A complex web of signal-dependent trafficking underlies the triorganellar distribution of P-selectin in neuroendocrine PC12 cells

By analyzing the trafficking of HRP-P-selectin chimeras in which the lumena l domain of P-selectin was replaced with horseradish peroxidase, we determi ned the sequences needed for targeting to synaptic-like microvesicles (SLMV ), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. W ithin the cytoplasmic domain of P-selectin, Tyr777 is needed for the appear ance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and (DPSP789)-D-786) which are responsible for movement through endosomes en r oute to the SLMV. Leu768 also mediates transfer from early transferrin (Trn )-positive endosomes to the lysosomes; i.e., operates as a lysosomal target ing signal. Furthermore, SLMV targeting of HRP-P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be deli vered to SLMV directly from the plasma membrane, is a Brefeldin A-sensitive pro cess. Together, these data are consistent with a model of SLMV biogene sis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a conc omitant increase in lysosomal delivery, illustrating the entwined relations hips between routes leading to regulated secretory organelles (RSO) and to lysosomes.