Cleavage of amyloid-beta precursor protein (APP) by alpha-,beta-, and gamma-secretases releases an extracellular fragment called APPS, small Abeta peptides, and a short APP intracellular domain that may provide a transcriptional signal analogous to the Notch intracellular domain. Notch cleavage is activated by extracellular ligands on the cell surface, but the cellular localization of APP cleavage remains unclear. We now show that in transfected cultured cells, the plasma membrane SNARE protein syntaxin 1A, when expressed as a full-length protein, disrupts the Golgi apparatus and blocks trans-Golgi traffic and exocytosis. In contrast, truncated syntaxin 1A1-243 selectively abolishes exocytosis but has no apparent effect on trans-Golgi traffic or Golgi structure, whereas further truncated syntaxins 1A1-236 and 1A1-230 have no effect on either exocytosis or Golgi traffic. Using these syntaxin 1A fragments, we demonstrated that blocking trans-Golgi traffic greatly impairs APP cleavage and AICD-dependent nuclear signaling, whereas blocking exocytosis alone does not affect either process, even though secretion of APPS and Abeta40 peptide is abolished. Our data suggest that, different from Notch, cleavage of APP is independent of cell surface regulation by extracellular ligands but may be controlled by intracellular signaling.