Protein translocation across planar bilayers by the colicin Ia channel-forming domain: where will it end?
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 approximately 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.
Kienker
P K
PK
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. kienker@aecom.yu.edu
Jakes
K S
KS
Finkelstein
A
A
D000818:Animals; D003087:Colicins / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli; D007202:Indicators and Reagents / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics*; D008051:Lipid Bilayers; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D019809:Streptavidin / Q000494:pharmacology
D003087:Colicins; D007202:Indicators and Reagents; D007473:Ion Channels; D008051:Lipid Bilayers; D019809:Streptavidin