Proteomic analysis of the 14-3-3 family in Arabidopsis.
In this study, various proteomics-based methods were utilized to examine the 14-3-3 protein family in Arabidopsis thaliana. A protein extract was prepared from an Arabidopsis hypocotyl suspension culture and analyzed by two-dimensional gel electrophoresis and immunoblotting with a 14-3-3 monoclonal antibody that recognizes multiple Arabidopsis isoforms. Protein spots that cross-reacted with the monoclonal antibody as well as the surrounding spots were analyzed by high performance liquid chromatography in conjunction with electrospray-tandem mass spectrometry. Nine separate spots contained 14-3-3s and each spot contained multiple 14-3-3 isoforms. Every isoform observed was verified by the identification of at least one isoform-specific peptide. Further analysis by mass spectrometry revealed that the isoforms Chi, Upsilon, Omega, Phi, and Lambda were acetylated on their N termini and no non-acetylated N termini were recovered. These data, together with the distribution of isoforms and the confirmation that 14-3-3s are not complexed during urea denaturing isoelectric focusing, supports the conclusion that Arabidopsis 14-3-3s are acetylated in vivo and are significantly affected by other post-translational modifications.
Fuller
Brian
B
Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, University of Florida, Gainesville, FL 32601, USA.
Stevens
Stanley M
SM
Sehnke
Paul C
PC
Ferl
Robert J
RJ
D048948:14-3-3 Proteins / Q000378:metabolism*; D000107:Acetylation; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D018546:Hypocotyl / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D040901:Proteomics
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D020033:Protein Isoforms