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A Second Protein L-Isoaspartyl Methyltransferase Gene inArabidopsis Produces Two Transcripts Whose ProductsAre Sequestered in the Nucleus1[w]Qilong Xu, Marisa P. Belcastro2, Sarah T. Villa, Randy D. Dinkins, Steven G. Clarke, and A. Bruce Downie*Department of Horticulture (Q.X., A.B.D.), University of Kentucky Agriculture Biotechnology UndergraduateProgram (M.P.B.), Department of Agronomy (R.D.D.), Plant Physiology/Biochemistry/Molecular BiologyProgram (Q.X.), Seed Biology Program (Q.X., A.B.D.), and University of Kentucky Agriculture ExperimentStation, S129, Agriculture Science Center North (Q.X., R.D., A.B.D.), University of Kentucky, LexingtonKentucky, 40546–0312; and Department of Chemistry and Biochemistry, University of California, Los Angeles,California 90095–1569 (S.T.V., S.G.C.)The spontaneous and deleterious conversion of L-asparaginyl and L-aspartyl protein residues to L-iso-Asp or D-Asp occurs asproteins age and is accelerated under stressful conditions. Arabidopsis (Arabidopsis L. Heynh.) contains two genes (At3g48330and At5g50240) encoding protein-L-isoaspartate methyltransferase (EC 2.1.1.77; PIMT), an enzyme capable of correcting thisdamage. The gene located on chromosome 5 (PIMT2) produces two proteins differing by three amino acids through alternative3# splice site selection in the first intron. Recombinant protein from both splicing variants has PIMT activity. Subcellularlocalization 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 transcriptis constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasingendogenous abscisic acid (ABA). Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA andapplied stress presumably through an ABA-dependent pathway. Furthermore, cleaved amplified polymorphic sequences fromPIMT2 amplicons determined that ABA preferentially enhances the production of PIMT2v transcript in leaves and possibly intissues other than germinating seeds.The proteome is subject to deleterious alterationover time through aging or due to stressful conditions.Aging is a conflict between the unrelenting productionof undesirable side products of metabolism or aber-rant molecules through spontaneous modificationversus an organism’s ability to eliminate, repair, ortolerate the change (Clarke, 2003). Protein damage canbe of two general types: (1) conformational damage tothree-dimensional structure or (2) covalent damage toprimary structure (Galletti et al., 1995; Visick andClarke, 1995). These detrimental conversions can leadto the recognition, tagging, and destruction of thealtered peptides, requiring that they be resynthesizedde novo. It is, therefore, orders of magnitude moreefficient to rectify damaging protein modifications ifpossible (Reissner and Aswad, 2003). Hence, organ-isms encode and deploy a plethora of damage repairsystems that are capable of recognizing and repairingspecific changes to polypeptides at a fraction of the‘‘cost’’ associated with protein degradation and syn-thesis (Schumacher et al., 1996; Zou et al., 1998; Pliyevand Gurvits, 1999; Hoshi and Heinemann, 2001). Pro-tein methyltransferase is one such system, capable ofrepairing abnormal isoaspartyl (isoAsp) residues,a predominate form of protein damage accrued undernormal physiological conditions (Lowenson andClarke, 1992).Protein-L-isoaspartate methyltransferase (EC2.1.1.77; PIMT) is a repair enzyme that catalyzes theS-adenosylmethionine (AdoMet)-dependent methylesterification of the a-carboxyl group ofL-isoAspresidues (as well as, in some instances, the b-carboxylgroup ofD-aspartyl residues). L-IsoAsp is an unen-coded amino acid arising from the spontaneous deg-radation of Asn and Asp in proteins throughdeamidation and isomerization in the former, anddehydration and isomerization in the latter, instance(Geiger and Clarke, 1987; Bhatt et al., 1990; Aswadet al., 2000). Formation of isoAsp in the proteinintroduces an extra carbon into the peptide backbone,which now proceeds through the R-group carboxy1This work was supported by the University of Kentucky De-partment of Horticulture (stipend to Q.X.) and by the NationalInstitutes of Health (grant nos. GM26020 and AG18000 to S.G.C.).2Present address: University of Kentucky Medical School, 800Rose Street, Lexington, KY, 40536.* Corresponding author; e-mail [email protected]; fax 859–257–7874.[w]The online version of this article contains Web-only data.Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.104.046094.2652 Plant Physiology, September 2004, Vol. 136, pp. 2652–2664, www.plantphysiol.org Ó 2004 American Society of Plant Biologiststerminus and produces a kink in the secondary struc-ture of the protein. These alterations are frequentlydetrimental to enzymatic function (Szymanska et al.,1998; Mamula et al., 1999; Esposito et al., 2000; Tarcsaet al., 2000). Repair begins with an enzyme-mediatedmethylation reaction followed by nonenzymaticsteps that, through reiteration of the methylation/demethylation process, eventually result in the net con-version ofL-isoAsp residues to L-Asp (Aswad et al.,2000) and, usually, a reconstitution of normal proteinfunction (Brennan et al., 1994; Clarke, 1999).Found in nearly all eukaryotic cells as well asmost archaebacteria and gram-negative eubacteria(Johnson et al., 1991; Kagan et al., 1997a), PIMT activ-ity is vitally important in those organisms employingit (Kagan et al., 1997b; Kim et al., 1997; Visick et al.,1998; Yamamoto et al., 1998; David et al., 1999).Nonetheless, although several isoforms of PIMT havebeen identified in a variety of organisms, only a singlegene encoding PIMT has been reported per eukaryoticorganism studied to date (Kagan et al., 1997a). PIMTisoforms in human erythrocytes, bovine brain, mousetestis, and wheat germ are proposed to be alternativesplicing variants (Potter et al., 1992; Romanik et al.,1992; Mudgett and Clarke, 1993). In plants, protein-L-iso-Asp methyltransferase, but not


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