Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning
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 seed...
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Published in | The Journal of biological chemistry Vol. 285; no. 48; pp. 37281 - 37292 |
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Main Authors | , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
26.11.2010
American Society for Biochemistry and Molecular Biology |
Subjects | |
Online Access | Get full text |
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Summary: | 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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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 Present address: Dept. of Plant Molecular Biology, University of Delhi, New Delhi 110021, India. Present address: Microbiology Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530007, China. |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M110.157008 |