Insertion of an uncharged polypeptide into the mitochondrial inner membrane does not require a trans-bilayer electrochemical potential: effects of positive charges.

Mitochondria with a ruptured outer membrane exhibited impaired import into this membrane of an outer membrane fusion protein containing the signal-anchor sequence of Mas70p. However, the Mas70p signal-anchor efficiently targeted and inserted the protein directly into exposed regions of the inner membrane. Import into the inner membrane was dependent on delta psi and this dependence was due to the presence of the positively-charged amino acids located at positions 2, 7, and 9 of the signal-anchor. In contrast to wild-type signal-anchor, mutants lacking the positively-charged residues mediated import into the inner membrane in both the presence and absence of delta psi. The results suggest two conclusions: (1) delta psi-dependent import of the signal-anchor sequence was due exclusively to an effect of delta psi on the positively-charged domain of the signal-anchor, rather than to an effect of delta psi on a property of the inner membrane import machinery; (2) in the absence of delta psi, the positively-charged domain of the signal-anchor prevented the otherwise import-competent signal-anchor from inserting into the membrane. This suggests that the positively-charged domain leads import across the inner membrane, and that delta psi is required to vectorially clear this domain in order to allow the distal region of the signal-anchor to enter the translocation pathway. The implications of these findings on the mechanism of import into the mitochondrial inner membrane and matrix are discussed.