Steenaart N A, Silvius J R, Shore G C
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Biochemistry. 1996 Mar 26;35(12):3764-71. doi: 10.1021/bi9528053.
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.
Mas70p通过其氨基末端信号锚序列，以N（in）-C（cyto）方向靶向并插入线粒体外膜。该信号锚由两个结构域组成：一个带正电荷的氨基末端亲水区（氨基酸1-10），接着是预测的跨膜片段（氨基酸11-29）。用基质靶向信号取代氨基末端亲水区，导致信号锚在膜中采用反向取向（N（cyto）-C（in））。这种取代导致亲水区的净正电荷从+4增加到+8。与内质网和细菌内膜不同，在这些膜中净正电荷是决定蛋白质在脂质双层中拓扑结构的重要因素，我们在此表明Mas70p信号锚的反向并非由于亲水区碱性氨基酸数量和位置的差异。然而，通过将精氨酸侧翼的非极性氨基酸转化为极性残基，得到的含精氨酸的预测两亲性螺旋的疏水矩降低，导致原本N（cyto）-C（in）的信号锚重新采用Mas70p原来的N（in）-C（cyto）取向。氨基末端疏水矩的降低显著降低了该结构域与脂质组成反映外膜脂质组成的合成脂质体结合的能力。这些结果表明两亲性是导致线粒体信号锚的氨基末端保留在外膜胞质侧的重要决定因素。除了该结构域与输入机制的胞质暴露成分之间的潜在相互作用外，这种保留也可能是由于氨基末端与周围膜表面的相互作用所致。
