Figure 4.

Effect of different levels of expression on targeting of HRP-P-selectin chimeras to late endosomes. (A) Titration of HRP expression. PC12 cells were transiently transfected by electroporation using increasing amounts of cDNA for ssHRP^P-selectin (WT), ssHRP^{P-selectinKCPL} (KCPL), or ssHRP^{P-selectinDPSP} (DPSP). Cells were washed with HB, scraped, and homogenized. HRP activity (shown in arbitrary units [A.U.]) was measured in each homogenate and normalized to total protein within the homogenate. The name of chimera and the amount of DNA used (starting from 0.5 μg) are shown along the abscissa. (B) Efficiency of late endosomal targeting at different expression levels. PC12 cells transiently expressing ssHRP^P-selectin, ssHRP^{P-selectinKCPL}, or ssHRP^{P-selectinDPSP} at different levels were processed by subcellular fractionation for isolation of late endosomes as described in the legend for Figure 3. The amount of HRP activity (shown in A.U. along the ordinate) within the late endosomal peak was measured, divided by that in the homogenate, and normalized for NAGA recovery. The amount of DNA used is shown before the name of the chimera. Note that the level of constitutive targeting of tailless ssHRP^{P-selectin763} to late endosomes, which usually comprises 25% of the level found for the wild-type chimera, has not been subtracted from the values of targeting efficiency for chimeras analyzed in this experiment.