Translocation of N-terminal tails across the plasma membrane.

Abstract

Previously we have shown that the first hydrophobic domain of leader peptidase (lep) can function to translocate a short N‐terminal 18 residue antigenic peptide from the phage Pf3 coat protein across the plasma membrane of Escherichia coli. We have now examined the mechanism of insertion of N‐terminal periplasmic tails and have defined the features needed to translocate these regions. We find that short tails of up to 38 residues are efficiently translocated in a SecA‐ and SecY‐independent manner while longer tails are very poorly inserted. Efficient translocation of a 138 residue tail is restored and is Sec‐dependent by the addition of a leader sequence to the N‐terminus of the protein. We also find that while there is no amphiphilic helix requirement for N‐terminal translocation, there is a charge requirement that is needed within the tail; an arginine and lysine residue can inhibit or completely block translocation when introduced into the tail region. Intriguingly, the membrane potential is required for insertion of a 38 residue tail but not for a 23 residue tail.

N-terminal Tail Export from the Mitochondrial Matrix

“…This was found to be in agreement with the positive-inside rule of prokaryotic membrane protein topology, where positively charged amino acids tend to be more prevalent in the cytoplasmic than periplasmic regions flanking the transmembrane domains (7)(8)(9)(10)(11). Furthermore, introduction of positively charged amino acid residues into the N-tails of these bacterial proteins led to an inhibition of their export from the cytoplasm (12,13).…”

“…preSu9(1-112) E70K,E77K,R111D,N112D -DHFR (left panel) and preSu9(1-112)-DHFR (right panel) were imported into isolated mitochondria in the presence of EDTA ϩ o-phe at 12°C for the times indicated. Mitochondria were converted to mitoplasts, and where indicated, treated with proteinase K, which gave rise to the matrix-protected N-terminal 12-kDa fragment, f 12 . p, precursor.…”

“…As the reversal of the topology would result in the N-terminal domain being localized in the matrix (N in -C out ), we performed import under conditions where MPP processing was inhibited by the addition of the chelators EDTA/o-phe. By doing so, an N-terminal fragment located in the matrix would be large enough to be identified easily by SDS-polyacrylamide gel electrophoresis (because of the presence of the uncleaved presequence) (12 kDa, f 12 ).…”

Oxa1p, an essential component of the N-tail protein export machinery in mitochondria

“…In mitochondria and bacteria, this export step is thought to be facilitated by an until now unidentified translocation machinery (4,5). Export of N-terminal tails and of prokaryotic polytopic proteins has been reported to occur in a Secindependent but membrane potential-dependent mechanism (5)(6)(7)(8)(9). Involvement of a bacterial Sec-type machinery appears to be unlikely as there is no indication, at least in yeast, of the existence of a mitochondrial Sec machinery (10).…”

Conserved Negative Charges in the Transmembrane Segments of Subunit K of the NADH:Ubiquinone Oxidoreductase Determine Its Dependence on YidC for Membrane Insertion

“…It could therefore be the inability of the IMVs to generate a PMF that causes the observed reduction in NuoK insertion in YidC Ϫ IMVs. The PMF has been shown to play an essential role in the insertion of membrane proteins such as FtsQ (10) and the phage coat proteins M13 and Pf3 (30,31). The role of the PMF in the insertion of in vitro synthesized NuoK was examined by the addition of the ionophores nigericin and valinomycin, which collapse the PMF (Fig.…”