Regulation of biosynthesis of hypusine in Chinese hamster ovary cells. Evidence for eIF-4D precursor polypeptides.

The effects of spermidine depletion and the effects of translation inhibition on hypusine biosynthesis were studied in Chinese hamster ovary cells. Upon depletion of cellular spermidine by treatment with DL-alpha-difluoromethylornithine for 42 h or longer, both the rate of deoxyhypusine + hypusine synthesis and the content of protein-bound hypusine were significantly reduced. Cycloheximide caused complete inhibition of deoxyhypusine + hypusine synthesis in untreated cells and in cells in which the spermidine level was reduced to approximately 10% that of the untreated cells by incubation with DL-alpha-difluoromethylornithine for 24 h. In contrast, the initial synthesis of deoxyhypusine + hypusine was not arrested by cycloheximide in cells depleted of spermidine by treatment with DL-alpha-difluoromethylornithine for 42 h. The initial rate of deoxyhypusine + hypusine production in these spermidine-depleted cells increased 5- to 10-fold when the cellular spermidine level was restored through addition of this polyamine to the culture medium. These findings suggest that in control Chinese hamster ovary cells and in cells containing approximately 10% of the control level of spermidine, deoxyhypusine + hypusine synthesis occurs during or immediately after eukaryotic initiation factor 4D precursor translation. However, in cells during depletion of spermidine, there is an accumulation of an eukaryotic initiation factor 4D precursor that contains no hypusine or deoxyhypusine, and in these cells deoxyhypusine + hypusine synthesis is mainly regulated by the cellular level of spermidine.