The C99 Domain Of The Amyloid Precursor Protein Resides In The Disordered Membrane Phase

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer's disease. The cleavage of APP by beta-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by alpha-secretase to yield amyloid-beta (A beta) peptides are essential steps in this pathway. Biochemical evidence suggests that amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquidordered phase membrane rafts. However, direct evidence that C99 preferentially associates with these rafts has remained elusive. Here, we tested this by quantifying the affinity of C99GFP for raft domains in cell-derived giant plasma membrane vesicles (GPMVs). We found that C99 was essentially excluded from ordered domains in vesicles from HeLa cells, undifferentiated SH-SY5Y cells, or SH-SY5Y-derived neurons; instead, similar to 90% of C99 partitioned into disordered domains. The strong association of C99 with disordered domains occurred independently of its cholesterol-binding activity or homodimerization, or of the presence of the familial Alzheimer disease Arctic mutation (APP E693G). Finally, through biochemical studies we confirmed previous results, which showed that C99 is processed in the plasma membrane by alpha-secretase, in addition to the well-known alpha-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying that either i) amyloidogenic processing of the protein occurs in disordered regions of the membrane, ii) processing involves a marginal subpopulation of C99 found in rafts, or iii) as-yetunidentified protein-protein interactions with C99 in living cells drive this protein into membrane rafts to promote its cleavage therein.