Genetic mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early-onset familial AIzheimer's disease (FAD). Extensive studies in the past few years focusing on the mechanisms by which mutant PS promote AD pathogenesis have demonstrated that PS1 is a key component of gamma-secretase complex, an unusual protease catalyzing the intramembrane cleavage of amyloid precursor protein (APP) and generating pathogenic small peptide Abeta. Nascent full length PS1 polypeptides undergo endoproteolytic cleavage to produce N-terminal and C-terminal fragments (NTF and CTF) which associate with each other to form functional heterodimer. Early studies about PS1 metabolism suggest that unknown cellular factors tightly regulate biogenesis of PS1 heterodimer. However, identity of these limiting factors as well as other members of gamma-secretase complex has remained elusive. Through biochemical and genetic approaches, recent discoveries of three essential proteins (Nct, PEN-2 and APH-1) for gamma-secretase activity, provide us candidates for those hypothesized factors. Indeed, three of them have been demonstrated to be components of high molecular weight gamma-secretase complex, while their precise functions are unclear. My preliminary results demonstrated the regulatory roles of PEN-2 and APH-1 in PS1 cleavage and Nct maturation. This proposal is designed to further investigate the biological functions of PEN-2 and APH-1 in regulating PS1/gamma-secretase complex formation via examining PEN-2 and APH-1 structure/function relationship (Specific Aim 1), their impact on protein trafficking (Specific Aim 2), as well as protein folding and intermolecular interactions (Specific Aim 3) of PS1 and Nct.