Characterization of hypusine-containing 21,000-dalton protein in Neurospora crassa

The deoxyhypusine/hypusine-containing 21,000-dalton protein was labeled by [3H]spermidine both in vitro and in vivo in polyamine depleted Neurospora crassa arg-12 ota aga and aga mutants. The in vitro labeling of the 21,000-dalton protein could be dramatically stimulated by NAD+ and NADP+ but not by FMN or FAD. The in vitro labeled 21,000-dalton protein contained the radioactivity of deoxyhypusine and hypusine with a ratio of 2 to 1. The in vivo labeled protein resulted only in hypusine. Three isoform structures of the in vitro labeled 21,000-dalton protein were found with pl values ranging from 5.2 to 6.5. In contrast, the 21,000-dalton protein metabolically labeled in vivo with [3H]ornithine gave only one spot with a pl value of 3.5. The deoxyhypusine-modifying enzyme is heat labile and has a half life of 40 min. The complete inhibition of deoxyhypusine-modifying enzyme by NEM and pCMBS suggests that the sulfhydryl group is required for the activity. The unmodified 21,000-dalton protein is heat stable and has a half life of more than 5 hrs. In order to determine the functional role of hypusine modification of 21,000-dalton protein, a protein synthesizing cell-free system has been established to translate endogenous mRNA to discrete polypeptides ranging up to 200,000-dalton. The inhibitory effect of spermidine on protein synthesis in cell-free system of unmodified 21,000-dalton protein is in contrast to the stimulatory effect on protein synthesis in cell-free system of modified protein, suggesting that unmodified 21,000-dalton protein might be responsible for the inhibitory effect of spermidine on protein synthesis in cell-free system containing unmodified 21,000-dalton protein