Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an z 68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three. key words: voltage-gated channels • streptavidin • His-tag antibody • trypsin • single-channel conductance I N T R O D U C T I O N Colicin Ia belongs to a family of water-soluble bactericidal proteins that consist of three domains: the central R domain and the amino-terminal T domain are responsible for receptor-binding and translocation of the colicin across the outer membrane of the target cell, respectively, and the carboxy-terminal C domain forms a channel in the inner membrane to kill the cell (for general review, see Cramer et al., 1995). The crystal structure of the water-soluble form of colicin Ia reveals that the three domains are separated by two long a -helices in a coiled-coil, making a “Y”-shaped molecule (Wiener et al., 1997). Aside from the hydrophobic hairpin formed by helices 8 and 9 of the C domain, the rest of the molecule is highly charged ( . 30% of the residues) (Mankovich et al., 1986). Whole colicin or isolated C domain can also form channels in planar lipid bilayer membranes (Nogueira and Varanda, 1988; Ghosh et al., 1993). When whole colicin Ia associates with a planar bilayer, it undergoes a series of conformational changes: after the hydrophobic hairpin inserts into the membrane (Kienker et al., 1997), the conducting channel is formed by the voltage-dependent insertion of two additional segments, with portions of helix 1 and helices 6–7 spanning the membrane, and helices 2–5 translocated completely across the membrane (Qiu et al., 1996) (Fig. 1). The T and R domains presumably remain on the cis side (the side to which the colicin was added). We have shown that colicin Ia can still form channels (albeit somewhat aberrantly) when residues, located in helices 1–5, that normally move into or across the membrane are forced to stay on the cis side (Qiu et al., 1996). In addition, foreign sequences inserted between helices 3 and 4 move across the membrane, along with the normally translocated segment (Jakes et al., 1998). Apparently, the precise identities of the translocated segment and of the “upstream” membrane-spanning segment are not critically important. This led us to wonder why translocation normally stops where it does; that is, why doesn’t all of helix 1 (residues 359–467) move across the membrane? We suspected that the T and R domains Portions of this work were previously published in abstract form (Kienker, P., S. Slatin, K. Jakes, and A. Finkelstein. 1999. Biophys. J.