BIOPHYSICAL STUDIES OF MEMBRANE TOPOLOGY OF HUMAN PRESENELIN-1

Purpose: Alzheimer's disease (AD) afflicts ~5 million Americans. Amyloid (Abeta) plaques accumulate in the brain of patients during AD leading to neurodegeneration. Presenelin-1 (PS1), a trans-membrane protein, acts as the catalytic subunit of gamma-secretase enzyme to cleave amyloid precursor protein (APP) and produce Abeta peptides. Mutations in the PS1 gene have been linked to the pathogenesis of early onset of familial AD (FAD). Recently, it has been reported that PS1 also acts as Ca2+ leak channel on the membrane of endoplasmic reticulum (ER). Currently there are divergent views in the literature on the subunit association and the correct membrane topology of PS1 protein. Considering the several physiological functions and the critical role of PS1 in the pathogenesis of AD, we sought to investigate the membrane topology of PS1 protein through various biophysical studies. Methods: Neuroblastoma (SK-N-SH) cells expressing PS1 protein with NH2-terminal tagged yellow fluorescent protein (YFP-PS1) and COOH-terminal tagged cyano fluorescent protein (PS1-CFP) were used as a model in our studies. Membrane localization and subunit association of PS1 were determined by biophysical assays. Expression and colocalization of YFP-PS1 and PS1-CFP proteins were assessed by confocal imaging. Localization of the N-terminal and C-terminal of PS1 was assessed by fluorescence correlation spectroscopy (FCS). Finally subunit aggregation of PS1 protein was determined by Forster Resonance Energy Transfer (FRET) assay. Results: When cells expressing both PS1-CFP and YFP-PS1 proteins were independently excited and imaged, their respective fluorescence overlapped suggesting co-expression and co-localization of the PS1 subunits. The diffusion coefficient of the PS1 protein in the transmembrane was the same (~0.15µm2/s) when FCS was measured in cells with and without 80% glycerol in 1XPBS indicating that the NH2 and COOH termini are facing the cytosolic side of the plasma-membrane. The quench in the fluorescence lifetime of CFP (donor) in the presence of YFP (acceptor) in FRET assay demonstrates the protein-protein interaction between PS1-CFP and YFP-PS1. Conclusions: Both YFP-PS1 and PS1-CFP chimeric proteins are expressed on the plasma membrane and intracellular membranes with NH2-terminal and COOH-terminal oriented towards the cytosolic side of the membrane. PS1 is a dynamic transmembrane protein which associates as dimer or multimer to form ERCa2+ channel and thus regulates intracellular calcium signaling.