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 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.
Chen
Tingsu
T
Department of Horticulture, University of Kentucky, Lexington, Kentucky 40546-0312, USA
Nayak
Nihar
N
Majee
Susmita Maitra
SM
Lowenson
Jonathan
J
Schäfermeyer
Kim R
KR
Eliopoulos
Alyssa C
AC
Lloyd
Taylor D
TD
Dinkins
Randy
R
Perry
Sharyn E
SE
Forsthoefel
Nancy R
NR
Clarke
Steven G
SG
Vernon
Daniel M
DM
Zhou
Zhaohui Sunny
ZS
Rejtar
Tomas
T
Downie
A Bruce
AB
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
D029681:Arabidopsis Proteins; D019151:Peptide Library; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase