AN AMPHIPHILIC LIPID-BINDING DOMAIN INFLUENCES THE TOPOLOGY OF A SIGNAL-ANCHOR SEQUENCE IN THE MITOCHONDRIAL OUTER-MEMBRANE

Mas70p is targeted and inserted into the mitochondrial outer membrane in the N-in-C-cyto orientation, via an NH2-terminal signal-anchor sequ ence. The signal-anchor is comprised of two domains: an NH2-terminal h ydrophilic region which is positively charged (amino acids 1-10), foll owed by the predicted transmembrane segment (amino acids 11-29). Subst itution of the NH2-terminal hydrophilic domain with a matrix-targeting signal caused the signal-anchor to adopt the reverse orientation in t he membrane (N-cyto-C-in). This substitution resulted in an increase i n the net positive charge of the hydrophilic region, from +4 to +8. In contrast to the endoplasmic reticulum and the bacterial inner membran e, where the net positive charge is an important determinant in confer ring protein topology in the lipid bilayer, we show here that the reve rsal of the Mas70p signal-anchor was not due to differences in the num ber and positions of basic amino acids in the hydrophilic domain. Howe ver, a reduction in the hydrophobic moment of predicted amphiphilic he lices containing an arginine, obtained by converting the apolar amino acids flanking the arginine to polar residues, caused the otherwise N- cyto-C-in signal-anchor to re-adopt the original N-in-C-cyto orientati on of Mas70p. The reduced hydrophobic moment at the NH2-terminus signi ficantly reduced the ability of this domain to bind to synthetic lipos omes whose lipid composition reflected that of the outer membrane. The se results identify amphiphilicity as an important determinant in caus ing retention of the NH2-terminus of a mitochondrial signal-anchor on the cytosolic side of the outer membrane. In addition to potential int eractions between this domain and cytosolic-exposed components of the import machinery, this retention may result as well from interaction o f the NH2-terminus with the surrounding membrane surface.