Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells

Abstract

The Alzheimer beta/A4 amyloid precursor protein (APP) can be proteolytically processed by at least two separate pathways in PC12 cells: chloroquine-insensitive secretory cleavage and chloroquine-sensitive intracellular degradation, presumably in the endosomal/lysosomal system. To further investigate the possibility of APP processing in the endosomal/lysosomal system, we have examined whether APP is present in clathrin-coated vesicles (CCVs), which mediate the transport of many proteins to the endosomal compartment. Using a procedure derived from established protocols for the purification of CCVs from mammalian organs, we obtained from PC12 cells highly purified CCVs that displayed the same morphological features as described for CCVs purified from other sources. The CCVs were enriched in full-length mature (fully post-translationally modified) forms of APP, as well as in the carboxyl-terminal APP fragment produced by the secretory cleavage pathway. As CCVs are known to be involved in only two intracellular pathways (trafficking from the plasma membrane to early endosomes, and from the trans-Golgi network to late endosomes/prelysosomes), these findings provide direct evidence that APP is transported to the endosomal/lysosomal system. Furthermore, the presence in CCVs of the carboxyl-terminal fragment resulting from APP secretory cleavage suggests that APP secretory processing occurs in a pre-CCV compartment.