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.
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This work was supported in part by National Science Foundation Molecular and Cellular Biochemistry Grant 0449649 (to A. B. D. and S. G. C.). This work was also supported by University of Kentucky undergraduate research and creativity Grant 14-28 (to A. B. D. and T. D. L.).
Chen, Tingsu; Nayak, Nihar; Majee, Susmita Maitra; Lowenson, Jonathan; Schäfermeyer, Kim R.; Eliopoulos, Alyssa C.; Lloyd, Taylor D.; Dinkins, Randy; Perry, Sharyn E.; Forsthoefel, Nancy R.; Clarke, Steven G.; Vernon, Daniel M.; Zhou, Zhaohui Sunny; Rejtar, Tomas; and Downie, A. Bruce, "Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning" (2010). Horticulture Faculty Publications. 6.