Proteolytic Processing of Amyloid-β Precursor Protein by Secretases Does Not Require Cell Surface Transport

Mikhail Khvotchev,Thomas C Südhof

doi:10.1074/jbc.m408474200

Abstract

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.

Highlights

Amyloid-␤ precursor protein (APP)1 of Alzheimer’s disease is a ubiquitous membrane protein that is physiologically processed by site-specific proteolysis [1,2,3,4]
Stage-specific Inhibitors of Secretory Membrane Traffic Based on Syntaxin 1A—syntaxin 1A is a SNARE protein of the plasma membrane that mediates Ca2ϩ-induced synaptic vesicle exocytosis and possibly other forms of exocytosis [46, 47]. syntaxin 1A is composed of an N-terminal three-helical Habc domain, a SNARE motif that participates in core complex formation with other SNARE proteins, and a C-terminal transmembrane region (Fig. 1A)
We found that full-length syntaxin 1A1–288 or C-terminally truncated syntaxin 1A1–243 severely inhibited constitutive human growth hormone (hGH) secretion in HEK293 and HeLa cells, whereas truncated syntaxin 1A1–236 and syntaxin 1A1–230, which contain only 7 or 13 fewer residues, respectively, than syntaxin 1A1–243, had no significant effect (Fig. 1B and data not shown)

Summary

Introduction

Amyloid-␤ precursor protein (APP)1 of Alzheimer’s disease is a ubiquitous membrane protein that is physiologically processed by site-specific proteolysis [1,2,3,4]. We 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 transGolgi traffic and exocytosis.

Results

Conclusion