Amyloid precursor protein, although partially detergent-insoluble in mousecerebral cortex, behaves as an atypical lipid raft protein

Lipid rafts are regions of the plasma membrane that are enriched in cholest erol, glycosphingolipids and acylated proteins, and which have been propose d as sites for the proteolytic processing of the Alzheimer's amyloid precur sor protein (APP). Lipid rafts can be isolated on the basis of their insolu bility in Triton X-100 at 4 degrees C, with the resulting low-density, dete rgent-insoluble glycolipid-enriched fraction (DIG) being isolated by flotat ion through a sucrose density gradient. The detergent-insolubility of APP i n mouse cerebral cortex relative to a variety of DIG marker proteins (arkal ine phosphatase, flotillin, F3 protein and prion protein) and non-DIG prote ins (alkaline phosphodiesterase I, aminopeptidase A and clathrin) has been examined. Alkaline phosphatase, flotillin, F3 protein and the prion protein were present exclusively in the DIG region of the sucrose gradient over a range of protein/detergent ratios used to solubilize the membranes and disp layed a characteristic enrichment in the low-density fraction as the protei n/detergent ratio was decreased. In contrast, most of the APP, alkaline pho sphodiesterase I, aminopeptidase A and clathrin was effectively solubilized at all of the protein/detergent ratios examined. However, a minor proporti on of these latter proteins was detected in DIGs at levels which remained c onstant irrespective of the protein/detergent ratio. When DIGs were isolate d from the sucrose gradients and treated with excess Triton X-100, both the DIG marker proteins and APP, alkaline phosphodiesterase I and clathrin wer e predominantly resistant to detergent extraction at 37 degrees C. These re sults show that, although a minor proportion of APP is present in DIGs, whe re it is detergent-insoluble even at 37 degrees C, it behaves as an atypica l lipid raft protein and raises questions as to whether lipid rafts are a s ite for its proteolytic processing.