Targeting Peptides Transiently Block a Mitochondrial Channel

Abstract

The effects of synthetic targeting peptides on the activity of the multiple conductance channel (MCC) of mouse and yeast mitochondria were investigated using patch-clamp techniques. Amino-terminal targeting peptides of two inner membrane proteins reversibly decreased the open probability and mean open time of MCC. One of these targeting peptides had no effect on two other voltage-dependent mitochondrial channels. Furthermore, the effects induced by the two targeting peptides on MCC were not elicited by two peptides of an outer membrane protein. The specific interactions of targeting peptides with MCC suggest that this channel may be involved in protein import across the inner mitochondrial membrane.

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“…2a), supporting the conclusion that reconstituted Tim23 has an asymmetric orientation. The characteristic multiple conductance state, with subconductances and a main conductance frequently present in multiples of three, was seen both for the inner membrane activity 8,11,23 and for reconstituted Tim23 (Fig. 1b) in a comparable pattern.…”

“…The successful reconstitution of purified Tim23 into a lipid bilayer described here has allowed a direct measure of its properties and, together with the use of mutants, an assessment of its role in mitochondria. The reconstituted channel exhibits all the characteristics of the physiological channel, including high cation selectivity, large multiple conductance 8,11 and an effective pore size that requires unfolding of the polypeptide chain during translocation. Bauer et al 21 reported that a membrane potential promotes dimerization of the N-terminal domains of Tim23 and an interaction with presequences.…”

“…We detected several channel activities, the most frequent being a high-conductance anion channel with two conductance states of 200 pS and 600 pS that specifically responded to inhibitors of the ADP-ATP carrier, the most abundant protein of the mitochondrial inner membrane (not shown). A channel activity resembling the multiple conductance channel 8,11 displayed a high cation selectivity and conductance much like the reconstituted Tim23 channel and was the only inner membrane activity sensitive to a mitochondrial presequence (Fig. 4b,c).…”

“…Other known Tom components function as receptors for preprotein recognition or in the assembly and stability of the TOM complex. Preproteins are translocated through the inner membrane by the TIM23 complex, which consists of the essential integral membrane proteins Tim23 and Tim17, the membrane-bound Tim44 with the associated matrix heat shock protein mtHsp70, and probably additional translocation components [1][2][3][4][7][8][9][10][11][12] . Translocation of the presequence requires a membrane potential (∆ψ) across the inner membrane, whereas an ATP-dependent motor system that includes mtHsp70 and its membrane anchor Tim44 drives the translocation of the mature portions of preproteins into the matrix [1][2][3][4] .…”

“…Translocation of the presequence requires a membrane potential (∆ψ) across the inner membrane, whereas an ATP-dependent motor system that includes mtHsp70 and its membrane anchor Tim44 drives the translocation of the mature portions of preproteins into the matrix [1][2][3][4] . Patch-clamp analysis has suggested that the mitochondrial inner membrane has a presequence-sensitive channel 8,11 . The identity of the channel-forming Tim protein(s) is not known, however, and thus the properties of the channel have not been characterized at the molecular level.…”