Translational Modification of an 18 Kilodalton Polypeptide by Spermidine in Rice Cell Suspension Cultures

Abstract

When rice (Oryza sativa) cell suspension cultures are grown in the presence of [terminal methylenes-3H]spermidine, label is incorporated in a single polypeptide with a molecular mass of 18 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of cell cultures with polyamine biosynthesis inhibitors difluoromethylarginine and difluoromethylornithine, resulted in increased incorporation of the label into the 18 kilodalton polypeptide. In cells in which protein synthesis was arrested by cycloheximide, no label was detected in the 18 kilodalton polypeptide, suggesting a requirement for de novo protein synthesis.The diamine Put' and polyamines Spd and Spm are ubiquitous in living cells and appear to play an important role in plant grow-,h and development (6,8,21,22). Considerable evidence indicates that polyamines regulate such basic plant processes as cell division, morphogenesis, senescence, and responses to stress (6,8). One mechanism by which such regulation might be achieved involves polyamine binding to specific regulatory proteins. An enzyme that catalyzes covalent binding of polyamines to proteins, transglutaminase, has indeed been reported in plants (9).Recent evidence suggests a possible role for polyamines, especially Spd, in reproductive differentiation in various organisms (2,7,10,11,15 eluted with a protein fraction having a molecular mass in excess of 45,000 in oat and petunia protoplasts (14). Because there was a large difference in the relative molecular mass in the polypeptides from tobacco thin-layer culture and oat and petunia protoplasts, a need exists to establish unambiguously the size of proteins that are labeled by Spd, as was also suggested by Mizrahi et al. (14).Anther-derived rice cell suspension cultures (19) offer a good model system for important agricultural crops, particularly the cereals, for metabolic studies. Previously, Schaeffer and Sharpe (19) recovered rice mutants from tissue cultures with inhibitor selection techniques. From these tissue cultures, plants were regenerated which had higher lysine and protein levels than the corresponding controls (20). Using two ofthese rice cell lines (Calrose 76 and the lysine-rich 4C mutant), we identify an 18 kD polypeptide that is translationally modified by Spd and influenced by endogenous polyamine levels. MATERIALS AND METHODS Plant

Identification of Posttranslationally Modified 18-Kilodalton Protein from Rice as Eukaryotic Translation Initiation Factor 5A

“…Using anther-derived rice (Oryza sativa L.) cell-suspension cultures, we have identified an 18-kD protein that is posttranslationally modified by spermidine, and the modification appears to be influenced by endogenous polyamine levels (Mehta et al, 1991a). We now identify the modified residue as the unusual amino acid hypusine and demonstrate a direct precursorial role for spermidine in its biosynthesis.…”

“…Preparation from suspension-culture cells of a prc'tein fraction containing the radiolabeled 18-kD protein for use as a tracer in the purification was camed out essentially as described by Mehta et al (1991a). The 40 to 75% ammonium sulfate fraction employed as the tracer was found to contain essentially all of its radioactivity in the 18-kD protein.…”

“…Radiolabeled cells were harvested, homogenized, and analyzed by one-and twodimensional gel electrophoresis as previously described (Mehta et al, 1991a(Mehta et al, , 1991b. The 18-kD protein band from one-dimensional polyacrylamide gels, as well as the single spot from two-dimensional gel electrophoresis, were excised and analyzed for the modified amino acid residue.…”

“…This eluate was taken to dryness under vacuum and the residue was dissolved in water. Dansylation was conducted on aliquots of these solutions as described previously (Mehta et al, 1991a). The samples were filtered through a nylon 66 membrane (pore size 0.22 pm; Rainin) prior to HPLC analyses.…”

“…The identity of hypusine was determined by mixing the dansyl derivative of hypusine with dansylated samples of HPLC fractions and running the resulting mixture on HPLC using the same column and solvent gradient as for nondansylated samples. Hypusine derivatives were detecied by an on-line fluorescence spectrophotometer as described previously (Mehta et al, 1991a).…”