Comparison of the TIM and TOM channel activities of the mitochondrial protein import complexes

Abstract

Water-filled channels are central to the process of translocating proteins since they provide aqueous pathways through the hydrophobic environment of membranes. The Tom and Tim complexes translocate precursors across the mitochondrial outer and inner membranes, respectively, and contain channels referred to as TOM and TIM (previously called PSC and MCC). In this study, little differences were revealed from a direct comparison of the single channel properties of the TOM and TIM channels of yeast mitochondria. As they perform similar functions in translocating proteins across membranes, it is not surprising that both channels are high conductance, voltage-dependent channels that are slightly cation selective. Reconstituted TIM and TOM channel activities are not modified by deletion of the outer membrane channel VDAC, but are similarly affected by signal sequence peptides.

FIGURE 1

Purity of the mitochondrial inner and outer membrane preparations is high from VDACless yeast. (A) Western blot of mitochondrial outer membranes verify the M22-2 strain is VDAC deficient at comparable Tom40p levels. WT is wild-type. (B) Western blots using antisera against Tom40p and ATP-IV, proteins of the outer (MOM) and inner (MIM) membranes, show minimal cross-contamination of the inner and outer membrane preparations from VDACless M22-2 yeast. In strains that are not VDAC deficient, VDAC antisera was used in lieu of Tom40p to determine purity (e.g., as done in Fig. 8).

FIGURE 2

Single-channel properties of TIM and TOM channels are similar. Typical single channel current traces of TIM (A) and TOM (B) channels are shown at various voltages with 2 kHz filtration in symmetrical 150 mM KCl 5 mM HEPES, pH 7.4. Typical current-voltage relationship of single TIM (C) and TOM (D) channels reveal similar conductance levels and voltage dependence. Voltage ramps are computer generated through Clampex (Version 8.1.0.12, Axon Instruments) at 5000 samples/250 s episode, from −75 to +75 mV. O, S, and C correspond to the open (1000 pS), half-open substate (500 pS), and closed conductance levels.

FIGURE 3

TIM and TOM channels are voltage dependent. The open probability (Po) of TIM (A) and TOM (B) channels were determined for recordings of 30-s duration at various voltages from total amplitude histograms (not shown). Gating charge is proportional to the slope of Ln(Po/(1−Po)) versus voltage plots and show that the gating charge of TIM (C) and TOM (D) are similar. Po was determined from the occupation of the 1000 pS level (O) of total amplitude histograms. Typically, points are the mean ± SD of at least eight independent patches.

FIGURE 4

Dwell-time distributions for TIM and TOM channels. Dwell-time distributions were fit to 5000–6000 events at voltages favoring the occupation of the state examined for a typical single channel activity. Open dwell-time distributions recorded at +20 mV for typical single TIM (A) and TOM (D) channels are fit with the sum of two exponentials with 2 kHz filtration and 5 kHz sampling rate. Substate time distributions are fit with a single exponential function for the TIM channel (B) at +40 mV and the TOM channel at +50 mV (E) at 5 kHz filtration and 10 kHz sampling rate. Closed-time distributions are fit with two exponential functions for the TIM (C) and TOM channels at +60 mV (F). Fits are shown in gray and data is black. Dwell-time constants (τ) are as indicated in each plot with % contribution in parenthesis.

FIGURE 5

Total amplitude histograms for TIM and TOM channels can deviate from predicted binomial distributions. Total amplitude histograms are shown for TIM (A at +20 mV; B, +40 mV) and TOM (C, +20 mV; D, +30 mV) channels for experimental data (black) and data simulations (gray) fit to the probability of occupying the 1 nS level, assuming two independent channels and a binomial distribution. Simulations were generated by Electrophysiology Data Recorder V-2.2.3.software (J. Dempster, University of Strathclyde) after providing Po, transition amplitude, τ_open, τ_closed with five openings per burst for each data set (see Methods section for further details). Interval durations were binned at a resolution of 200–500 bins. Histograms are not leak subtracted.

FIGURE 6

Signal peptides modulate the flicker frequency of the TIM and TOM channel activities. Typical current traces are shown at +20 mV for TIM (A) and TOM (B) channels in excised patches before (control, no peptide) and after sequential perfusions of the bath with 50 μM of the control peptide SynB2 and then with 50 μM of the signal peptide yCOX-IV_1–13. (C) The histograms of flicker rates (number of transition events per second) in the absence (control) and presence of SynB2 or yCOX-IV_1–13 are similar for the TIM and TOM channels. (D) The dose dependence of the flicker rate on the concentration of peptide yCOX-IV_1–13 is shown for TIM (o) and TOM (•) channels. Other conditions are as in Fig. 2.

FIGURE 7

Voltage dependence of the open probability of the TIM and TOM channels is modified by signal peptides. Current voltage curves are shown for a single TIM (A, C, E) and TOM (B, D, F) channel in the absence and presence of 50 μM yCOX-IV_1–13 in the bath as indicated. Note that the channels show a decrease in open probability (C, D) as well as rapid flickering (E, F) in the presence of signal peptide at positive but not negative potentials. Open probability voltage curves are shown for TIM (G) and TOM (H) in the absence (•) and presence (○) of yCOX-IV_1–13. Total amplitude histograms are shown for TIM (I) and TOM (J) channels in the absence (control) and presence of yCOX-IV_1–13 peptide at +40 mV.

FIGURE 8

Mutation in Tim23p distinguishes TIM from TOM channel activity. (A) Current traces of TIM and TOM channels are shown in the presence and absence of 50 μM yCOX-IV_1–13 and illustrate the decrease in peptide sensitivity of TIM but not TOM in the tim23.1 strain. (B) Histograms show the peptide sensitivity as indicated by flicker rate of the TIM channel is reduced; the TOM channel of this strain is identical to that of wild-type yeast. (C) Western blots show the cross-contamination of the preparations is low when probing with VDAC antisera for the outer membrane and ATP-IV antisera for the inner membrane.