Newly formed, but not mature, FECH protein is susceptible to regulation by iron availability. (A) To test for changes in the instantaneous synthesis rate of FECH protein under iron-depleted conditions, MEL cells were differentiated for 24 hours in the presence or absence of DFO, and harvested immediately after a rapid 10-minute pulse with ³⁵S-Cys and ³⁵S-Met. Radiolabeled FECH was visualized by autoradiography after immunoprecipitation and SDS-PAGE (top panel), whereas total protein levels were measured by Western blot (bottom panel). (B) The effect of iron limitation on mature, Fe-S cluster-containing FECH was assessed by metabolic labeling of cells for 1 hour with ³⁵S-Cys and ³⁵S-Met 4 hours before the onset of 24 hours of differentiation and DFO treatment. After 24-hour incubation, cells were harvested and analyzed for radiolabeled and total protein levels as in panel A. (C) A pulse-chase experiment was performed to follow the fate of newly formed FECH protein during normal and iron-limited growth conditions. After 24-hour differentiation in the presence or absence of DFO, cells were pulsed for 40 minutes with ³⁵S-Cys and ³⁵S-Met, followed by incubation for various periods of time in differentiation medium with or without DFO. A representative autoradiogram is shown; the results of 2 experiments are quantified and plotted in panel D.