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United States Department of Agriculture

Agricultural Research Service

Research Project: FORAGE SYSTEMS FOR SUSTAINABLE ANIMAL PRODUCTION IN THE MID-SOUTH

Location: Forage-Animal Production Research

Title: Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferasel identified using phage display and biopanning

Authors
item Chen, Tingsu -
item Martin, David -
item Nayak, Nihar -
item Majee, Susmita -
item Lowenson, Jonathan -
item Schafermeyer, Kim -
item Eliopoulos, Alyssa -
item Lloyd, Taylor -
item Villa, Sarah -
item DINKINS, RANDY
item Perry, Sharon -
item Forsthoefel, Nancy -
item Clarke, Steven -
item Vernon, Daniel -
item Zhou, Zhaouhui 'sunny -
item Rejtar, Tomas -
item Downie, Bruce -

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 24, 2010
Publication Date: September 24, 2010
Citation: Chen, T., Martin, D., Nayak, N., Majee, S.M., Lowenson, J., Schafermeyer, K.R., Eliopoulos, A.C., Lloyd, T.D., Villa, S., Dinkins, R.D., Perry, S.E., Forsthoefel, N.R., Clarke, S.G., Vernon, D.M., Zhou, Z., Rejtar, T., Downie, B. 2010. Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferasel identified using phage display and biopanning. Journal of Biological Chemistry. 285:37281-37292.

Interpretive Summary: The role of PROTEIN ISOASPARTYL-METHYLTRANSFERASE (PIMT) in repairing damaged proteins in-planta has been hindered by large amounts of isoaspartate-containing storage proteins in the seed. In order to circumvent this problem . a seed phage display library was constructed and tested under condition s that bind PIMT to the protein fragments. This approach allowed for the isolation of 17 putative in frame targets of PIMT. Five targets were recovered independently in different experiments. Two in-frame targets were produced in E. coli as recombinant protein and shown, by on blot methylation experiments acquire isoAspartate, thus becoming a PIMT target. Mutant analysis of plants deficient in three of the in-frame PIMT targets resulted in demonstrable phenotypes during seed development and/or germination. These results suggest that impaired PIMT activity would hinder protein function in these various targets, possibly resulting poor seed performance (quality).

Technical 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 specific 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 storage protein abundance. Inclusion of the PIMT co-substrate, 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 AdoHcy. At 4 rounds, hitherto increasing phage titer plateaued in AdoMet containing pans (consolidating around a few, highly represented sequences) while 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 (OBM). All recovered phage, regardless of reading-frame, had at least one susceptible D or N residue. Five targets were recovered independently in separate pans, as different clones, and/or from different libraries. Two in-frame targets were produced in E. coli as recombinant protein and shown, by OBM, to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution (one to a high titer) 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 sub-group for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would simultaneously hinder protein function in these various targets, possibly resulting in a malaise and poor seed performance (quality).

Last Modified: 8/27/2014
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