Presenilin (PS1͞PS2) is a major component of ␥-secretase, the activity that mediates proteolysis of -amyloid precursor protein to generate -amyloid (A). Here we demonstrate that PS1, through its loop region, binds to phospholipase D1 (PLD1), thereby recruiting it to the Golgi͞trans-Golgi network. Overexpression of wildtype PLD1 reduces A generation. Conversely, down-regulation of endogenous PLD1 by small hairpin RNA elevates A production. The A-lowering effect of PLD1 is independent of its ability to promote vesicular budding of -amyloid precursor protein. The data indicate that overexpression of PLD1 decreases, and downregulation of PLD1 increases, the catalytic activity, and the association of the subunits, of ␥-secretase.-amyloid precursor protein ͉ ␥-secretase complex activity ͉ negative regulator ͉ protein interaction ͉ trans-Golgi network M ost early-onset, familial Alzheimer's disease (FAD) cases are linked to mutations in presenilin (PS1͞PS2) genes (1). PS is a major component of ␥-secretase, the activity that mediates proteolysis of -amyloid precursor protein (APP) to generate -amyloid (A) (2, 3). Expression of autosomal dominant variants of PS results in increased A 42 production, leading to amyloid plaque deposition in the brains of patients and early onset of disease (3-5). PS interacts with three other membrane proteins (nicastrin, APH1, and PEN2) to form high-molecularweight complexes with ␥-secretase activity that cleave type I membrane proteins including APP and Notch-1 (5-11). The demonstration that coexpression of these four proteins in Saccharomyces cerevisiae, which lacks endogenous ␥-secretase activity, is sufficient to reconstitute ␥-secretase activity (12) confirms that these four molecules are the major core components of the ␥-secretase complex. Much attention in the Alzheimer's disease field is being devoted to the characterization of ␥-secretase and identification of factors that regulate its activity.The lipid composition of cellular membranes appears to regulate the production of A species (13,14). For example, it was reported that phosphatidylcholine and sphingomyelin increase ␥-secretase activity without changing cleavage specificity (13). Interestingly, disordered metabolism of membrane phospholipids has been reported in Alzheimer's disease (15,16). In the present study, a possible role for phospholipase D1 (PLD1), a phospholipid-modifying enzyme, in regulating ␥-secretase activity has been explored.
Results
PS1, Through Its Loop Region, Interacts with and Recruits PLD1 to the
Golgi͞Trans-Golgi Network (TGN).To investigate the modulation of PS1-regulated APP processing and trafficking (5-11, 17-21), a variety of factors known to be important in regulating protein trafficking were examined, including Rab11, RhoA, and PLD family and Arf family members. Among these factors, only PLD1 showed an interaction with PS1. The interaction between PS1 and PLD1 was demonstrated by coimmunoprecipitation (co-IP) from wild-type (wt) ES cell lysates (Fig. 1a). To confirm the specif...
