the flanking region of the right border ( Figure 2B). This 13-bp deletion encompasses the predicted MT1a ABA-responsive element (ABRE) that was previously suggested to be in the Arabidopsis PIMT1 gene (Mudgett and Clarke, 1996) and that exhibits features of the Em1a ABRE motif of the wheat Em promoter (Marcotte et al, 1989). The selfprogeny of the hemizygous T1 plants segregated the kanamycin resistance marker with a 3:1 segregation ratio (153 kanamycin resistant:47 kanamycin sensitive; x 2 = 0.24) testifying to a single Mendelian locus for the T-DNA insertion.…”

“…Thus, the different levels of PIMT1 mRNA in wild-type and pimt1-1 mutant seeds ( Figure 2C) correlated well with respective protein levels. Since the Arabidopsis genome contains two PIMT genes, namely, AT3G48330 and AT5G50240, encoding PIMT1 and PIMT2, respectively (Mudgett and Clarke, 1996;Xu et al, 2004), it was important to distinguish between these two proteins in protein gel blotting experiments. To this end, in addition to the PIMT1-specific antibodies, we prepared PIMT2-specific antibodies (see Supplemental Figure 3 online).…”

“…The sequence deletion in PIMT1 caused by the insertion of the T-DNA is underlined. The three putative ABRE motifs previously described by Mudgett and Clarke (1996) are highlighted in gray, and the first motif that encompasses the 13-bp deleted sequence is called MT1a (Mudgett and Clarke, 1996). (C) RT-PCR comparison of PIMT1 transcript accumulation in the wild type (black) and pimt1-1 mutant (gray) freshly harvested dry mature seeds and in 5-d-old seedlings cultivated on water (ÀABA) or on water for 4 d and transferred the fourth day to water containing 100 mM ABA for an additional day (+ABA).…”

“…In Arabidopsis thaliana, there are two PIMT genes, PIMT1 and PIMT2 (Mudgett and Clarke, 1996;Xu et al, 2004), whereas animal or bacterial genomes contain only one such gene. While PIMT1 mRNA accumulates to relatively high levels in a range of tissues, PIMT2 transcripts are detected at low levels and only during seed maturation (Xu et al, 2004).…”