Translocation of inserted foreign epitopes by a channelforming protein

Abstract

Certain bacterial protein toxins are able to insert themselves into, and at least partially across, lipid bilayer membranes in the absence of any auxiliary proteins, by using unknown mechanisms to overcome the high energy barrier presented by the hydrophobic bilayer core. We have previously shown that one such toxin, colicin Ia, translocates a large, hydrophilic part of itself completely across a lipid bilayer in conjunction with the formation of an ion-conducting channel. To address the question of whether the colicin can translocate any arbitrary amino acid sequence, we have altered the translocated segment by inserting, singly, two different foreign epitopes. Colicins containing either epitope retain significant bactericidal activity and form channels of normal conductance in planar bilayers. Furthermore, antibodies added on the side of the bilayer opposite that to which the colicin was added interact specifically with the corresponding epitopes, producing an inhibition of channel closing. Thus, the inserted epitopes are translocated along with the rest of the segment, suggesting that a surprisingly small part of colicin Ia, located elsewhere in the molecule, acts as a nonspecific protein translocator.

Adventures in Membrane Protein Topology

“…the membrane surface. This suggests that the protein unfolds and spreads out on the surface of the membrane, which is in agreement with the previous proposals for colicin A (26 -28) E1 (29,31,(47)(48)(49) and I a (25,(41)(42)(43). The em max values for W-355 and W-367 single Trp proteins indicate that both helices 1 and 2 are membrane-associated under the conditions used in this study but that the corresponding Trp residues are in a more polar environment than those found within the hydrophobic anchor domain of the channel protein (Table I).…”

“…The em max data for the contingent of single Trp mutant and WT proteins strongly suggest that the polarity of the Trp residues (at 11 of the 14 different Trp sites within the peptide) increases when the protein associates with (51); biotinylation (41,42,53); epitope mapping (43); saturation mutagenesis (55); protease accessibility (28,47); depth-dependent fluorescence quenching (29,31); cysteine-scanning mutagenesis (56); FRET (27); CD, Fourier transform infrared radiation, and differential scanning calorimetry (48,49); time-resolved spin labeling (57); and acrylamide quenching and other fluorescence data presented in this work. The initial surface-bound structure adopts at least two states with state 2 being the most populated.…”

“…The segments designated TM1, 2, 3, and 4 represent TM segments whose existence has been implied from various topological mapping experiments (see "Discussion"). The segment TLP refers to the translocating segment that has been located on the trans side of the membrane by biotinylation and epitope-mapping experiments (41)(42)(43). The membrane disposition of this peptide segment in the open channel state is highly controversial and is currently being investigated.…”

Swimming through the hydrophobic sea: New insights in protein translocation

“…In a paper appearing in this issue of Proceedings, Jakes et al Jakes et al (1) found that two inserted peptide epitopes are indeed dragged across the lipid bilayer. They chose the site of insertion for these epitopes based on the crystal structure of colicin Ia in solution (10), placing the heterologous sequences in the loop connecting two ␣-helices in the translocated segment.…”

Translocation of a functional protein by a voltage-dependent ion channel

“…7). The observation that localized mutations in this transported segment, including insertions of up to 12 residues, do not block its movement (8) suggested that this system might be recruited to translocate suitably attached exogenous proteins.…”