Among the many important changes which occur in the physiology of serum- starved cells is the accelerated lysosomal proteolysis of many cytoplasmic proteins. It has been demonstrated that ferritin is spared this fate in serum-deprived K562 erythroleukemia cells. The absence of a specific recognition sequence which codes for protein uptake by lysosomes during serum starvation may account for the phenomenon. The proposed work will directly test this hypothesis by detailed evaluation of ferritin physiology with serum deprivation and by the creation of site-directed mutants of ferritin which can be used in cell transfection experiments. The normal steady-state uptake of ferritin into lysosomes may mediated by a specific receptor protein on the organelle. They will construct modified ferritin cDNAs which contain the amino acid sequence KFERQ which enhances the uptake of ribonuclease A from cytoplasm into lysosomes with serum deprivation, determine whether any relationship exists between ascorbic acid and serum starvation as concerns ferritin degradation, and use isolated ferritin and lysosomes to explore the effects of serum starvation on variables important to ferritin turnover such as cytoplasmic aggregation of the protein and the interaction of ferritin aggregates with lysosomes. Also, purification of a lysosomal membrane receptor for ferritin will be attempted.
