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© The Rockefeller University Press, 0021-9525/1999//1419 $5.00
The Journal of Cell Biology, Volume 145, Number 7, , 1999 1419-1433

Regular Articles

A Complex Web of Signal-dependent Trafficking Underlies the Triorganellar Distribution of P-Selectin in Neuroendocrine PC12 Cells



Anastasiya D. Blagoveshchenskaya, Eric W. Hewitt, and Daniel F. Cutler

MRC Laboratory for Molecular Cell Biology, and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom

By analyzing the trafficking of HRP–P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP–P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A–sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.

Key Words: P-selectin • PC12 cells • lysosomes • dense core granules • synaptic-like microvesicles

Abbreviations used in this paper: AP, adaptor protein; ARF1, adenosine ribosylation factor 1; BFA, Brefeldin A; DCG, dense core granules; iDCG, immature dense core granules; mDCG, mature dense core granules; GTI, granule targeting index; HB, homogenization buffer; LTI, lysosomal targeting index; NAGA, N-acetyl-β-D-glucosaminidase; RSO, regulated secretory organelles; SLMV, synaptic-like microvesicles; SLMV-TI, SLMV targeting index; SSV, small synaptic vesicles; Trn, transferrin; TrnR, Trn receptor; VAMP, vesicle-associated membrane protein.


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