Role of the sterol carrier protein-2 N-terminal membrane binding domain in sterol transfer.

Abstract

Previous studies showed that the N-terminal 32 amino acids of sterol carrier protein-2 ((1-32)SCP(2)) comprise an amphipathic alpha-helix essential for SCP(2) binding to membranes [Huang et al. (1999) Biochemistry 38, 13231]. However, it is unclear whether membrane interaction of the (1-32)SCP(2) portion of SCP(2) is in itself sufficient to mediate intermembrane sterol transfer, possibly by altering membrane structure. In this study a fluorescent sterol exchange assay was used to resolve these issues and demonstrated that the SCP(2) N-terminal peptide (1-32)SCP(2) did not by itself enhance intermembrane sterol transfer but potentiated the ability of the SCP(2) protein to stimulate sterol transfer. Compared with SCP(2) acting alone, (1-32)SCP(2) potentiated the sterol transfer activity of SCP(2) by increasing the initial rate of sterol transfer by 2.9-fold and by decreasing the half-time of sterol transfer by 10-fold (from 11.6 to 1.2 min) without altering the size of the transferable fractions. The ability of a series of SCP(2) mutant N-terminal peptides to potentiate SCP(2)-mediated sterol transfer was directly correlated with membrane affinity of the respective peptide. N-Terminal peptide (1-32)SCP(2) did not potentiate intermembrane sterol transfer by binding sterol (dehydroergosterol), altering membrane fluidity (diphenylhexatriene) or membrane permeability (leakage assay). Instead, fluorescence lifetime measurements suggested that SCP(2) and (1-32)SCP(2) bound to membranes and thereby elicited a shift in membrane sterol microenvironment to become more polar. In summary, these data for the first time showed that while the N-terminal membrane binding domain of SCP(2) was itself inactive in mediating intermembrane sterol transfer, it nevertheless potentiated the ability of SCP(2) to enhance sterol transfer.