title: Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferase 1 identified using phage display and biopanning.
abstract: 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 on-blot 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 over-representation 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.
authors.affiliation: Department of Horticulture, University of Kentucky, Lexington, Kentucky 40546-0312, USA
authors.forename: Tingsu
authors.initials: T
authors.lastname: Chen
authors.forename: Nihar
authors.initials: N
authors.lastname: Nayak
authors.forename: Susmita Maitra
authors.initials: SM
authors.lastname: Majee
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lowenson
authors.forename: Kim R
authors.initials: KR
authors.lastname: Schäfermeyer
authors.forename: Alyssa C
authors.initials: AC
authors.lastname: Eliopoulos
authors.forename: Taylor D
authors.initials: TD
authors.lastname: Lloyd
authors.forename: Randy
authors.initials: R
authors.lastname: Dinkins
authors.forename: Sharyn E
authors.initials: SE
authors.lastname: Perry
authors.forename: Nancy R
authors.initials: NR
authors.lastname: Forsthoefel
authors.forename: Steven G
authors.initials: SG
authors.lastname: Clarke
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Vernon
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
authors.forename: Tomas
authors.initials: T
authors.lastname: Rejtar
authors.forename: A Bruce
authors.initials: AB
authors.lastname: Downie
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D019151:Peptide Library; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D019151:Peptide Library; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase