Limits...
Protein translocation across planar bilayers by the colicin Ia channel-forming domain: where will it end?

Kienker PK, Jakes KS, Finkelstein A - J. Gen. Physiol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. kienker@aecom.yu.edu

ABSTRACT
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.

Show MeSH
Single channels formed by the colicin Ia carboxy-terminal fragment, 282–626. Before the start of each record, 46 ng of mutant “−3” C/CT-XL (without a His-tag) were added to the cis compartment. (The colicin mutant was biotinylated via a maleimide linker, but streptavidin was not used for these experiments.) (A) A single channel opens to the normal conductance (44 pS), and then drops to a low-conductance state (9 pS). The voltage was held at +80 mV for the duration of this record. (B) A channel that has already passed through the normal conductance state is shown in the low-conductance state (8 pS) at the beginning of the record. When the voltage was switched to −80 mV, the channel flickered once (the conductance at this voltage is only 1.6 pS) and closed. After the return to +80 mV, the channel reopened directly to the low-conductance state, apparently without passing through the normal conductance state. The solution on both sides of the membrane was the same as in Fig. 5, with the addition of 2.6 mM tris(2-carboxyethyl)-phosphine. The records were filtered at (A) 30 Hz and (B) 10 Hz.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230624&req=5

Figure 6: Single channels formed by the colicin Ia carboxy-terminal fragment, 282–626. Before the start of each record, 46 ng of mutant “−3” C/CT-XL (without a His-tag) were added to the cis compartment. (The colicin mutant was biotinylated via a maleimide linker, but streptavidin was not used for these experiments.) (A) A single channel opens to the normal conductance (44 pS), and then drops to a low-conductance state (9 pS). The voltage was held at +80 mV for the duration of this record. (B) A channel that has already passed through the normal conductance state is shown in the low-conductance state (8 pS) at the beginning of the record. When the voltage was switched to −80 mV, the channel flickered once (the conductance at this voltage is only 1.6 pS) and closed. After the return to +80 mV, the channel reopened directly to the low-conductance state, apparently without passing through the normal conductance state. The solution on both sides of the membrane was the same as in Fig. 5, with the addition of 2.6 mM tris(2-carboxyethyl)-phosphine. The records were filtered at (A) 30 Hz and (B) 10 Hz.

Mentions: We now consider the channels formed by our longest carboxy-terminal fragment of colicin Ia, CT-XL, which includes all of the R and C domains, plus the long helix that connects them. These channels showed the drop in single-channel conductance that we believe is diagnostic of translocation of the amino terminus (Fig. 6 A). This did not depend on the presence of an amino-terminal His-tag or biotin. There was, however, a subtle difference between the CT-XL channels and the channels formed by the shorter fragments (CT-S, CT-M, and CT-L). For the shorter fragments, after the channels entered the low-conductance state, they could be turned off at negative voltage; when they subsequently reopened at positive voltage, they opened initially, as before, to the transient, higher-conductance state, before dropping to the low-conductance state. For CT-XL, channels in the low-conductance state could also be turned off at negative voltage; upon the return to positive voltage, however, the channels reopened directly to the low-conductance state (Fig. 6 B). This held true even when large negative voltages (−200 mV) were used to turn the channels off. Our interpretation of this result is that, although the amino terminus is translocated across the membrane to the trans side at positive voltage, it does not generally move back to the cis side at negative voltage. Thus, the closings that we observe reflect the entry into some new closed state, in which the amino terminus is still on the trans side.


Protein translocation across planar bilayers by the colicin Ia channel-forming domain: where will it end?

Kienker PK, Jakes KS, Finkelstein A - J. Gen. Physiol. (2000)

Single channels formed by the colicin Ia carboxy-terminal fragment, 282–626. Before the start of each record, 46 ng of mutant “−3” C/CT-XL (without a His-tag) were added to the cis compartment. (The colicin mutant was biotinylated via a maleimide linker, but streptavidin was not used for these experiments.) (A) A single channel opens to the normal conductance (44 pS), and then drops to a low-conductance state (9 pS). The voltage was held at +80 mV for the duration of this record. (B) A channel that has already passed through the normal conductance state is shown in the low-conductance state (8 pS) at the beginning of the record. When the voltage was switched to −80 mV, the channel flickered once (the conductance at this voltage is only 1.6 pS) and closed. After the return to +80 mV, the channel reopened directly to the low-conductance state, apparently without passing through the normal conductance state. The solution on both sides of the membrane was the same as in Fig. 5, with the addition of 2.6 mM tris(2-carboxyethyl)-phosphine. The records were filtered at (A) 30 Hz and (B) 10 Hz.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230624&req=5

Figure 6: Single channels formed by the colicin Ia carboxy-terminal fragment, 282–626. Before the start of each record, 46 ng of mutant “−3” C/CT-XL (without a His-tag) were added to the cis compartment. (The colicin mutant was biotinylated via a maleimide linker, but streptavidin was not used for these experiments.) (A) A single channel opens to the normal conductance (44 pS), and then drops to a low-conductance state (9 pS). The voltage was held at +80 mV for the duration of this record. (B) A channel that has already passed through the normal conductance state is shown in the low-conductance state (8 pS) at the beginning of the record. When the voltage was switched to −80 mV, the channel flickered once (the conductance at this voltage is only 1.6 pS) and closed. After the return to +80 mV, the channel reopened directly to the low-conductance state, apparently without passing through the normal conductance state. The solution on both sides of the membrane was the same as in Fig. 5, with the addition of 2.6 mM tris(2-carboxyethyl)-phosphine. The records were filtered at (A) 30 Hz and (B) 10 Hz.
Mentions: We now consider the channels formed by our longest carboxy-terminal fragment of colicin Ia, CT-XL, which includes all of the R and C domains, plus the long helix that connects them. These channels showed the drop in single-channel conductance that we believe is diagnostic of translocation of the amino terminus (Fig. 6 A). This did not depend on the presence of an amino-terminal His-tag or biotin. There was, however, a subtle difference between the CT-XL channels and the channels formed by the shorter fragments (CT-S, CT-M, and CT-L). For the shorter fragments, after the channels entered the low-conductance state, they could be turned off at negative voltage; when they subsequently reopened at positive voltage, they opened initially, as before, to the transient, higher-conductance state, before dropping to the low-conductance state. For CT-XL, channels in the low-conductance state could also be turned off at negative voltage; upon the return to positive voltage, however, the channels reopened directly to the low-conductance state (Fig. 6 B). This held true even when large negative voltages (−200 mV) were used to turn the channels off. Our interpretation of this result is that, although the amino terminus is translocated across the membrane to the trans side at positive voltage, it does not generally move back to the cis side at negative voltage. Thus, the closings that we observe reflect the entry into some new closed state, in which the amino terminus is still on the trans side.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. kienker@aecom.yu.edu

ABSTRACT
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.

Show MeSH