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 sequence. The signal-anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10), followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NH2-terminal hydrophilic domain with a matrix-targeting signal caused the signal-anchor to adopt the reverse orientation in the membrane (N(cyto)-C(in)). This substitution resulted in an increase in the net positive charge of the hydrophilic region, from +4 to +8. In contrast to the endoplasmic reticulum and the bacterial inner membrane, where the net positive charge is an important determinant in conferring protein topology in the lipid bilayer, we show here that the reversal of the Mas70p signal-anchor was not due to differences in the number and positions of basic amino acids in the hydrophilic domain. However, a reduction in the hydrophobic moment of predicted amphiphilic helices 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) orientation of Mas70p. The reduced hydrophobic moment at the NH2-terminus significantly reduced the ability of this domain to bind to synthetic liposomes whose lipid composition reflected that of the outer membrane. These results identify amphiphilicity as an important determinant in causing retention of the NH2-terminus of a mitochondrial signal-anchor on the cytosolic side of the outer membrane. In addition to potential interactions between this domain and cytosolic-exposed components of the import machinery, this retention may result as well from interaction of the NH2-terminus with the surrounding membrane surface.