The corresponding molecular mass of the uncharged peptide (2139

The corresponding molecular mass of the uncharged peptide (2139.08?Da) closely matches the predicted monoisotopic mass of non-phosphorylated form of this peptide (2059.07?Da) plus that of a single phosphate group (79.97?Da). phosphorylated protein in seeds and FLICE that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, target for this enzyme or that regulates the protein kinase/phosphatase system required for cruciferin phosphorylation. (thale cress) mutant, cruciferin, mass spectrometry (MS), protein phosphorylation, seed storage proteins, two-dimensional electrophoresis Information Resource; TOF, time-of-flight; VPE, vacuolar processing enzymes INTRODUCTION During their development, plant seeds accumulate large amounts of proteins. These proteins provide the main source of nitrogen for seed germination (±)-Ibipinabant and for early seedling growth. In (thale cress) and other crucifers, the main seed proteins are the 12?S globulins (cruciferin) and the 2 2?S albumins (napin), which are localized to PSVs (protein storage vacuoles), and the oleosins, which are integral proteins of the oil-body membrane. Salt-soluble globulins are widely distributed in dicotyledons, monocotyledons and gymnosperms, and comprise the trimeric and predominantly glycosylated 7? S vicilins and the hexameric and generally non-glycosylated 11C12?S legumins [1]. The three-dimensional structures of several vicilin-type proteins, namely phaseolin [2], canavalin [3], -conglycinin [4] and germin [5], and the legumin-type proglycinin A1aB1b homotrimer [6] and glycinin A3B4 homohexamer [7], have revealed that seed storage globulins share striking similarities in secondary and tertiary structure with the cupin superfamily of prokaryotic and eukaryotic proteins [8,9]. Proteins in this family have been ascribed a wide variety of functions [8,9]. The characteristic cupin domain contains two conserved -barrel motifs separated by a lesser-conserved region comprising two -strands with an intervening loop of variable length. This compact jelly-roll -barrel structure is thought to confer on the cupin domain a high degree of thermal stability and resistance to protease digestion. Temporal and spatial factors influence the accumulation of seed storage proteins in the cotyledon and axis during seed development [10C12]. The latter is exemplified by the observation that antisense suppression of napin expression causes a corresponding increase (±)-Ibipinabant in cruciferin synthesis, thus neutralizing the overall impact on total seed protein content [13]. Cruciferin is synthesized in the rough endoplasmic reticulum as a 50?kDa precursor and is then transported to the PSV by either a Golgi-dependent [14] or Golgi-independent process [15C17]. Regardless of the translocation pathway used, the precursor is processed into a 30?kDa acidic -peptide and a 20?kDa basic -peptide, which are linked together by a single interchain disulfide (±)-Ibipinabant bond. The processing is accompanied by the organization of the mature peptides into a hexameric complex [18C20]. Four VPEs (vacuolar processing enzymes), which are cysteine-type proteinases with specificity toward conserved Asn-Gly motifs in both napin and cruciferin, have been identified in genome encodes more than 1300 types of protein kinases and phosphatases that perform such functions. The mapping of phosphorylation sites in proteins remains a significant challenge, due to the limited abundance of most phosphorylated proteins, low phosphorylation stoichiometry and phosphorylation-site heterogeneity. Considerable effort has been devoted to developing strategies for the enrichment and detection of phosphorylated proteins and peptides, and a variety of MS techniques, employing both electrospray ionization and MALDI (matrixassisted laser desorptionCionization) have been used to identify sites of stable and labile (i.e. histidine) phosphorylation [25C27]. Such methods were recently applied to the characterization of heat-stable phosphorylated proteins in mature seeds [28]. The present study was undertaken with the goal of identifying proteins that are phosphorylated during seed.