Volume 9, Issue 2, Pages 363-373 (February 2002) Tim22, the Essential Core of the Mitochondrial Protein Insertion Complex, Forms a Voltage-Activated and Signal-Gated Channel  Peter Kovermann, Kaye N Truscott, Bernard Guiard, Peter Rehling, Naresh B Sepuri, Hanne Müller, Robert E Jensen, Richard Wagner, Nikolaus Pfanner  Molecular Cell  Volume 9, Issue 2, Pages 363-373 (February 2002) DOI: 10.1016/S1097-2765(02)00446-X

Figure 1 Viability of S. cerevisiae in the Absence of TIM54 (A) Analytical PCR indicating fragment sizes expected for intact TIM54, including 612 bp of flanking region in wild-type cells (lane 1, 2049 bp); ADE2-disrupted TIM54 gene in tim54Δ cells, including 612 bp of flanking region (lane 2, 2852 bp); internal PCR fragment of TIM54 (TIM54*) in BG54-2 parent strain (lane 3, 421 bp) and following selection on 5-FOA (lane 4). (B) Growth of tim54Δ cells on YPD (glucose) is partially compromised at 24°C and completely compromised at 37°C compared with the parental strain (WT). tim54Δ cells do not grow on YPG (glycerol) at either temperature. However, growth of tim54Δ cells expressing Tim54 from a plasmid is restored to normal levels. Cells were pregrown in 2% glucose (YPD) at 24°C to an OD600 = 1, then diluted in 10-fold increments. Starting with 10-fold diluted material, 10 μl samples were spotted onto YPD or YPG plates and incubated at 24°C and 37°C for 5 days. (C) Immunodecoration of purified mitochondria illustrating the absence of Tim54 in tim54Δ mitochondria (lanes 2 and 4) compared with wild-type mitochondria (lanes 1 and 3). Mitochondrial proteins (25 μg, lanes 1 and 2; or 50 μg, lanes 3 and 4) were separated by SDS-PAGE, transferred to PVDF by Western blot, and then decorated with affinity-purified anti-Tim54 antibodies. (D) Protein composition of tim54Δ mitochondria. Isolated wild-type or tim54Δ mitochondria (25 μg of protein/lane) were treated as in (C) and decorated with antibodies as indicated. Molecular Cell 2002 9, 363-373DOI: (10.1016/S1097-2765(02)00446-X)

Figure 2 Tim22 Forms the Core of the Protein Insertion Complex (A) Separation of Tim proteins by blue native electrophoresis revealed a loss of structural organization of the protein insertion complex in tim54Δ mitochondria. Isolated wild-type and tim54Δ mitochondria were solubilized in 1% digitonin-containing buffer. Native membrane complexes separated by BN-PAGE were resolved into their constituent components by SDS-PAGE. Following electrophoresis, proteins were blotted to PVDF and then immunodecorated with a range of specific Tim antibodies as indicated (the Tim22 strip of tim54Δ was exposed three times longer than that of WT). 300K, protein insertion complex of wild-type mitochondria; 70K, complex of small Tim proteins; the asterisk indicates the migration of Tim22 in tim54Δ. (B) Suppression of growth defects of tim54Δ cells by overexpression of Tim22. tim54Δ cells received plasmids overexpressing Tim12, Tim18, or Tim22, or the empty plasmid as control. Cells were pregrown in 2% glucose (YPD) at 24°C to an OD600 = 1, then diluted in 10-fold increments. Starting with 10-fold diluted material, 10 μl samples were spotted onto YPD or YPG (glycerol) plates and incubated at 24°C and 37°C for 3 days (YPD) or 5 days (YPG). Molecular Cell 2002 9, 363-373DOI: (10.1016/S1097-2765(02)00446-X)

Figure 3 A Hydrophilic Channel Formed by Tim22 (A) Purification of recombinant Tim22 analyzed by Coomassie brilliant blue-stained SDS-PAGE. Lane 1, molecular size markers; lane 2, purified Tim22; and lane 3, mock control. (B) Current recordings from a bilayer containing up to three active Tim22 channels (indicated by hatched boxes on the right) at membrane potentials Vh from +150 to −150 mV with 250 mM KCl, 0.1 mM CaCl2, and 10 mM MOPS-Tris, pH 7.0, on both sides of the membrane. Time scale-magnified trace and its idealized current trace (bottom panels), horizontal lines indicate amplitudes of the subconductance states. The smallest subconductance unit at Vh = ±150 mV was 67 ± 5 pS. (C) Amplitude histogram of a Tim22 channel recorded as described for (B). (D) Current-voltage relationship of a bilayer containing one active Tim22 channel obtained from voltage increments of 1 mV/s from 0 to +150 mV and from 0 to −150 mV. The buffer on both sides of the membrane was 250 mM KCl, 0.1 mM CaCl2, and 10 mM MOPS-Tris, pH 7.0. (E) Current-voltage relationship of two different direct gating transitions (squares, fully open channel; triangles, three subconductance units) deduced from single-channel currents of the Tim22 channel in asymmetric buffers of 250 mM KCl/20 mM KCl (cis/trans), and 0.1 mM CaCl2 and 10 mM MOPS-Tris, pH 7.0. Data points are averages from three independent bilayers. (F) Purity of recombinant Tim18 and Tim54 as determined by Coomassie brilliant blue-stained SDS-PAGE. Molecular Cell 2002 9, 363-373DOI: (10.1016/S1097-2765(02)00446-X)

Figure 4 Estimation of the Tim22 Pore Size by the Polymer Exclusion Method (A) Values for the fully open Tim22 channel. (B) Values for the smallest subconductance unit. Upper panels, ratio of the Tim22 channel conductance without (Λ0) and after partition into the pore (Λ) of nonelectrolytes (NE) of increasing hydrodynamic radii. Smaller NE and a range of PEGs from PEG200 up to PEG6000 were used (Krasilnikov et al., 1992). Data points are averages of at least three independent bilayers. The line shows the fit of the data according to Bezrukov and Kasianowicz (1997). Lower panels, second derivative of the data fit from the upper panel revealing restriction radii for small and large NE in the inner and outer parts of the channel, respectively. The buffer was 20% (w/v) NE, 100 mM KCl, and 10 mM MOPS-Tris, pH 7.0. Molecular Cell 2002 9, 363-373DOI: (10.1016/S1097-2765(02)00446-X)

Figure 5 Fraction of Open Tim22 Subconductance Units in Relation to the Applied Voltage After application of different voltage steps at t = 0, mean currents were calculated for a time period of 10 s. The number of open subconductance units was calculated from these mean currents by division through the single unit current at the given voltage. Data were normalized with respect to the maximal mean current at Vh = 200 mV. Data points are averages of three independent bilayers. Molecular Cell 2002 9, 363-373DOI: (10.1016/S1097-2765(02)00446-X)

Figure 6 A Carrier-Derived Peptide Specifically Reacts with the Tim22 Channel Comparison of the current-voltage relationship from bilayers containing active Tim22 channels in the absence (control) or presence of peptides (P2, CoxIV, and SynB2). The buffer was 10 mM MOPS-Tris, pH 7.0, and 0.1 mM CaCl2 with a salt gradient of 250 mM KCl (cis)/20 mM KCl (trans) across the membrane. The sweep rate of the command voltage was 1 mV/s. (A and B) Carrier peptide P2 added to the trans side of the membrane at indicated concentrations. P2 peptide was derived from residues 211–223 of the yeast mitochondrial phosphate carrier (Brix et al., 1999). (C) P2 added to the trans side of the membrane. Quantification of the relative current reduction from three to eight independent experiments for each peptide concentration and membrane voltage. The standard deviations are <3% for each value. (D) P2 added to the cis side. The experiment was performed as for (A) and (B) except that 10 μM P2 were added on the cis side of the membrane and the salt gradient was reversed. (E) Addition of presequence peptide CoxIV to the trans side of the bilayer. (F) SynB2 added to the trans side of the bilayer. Molecular Cell 2002 9, 363-373DOI: (10.1016/S1097-2765(02)00446-X)

Figure 7 Activation of Tim22 by a Targeting Signal in the Presence of a High Membrane Potential (A) Single-channel recordings from a bilayer containing active copies of Tim22 channels at Vh = 125 or 150 mV in the absence and presence of 100 nM peptide P2. Bottom panels, time scale-expanded trace (left) and amplitude histogram (right) derived from the recording at 150 mV and 100 nM P2. (B) Quantification of the frequencies of direct gating transitions between the fully open state and closed state at the indicated membrane potentials in the presence or absence of 100 nM P2. The values and standard deviations are derived from three to eight independent experiments each. Molecular Cell 2002 9, 363-373DOI: (10.1016/S1097-2765(02)00446-X)