Electrophysiological Approaches to the Study of Protein Translocation in Mitochondria

Electrophysiological Approaches to the Study of Protein Translocation in Mitochondria Sergey M. Grigoriev,* Concepcio´n Muro,*,{ Laurent M. Dejean,* Maria Luisa Campo,{ Sonia Martinez-Caballero,* and Kathleen W. Kinnally* *College of Dentistry, Department of Basic Sciences, New York University, 345 East 24th Street, New York, New York 10010 Departamento de Bioquı´mica y Biologı´a Molecular y Gene´tica, Facultad de Veterinaria, Universidad de Extremadura, 10071 Ca´ceres, Spain

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Electrophysiological techniques have been integral to our understanding of protein translocation across various membranes, and, in particular, the mitochondrial inner and outer membranes. Descriptions of various methodologies (for example, patch clamp, planar bilayers, and tip dip, and their past and potential contributions) are detailed within. The activity of protein import channels of native mitochondrial inner and outer membranes can be studied by directly patch clamping mitochondria and mitoplasts (mitochondria stripped of their outer membrane by French pressing) from various genetically manipulated strains of yeast and mammalian tissue cultured cells. The channel activities of TOM, TIM23, and TIM22 complexes are compared with those reconstituted in proteoliposomes and with those of the recombinant proteins Tom40p, Tim23p, and Tim22p, which play major roles in protein translocation. Studies of the mechanism(s) and the role of channels in protein translocation in mitochondria are prototypes, as the same principles are likely followed in all biological membranes including the endoplasmic reticulum and chloroplasts. The ability to apply electrophysiological techniques to these channels is now allowing investigations into the role of mitochondria in diverse fields such as neurotransmitter release, long-term potentiation, and apoptosis. KEY WORDS: Protein import, Protein-translocating channels, Patch clamp, Mitochondria, TOM complex, TIM22 complex, TIM23 complex. ß 2004 Elsevier Inc.

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Copyright 2004, Elsevier Inc. All rights reserved.

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I. Introduction Compartmentalization of proteins within a cell is critical to maintaining homeostasis. Protein translocation across membrane barriers is important in many cellular functions including signaling, secretion, biogenesis of organelles, compartmentalization, and apoptosis. Because about half the proteins synthesized in a cell must cross at least one membrane before reaching their final destinations, protein translocation across membranes is a fundamental cellular process (Schatz and Dobberstein, 1996). Although mitochondria contain their own genome, more than 95% of the mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol, and imported into these double-membrane organelles. Singer and Nicolson (1972) described the essential structure of biological membranes in their fluid mosaic model, but Blobel and Dobberstein (1975) were the first to suggest that transiently formed tunnels or channels were involved in protein translocation across membranes. Several years later, Simon and Blobel demonstrated the involvement of a pore in the translocation of newly formed proteins across the endoplasmic reticulum and bacterial membranes (Simon and Blobel, 1991, 1992, 1993; Simon et al., 1989). Further evidence of the involvement of water-filled channels in protein translocation in the endoplasmic reticulum was provided by the Johnson laboratory, using elegant fluorescence techniques (Crowley et al., 1994; Hamman et al., 1997). Electrophysiological techniques were used to show that ion channels are integral to protein import across the mitochondrial inner membrane (Lohret and Kinnally, 1995b; Lohret et al., 1997) and across the outer membrane (Juin et al., 1997). The use of electrophysiological techniques to study protein import channels is the subject of this review. Mitochondria are double-membrane organelles, and newly synthesized proteins are targeted to their various final destinations by targeting sequences and/or internal signals within the precursor proteins. These locations include the outer and inner membranes, the matrix, and the intermembrane space. Most precursor proteins destined for the matrix space are targeted to mitochondria by a cleavable presequence or targeting sequence located at the amino terminus, whereas those destined for the inner membrane often have internal targeting sequences. The import routes can be direct or rather circuitous after interacting with one or more protein import complexes. The principal protein import complexes of mitochondria are called TIM23, TIM22, and TOM (Bauer et al., 2000; Jensen and Johnson, 2001; Pfanner and Geissler, 2001; Pfanner and Wiedemann, 2002; Schatz and Dobberstein, 1996) and are illustrated in Fig. 1. TIM and TOM are the translocases of the inner and outer membranes, respectively. Some proteins reach the intermembrane space after interaction of their bipartate signal with the TIM23 complex,

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FIG. 1 Protein translocation complexes of mitochondria: The three principal protein import machines in mitochondria are called the TOM, TIM23, and TIM22 complexes. Precursor proteins with mitochondrial targeting signals bind to specific receptors (e.g., Tom70p, Tom22p, and Tom20p), which direct import via Tom40p, the pore-forming protein of the translocase of the outer membrane. Precursor proteins are translocated as unfolded, linear polypeptides. Precursor proteins with N-terminal targeting sequences (black) are directed to the TIM23 complex for import across the inner membrane. The TIM23 complex is composed of Tim23p, Tim17p, Tim44p, Tim50p, and Tim14p. Translocation of precursor proteins across the inner membrane requires a membrane potential (
c) and the ATP-driven action of mtHsp70. Precursor proteins with internal targeting signals (purple) are guided to the TIM22 complex by chaperones in the intermembrane space (e.g., Tim9p and Tim10p). Tim22p forms the insertion pore, and Tim54p is required to maintain structural integrity. Insertion of membrane proteins by the Tim22 complex also requires a membrane potential.

whereas others reach this spot directly through the TOM complex without a presequence. Most of these precursors are translocated across one or both of the mitochondrial membranes, at least in part, because of the interaction of their cationic amphipathic targeting regions with the protein import machinery. Although the import machinery of the inner and outer membranes can operate independently, current models favor their transient linkage at contact sites ( junctions where the two membranes are closely apposed) (Glick et al., 1991; Lithgow et al., 1995). Furthermore, the inner and outer membranes can be ‘‘zippered’’ together by stalling precursors in the import machinery as shown in the elegant study of Schulke et al. (1997). Protein import into mitochondria is a multistep process involving import complexes that recognize, sort, translocate, and begin the processing of these preproteins. The TIM23, TIM22, and TOM complexes have receptors

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necessary for recognition and sorting of precursors, as well as a pore for selective translocation across the membranes. The channels of these complexes are referred to as the Tim23, Tim22, and Tom channels. Finally, the channel called MAC forms in the outer membrane early in apoptosis and is responsible for the export of cytochrome c from the intermembrane space across the outer membrane and into the cytosol. The protein import complexes are formed by a variety of proteins (Fig. 1). TIM23 and TOM complexes catalyze the import from the cytosol across the mitochondrial inner and outer membranes, respectively (Bauer et al., 2000; Jensen and Johnson, 2001; Pfanner and Geissler, 2001; Pfanner and Wiedemann, 2002; Schatz and Dobberstein, 1996). Components of the complexes are designated Tim or Tom, followed by their molecular weight. The principal protein components of the TOM complex are the pore protein Tom40p; the receptors Tom20p, Tom22p, and Tom70p; and the three smaller proteins Tom5p, Tom6p, and Tom7p. The proteins Tim23p, Tim17p, Tim50p, Tim14p, Tim44p, and mtHsp70 are the identified components of the TIM23 complex. Tim23p and Tim17p are integral membrane proteins that are thought to form the pore, whereas the amino-terminal portion of Tim23p is a receptor that recognizes presequences and anchors the TIM23 complex to the outer membrane. Tim50p recognizes presequences and passes the precursor proteins from the TOM complex to the Tim23 channel. The proteins Tim14p, Tim44p, and mtHsp70 are thought either to facilitate diVusion of the preproteins or to form the motor that pulls precursors through the import pore (Bauer et al., 1996; Berthold et al., 1995; Emtage and Jensen, 1993; Ryan and Jensen, 1993). Mge1p is a cochaperone of mtHsp70. A new cochaperone, Tim16p, has been identified (Kozany et al., 2004). Several laboratories suggest other members of the TIM23 complex have yet to be identified. For example, immunoprecipitation and cross-linking studies suggest the involvement of proteins with molecular masses of 20 and 33 kDa (Berthold et al., 1995; Blom et al., 1995; Kerscher et al., 1998, 2000). Whereas the outer membrane relies on a general insertion pore in the TOM complex for import, the inner membrane has machinery in addition to the TIM23 complex for translocating proteins. The TIM22 complex is responsible for inserting and folding proteins from the intermembrane space into the inner membrane and contains the essential proteins Tim18p, Tim22p, and Tim54p (Fig. 1). This TIM22 complex forms a system distinct from the Tim23p- and Tim17p-containing TIM23 complex but is responsible for import of both of these proteins as well as most of the family of mitochondrial carrier proteins, for example, phosphate carrier (Kerscher et al., 2000; Kinnally, 2002; Kovermann et al., 2002; Tokatlidis et al., 1996). These membrane proteins do not contain a cleavable presequence but do have an internal targeting sequence. The TIM22 complex recognizes this second class of targeting signals and does not interact with the classic presequences.

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There are also a variety of chaperones, for example, Tim9p, Tim10p, and Tim12p, that facilitate import through the TOM complex and hand oV the precursors to the TIM22 complex (Vasiljev et al., 2004). There are two additional protein translocation machines in mitochondria. The OXA1 complex inserts proteins (encoded in mitochondria) from the matrix space into the inner membrane. Finally, the channel called MAC (mitochondrial apoptosis-induced channel) forms in the outer membrane early in apoptosis (Pavlov et al., 2001). Hence, MAC is responsible for the export of cytochrome c, and possibly other proteins, from the intermembrane space to the cytoplasm during apoptosis (Guo et al., 2004). Studies of the mechanism(s) and the role of channels in protein translocation in mitochondria are prototypes, as the same principles are likely followed in all biological membranes. Therefore, understanding mitochondrial sorting and protein translocation can facilitate understanding of, for example, the same process in the endoplasmic reticulum and chloroplasts. Finally, the ability to patch-clamp mitochondria and identify their channels is now allowing investigations into the role of mitochondria in areas of study as diverse as enhancement of neurotransmitter release, long-term potentiation (possibly learning), programmed cell death, and viral infections. Sections describing the single-channel behavior of these protein translocation channels, with emphasis on the Tom and Tim23 channels, follow a summary of the fundamentals of electrophysiological methods and a description of the various channel preparations that have been studied.

II. Electrophysiological Methods A. Single-Channel Fingerprint Protein import channels have water-filled pores that allow for the flow of ions down their electrochemical gradients. As the channel opens and closes, the current increases and decreases stochastically. This ion flow, or current, is passive and often measured by electrophysiological approaches. Although Ohm’s law (V ¼ IR; where I is current, V is voltage, and R is resistance) is the usual standard, Sorgato and Moran (1993) have extended this so that the current, I, is defined as I ¼ GNPo ðV  Vi Þ ð1Þ where G is the single-channel conductance, N is the number of channels, Po is the probability the channel will be open, V is the imposed voltage, and Vi is the equilibrium potential for the conducting ion. Vi can be approximated from the concentration gradient, using the Nernst equation, and is zero under symmetrical conditions.

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Single-channel measurements of the current enable a close examination of a variety of parameters. Conductance is the reciprocal of the pore resistance; larger pores have bigger conductances and lower resistances to ion flow. In keeping with their function, protein translocation channels tend to have enormous conductances relative to most other ion channels. For example, yeast Tim23 and Tom channels have peak conductances of 1000 pS, whereas a typical sodium channel has a conductance of 10 pS. The conductance can be related to the size of the pore by the method of Hille (2001) and the following formula:
Rchannel ¼ r L=pa2 ð2Þ where R is resistance of the pore, r is the resistivity of the solution, L is pore length, and a is pore radius. A pore length of 5.5 nm can be used, assuming this length corresponds to the average thickness of the outer membrane (Mannella, 1981). Under these conditions, a channel with a conductance of 500 to 1000 pS is predicted to have a pore diameter of 1.7 to 2.4 nm. This estimation can be extended by adding in the access resistance as described by others (Guo et al., 2004; Hille, 2001). As discussed later, further probing of the pore size is routinely done with molecules of known dimensions, for example, dextrans and polyethylene glycols of various molecular weights. Ion selectivity is measured through the reversal potential, that is, the voltage necessary to eliminate the ion flow, or current, in the presence of a known concentration gradient. In the presence of a KCl concentration gradient, the polarity of the reversal potential indicates whether the channel is anion or cation selective. The magnitude of the potential indicates the degree of ion selectivity; larger concentration gradients yield larger potentials. The relative permeability of cations and anions is calculated on the basis of Eq. (3), which is derived from the Goldman–Hodgkin–Katz equation:


Er ¼ ðRT=zF Þln PKþ ½Kþ 1 þ PCl ½Cl 2 = PKþ ½Kþ 2 þ PCl ½Cl 1 ð3Þ where Er is the reversal potential (or zero current voltage), R is the gas constant, T is absolute temperature, z is the charge, F is the Faraday constant, PKþ and PCl are the permeabilities of Kþ and Cl, respectively, and [Kþ] and [Cl] are the concentrations on the two sides of the membrane (Sorgato and Moran, 1993). The protein translocation channels of mitochondria are typically slightly cation selective, with relative permeabilities of Kþ/ Cl (PK/PCl) of approximately 5, which means 5 potassium ions pass through the channel for every chloride ion. This ion selectivity can be considered quite minimal as the PNa/PK of a typical sodium channel is 400, that is, only 1 potassium ion passes for every 400 sodium ions.

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Kinetics and voltage dependence are revealed by closer examination of the currents flowing through these ion channels. Voltage dependence describes the probability that the channel will be open at various potentials and can be visualized by current voltage curves. Voltage dependence is quantified by two parameters. The Vo is the voltage at which the channel spends half its time open, and the gating charge is related to the steepness of the voltage dependence; that is, how many charges move across the membrane as the channel gates open and close. Finally, single-channel kinetics complete the fingerprint of each channel. The mean open and closed times are the average times the channel spends in the open and closed states, respectively, at a particular voltage. Further analyses of the distribution of these open and closed times are used to reveal diVerent kinetic states. These parameters, as well as the conductance and ion selectivity, then set the stage for a myriad of pharmacological and other eVector studies.

B. General Approaches There are three general approaches to apply electrophysiological techniques to mitochondria. These approaches include patch-clamp, tip-dip, and planar bilayer methodologies. Each approach has its advantages and shortcomings. All three approaches rely on voltage clamp techniques. Under these conditions, the amplifier injects current to maintain a constant applied voltage. It is these currents that are monitored to reveal the single-channel characteristics. Neher and Sakmann received the Nobel Prize in 1991 for their work in the development of patch-clamp techniques (Hamill et al., 1981). Patch-clamp techniques allow recording of the currents flowing through ion channels in their native membranes as well as after reconstitution in proteoliposomes (see following discussion). Micropipettes to be used on mitochondrial preparations are fabricated with a pipette puller so that the tip has a relatively low resistance (10–30 M) and a small opening of about 0.2–0.5 mm, befitting the size of a mitochondrion. The micropipettes are filled with a conducting solution, for example, 150 mM KCl; mounted on a micromanipulator; and connected to an amplifier through a silver/silver chloride wire. The microelectrode is moved adjacent to the mitochondria, and a tight seal is formed between the glass tip and the membrane either spontaneously or after application of a small negative pressure (see Fig. 2). The seal has high electrical resistance and is usually referred to as a gigaseal, as it has gigaohm resistance. There are a variety of patch-clamp modes, which include attached, excised (inside out), and whole mitoplast configurations (see Fig. 2). In the attached mode, a micropipette is sealed directly on the mitochondrion or mitoplast. Drawing the micropipette away from the mitochondrion after the gigaseal is formed results in the excised patch mode, in which a small patch of

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FIG. 2 Patch-clamp configurations on native membranes and proteoliposomes. A high-resistance seal (gigaseal) is needed for patch-clamp recordings; it is formed between a glass micropipette (10– 30 M, with a diameter of 0.2–0.5 mm) and the mitochondrial membranes by application of a slight negative pressure. Ion currents through channel proteins within the isolated membrane patch can then be recorded under voltage clamp conditions. (A) This mode is called attached patch configuration. Two other modes of measuring currents through ion channels can be achieved. The membrane patch can be excised from the surrounding membrane (B) by pulling the micropipette away from the rest of the membrane of, for example, a mitoplast. In this conformation, a mitoplast patch is said to be inside out as the matrix face of the inner membrane is now exposed to the bath. While in the attached mode on a mitoplast, the membrane patch can be ruptured to achieve a direct junction between the solution in the micropipette and the matrix. In this whole mitoplast configuration (C), the ion flux through all ion channels in the entire inner membrane can be recorded. Sequential phase-contrast images show seal formation between the microelectrode and a mitochondrion (D–F), and then a proteoliposome (G), to achieve an attached patch configuration. Images were taken with a SPOT camera through a 40 lens; scale bars are indicated.

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membrane is studied in isolation away from the mitochondrion. Under these conditions, the medium bathing both sides of the membrane patch can be controlled, and eVectors can be directly added to the channels. However, important internal eVectors may be lost under these conditions. The whole mitoplast mode is accomplished by disrupting the membrane inside the micropipette opening without losing the gigaseal while in the attached mode. This method enables the recording of many channels at once in the entire inner or outer membrane. Figure 2 also illustrates seal formation on a mitochondrion and a proteoliposome in attached mode. The tip-dip method allows for examination of organelle channels in an artificial bilayer within a microelectrode (Henry et al., 1989). Micropipette tips with the same dimensions as those used for patch-clamp experiments are submerged vertically with respect to the bath solution (often 1 well of a 96-well plate) as shown in Fig. 3A. Lipids in an organic solvent, for example, decane, are layered on top of the bath. The lipids arrange themselves as a monolayer on the surface with their hydrophobic tails in the air and polar head groups in the aqueous solution. The micropipette is brought out of the bath, and the lipid molecules then spontaneously form a monolayer at the tip. A bilayer forms as a sandwich when the micropipette is again submerged in the bath. Channels can be added to the bath as membrane vesicles, in a detergent-solubilized form, or in liposomes that spontaneously insert/fuse into this bilayer. Planar bilayer methods are similar to tip-dip methods in that they too rely on reconstitution of channels in artificial membranes. Two compartments are separated by a hole typically 100–200 mm in diameter in a Teflon cup. Planar bilayers are formed either by ‘‘painting’’ the hole with a lipid solution or by raising the fluid level of the baths above the hole after lipid monolayers have formed in the two compartments (Montal and Mueller, 1972). As in the tipdip method, channels spontaneously insert into the planar bilayer after their addition to the bath.

III. Channel Preparations A. Channel Activity in Native Membranes The characterization of channels in their native membranes and environment should be the ‘‘gold standard’’ and, at this time, can be done only by patchclamp techniques. However, access to the native mitochondrial membranes can present problems. For example, the plasma membrane presents a barrier that must be bypassed while the microelectrode tip is kept clean; a clean tip is essential for formation of a gigaseal between the glass tip and the membrane of interest. Jonas et al. (1999) were the first to record channel

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FIG. 3 The tip-dip method. Ion channels can be reconstituted into bilayers formed at the tip of a microelectrode by the tip-dip method. A microelectrode is submerged in a bath. Lipids (typically in an organic solvent such as decane) are layered on top of the bath and allowed to form a monolayer on the bath surface (A). The microelectrode is raised out of the bath (B). A bilayer is formed like a sandwich as the microelectrode is again returned to the bath (C). Channels, either in detergent, proteoliposomes, or membrane fractions, are added to the bath and insert spontaneously (not shown).

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activity from mitochondria within a cell by patch-clamp techniques. They developed a double-barrel tip in which the patch micropipette is protected as the plasma membrane is punctured. To our knowledge, no such recordings exist for the protein import channels at this time. Furthermore, singlechannel studies of the Tom channel in isolated mitochondria have been hindered by the abundance of another outer membrane channel, the voltagedependent anion-selective channel (VDAC). As shown, this diYculty has been overcome by directly patch-clamping mitochondria isolated from cells deficient in VDAC. The micropipette must have access to the inner membrane in order to record the Tim23 and Tim22 channels. Access to the native membrane is obtained by peeling away most of the outer membrane, by French pressing (Decker and Greenawalt, 1977; Lohret and Kinnally, 1995a) isolated mitochondria that have previously been osmotically shrunken. Alternatively, osmotic swelling of mitochondria ruptures the outer membrane to expose the intact inner membrane (Grigoriev et al., 2003; Zorov et al., 1992). Incubation of mitochondria in 10- to 30-mOsm medium for 10–20 min on ice is usually suYcient to rupture the outer membrane. Mitochondria in which the inner membrane is exposed are referred to as mitoplasts (Fig. 4). Extensive studies of the Tim23 channel have been carried out in mitoplasts (Lohret and Kinnally, 1995a,b; Lohret et al., 1996, 1997; Muro et al., 2003). At this time, there are no single-channel recordings of the Tim22 channel in the native inner membrane.

B. Reconstitution Miller (1986) aptly indicated that artificial model systems can often overcome the obstacles presented by studies of native membranes and provide a simpler system with which to study the behavior of channels. Whenever possible, comparisons of the channel activities recorded in model systems should be made with that of native membranes. Proteoliposomes formed by fusion of purified inner and outer membranes with liposomes are often studied by patch-clamp techniques. These preparations are perhaps most similar to native membranes as the channel activity is almost indistinguishable from that recorded from isolated mitochondria and mitoplasts. Methods to separate and purify mitochondrial inner and outer membranes are illustrated in Fig. 4 (Lohret et al., 1997; Muro et al., 2003). Isolated mitochondria are shrunken in a 0.6-Osm sucrose–mannitol solution in the presence of EGTA to minimize contact sites between the inner and outer membranes. The preparation is then French pressed to peel the outer membrane from the inner membrane (Decker and Greenawalt, 1977). Additional eVorts to detach the membranes include subsequent swelling and

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FIG. 4 Various configurations to patch mitochondrial membranes and prepare proteoliposomes. Mitochondria can be isolated from various sources (e.g., yeast, rat liver, tissue cultured cells) by previously described methods (Campo et al., 1992; Lohret et al., 1996; Muro et al., 2003). Mitochondria can be patch-clamped directly for the outer membrane channels. Mitoplasts are prepared from isolated mitochondria by the French press method (Decker and Greenawalt, 1977; Guo et al., 2004; Lohret et al., 1997; Pavlov et al., 2001) and can then be patch-clamped directly for inner membrane channels. At the same time, the mitochondrial outer membranes (MOMs) are harvested. The mitochondrial inner membranes (MIMs) can be further purified according to Mannella (1982). The purity of the membrane fractions can be analyzed by SDS–PAGE, Western blotting, and immunodecoration with antibodies against components of the inner (e.g., ATPase or cytochrome oxidase subunit IV) and outer (VDAC) membranes. As shown in Western blots, crosscontamination is typically <5%. Inner and outer membranes can be separately reconstituted into giant proteoliposomes by dehydration–rehydration as previously described (Muro et al., 2003), using lipids (e.g., soybean l-a-phosphatidylcholine) (Sigma type IV-S). These proteoliposomes are heterogeneous, but many are 3–5 mm in diameter and are easily patch-clamped to detect single-channel activity.

homogenization. The inner and outer membranes are then separated either on a sucrose or Ficoll gradient (Mannella, 1982). These approaches can generate membranes that have less than 5% cross-contamination as shown by Western blotting for inner and outer membrane proteins (Fig. 4). Reconstitution protocols depend on the channel activity of interest. Incorporation of Tom and Tim23 channels into giant proteoliposomes is best done by modifications of the dehydration–rehydration method of Criado and Keller (1987) as described by Lohret et al. (1997). Briefly stated, 10–50 mg of purified outer (for Tom) or inner (for Tim23) membranes is mixed with 1 mg of azolectin liposomes (previously prepared by sonication of lipids in water) and brought to a final volume of 50 ml of 5 mM Tris, pH 7.4. The mixture is dotted on a slide and dehydrated for about 3 h or until dry at 4 C.

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Rehydration is accomplished by covering each drop with 5 mM Tris for an

overnight incubation at 4 C. Although heterogeneous, many of the resulting proteoliposomes are several microns in diameter and are easily patch-clamped. Proteoliposomes are either used directly or harvested, aliquoted, and stored at

80 C. The preparation is most active when used immediately as freezing breaks about half the giant unilamellar proteoliposomes. However, these preparations, once made, can be used for several months with little detectable change in the single-channel behavior for Tim23 and Tom activities. Unlike Tom and Tim23 channels, the other outer membrane–resident channel, VDAC, is more eVectively reconstituted by a freeze–thaw protocol. The same mixture of small liposomes and purified membranes is rapidly frozen in liquid nitrogen and thawed on ice three times. The resulting proteoliposomes are smaller than those prepared by dehydration–rehydration, but channel activity is readily detected. The native orientations of the Tom and Tim23 channels in these membranes are, for the most part, maintained after reconstitution into proteoliposomes by the dehydration–rehydration method (Lohret et al., 1996). As shown in the Western blots of Fig. 5, trypsin treatment of mitochondria cleaved Tom70p, the accessibility of which to trypsin was similar in mitochondria and proteoliposomes containing mitochondrial outer membranes. Similarly, Tim23p was cleaved by trypsin in both mitoplasts and proteoliposomes containing mitochondrial inner membranes (Fig. 5). Calibration of the protease eVects by densitometry of Western blots indicated that 80–90% of the TIM and TOM complexes maintained their native conformation after reconstitution into proteoliposomes. Notably, the native asymmetry of voltage dependence is recorded from >95% of all Tim23 and Tom channels, a finding that strongly

FIG. 5 Proteoliposomes containing inner and outer membranes maintain the native orientation of Tim23 and Tom channels. Trypsin treatment (200 mg/mg protein for 5 min) of mitochondria and proteoliposomes containing mitochondrial outer membranes cleaves Tom70p, indicating accessibility to Tom70p by trypsin is similar in mitochondria and proteoliposomes. Similarly, Tim23p is cleaved by trypsin in both mitoplasts and proteoliposomes containing mitochondrial inner membranes with the same sensitivity. The amount of protein (in micrograms) and the presence and absence of trypsin are indicated.

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indicates the channels reproducibly maintain their native orientation in proteoliposomes. Importantly, the Tim23 channel activity recorded from the native membrane of mitoplasts, isolated from a yeast strain deficient in VDAC1, was identical to the channel activity reconstituted in proteoliposomes containing mitochondrial inner membranes of a double-deletion mutant of VDAC (data not shown) (Kinnally et al., 1996; Lohret and Kinnally, 1995a). Tom channel activity was originally discovered by the tip-dip method (Chich et al., 1991; ThieVry et al., 1987, 1988). The experimental approach was later verified by observation of the same activity when patching proteoliposomes and through incorporation into planar bilayers (ThieVry et al., 1992). The same is not the case for Tim23 channels. Whereas virtually indistinguishable activity is recorded from mitoplasts and proteoliposomes prepared by dehydration– rehydration with mitochondrial inner membranes, recombinant Tim23 channel activity recorded in planar bilayers is significantly diVerent (Truscott et al., 2001). These diVerences are discussed in Section IV.A. A powerful advantage of tip-dip and planar bilayer studies is the ability to control the number of channels incorporated during an experiment by simply adding more or removing material from the bath. The inclusion of a small amount of detergent (e.g., Triton X-100 or CHAPS) and/or a concentration gradient, as well as working at high ionic strength (i.e., 1 M salt), often facilitates channel insertions in both tip-dip and planar bilayer studies. Regulating the number of channels in proteoliposome patches is done by varying the protein-to-lipid ratio before freezing or dehydrating the mixture or by changing the tip diameter of the microelectrodes. Biochemical and molecular advances have enabled studies of purified TOM, TIM23, and TIM22 complexes and of recombinant Tom40p, Tim23p, and Tim22p proteins. For the most part, these preparations have relied on reconstitution into planar bilayers to establish their single-channel behaviors. As delineated previously, there are several electrophysiological approaches to studying protein translocation channels and a variety of reconstitution protocols, each with their own positive and negative attributes.

IV. Protein Translocation Channels A. Electrophysiological Characteristics of Tim23, Tim22, and Tom Channels Tim23 and Tom channels have almost identical single-channel characteristics. For this reason, the electrophysiological behavior of these channels is presented together for comparison after an introduction to both channels. If data are available, the comparisons are extended to the Tim22 channel.

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1. Tim23 Channel Activity as the Electrophysiological Manifestation of the Pore of the TIM23 Complex The TIM23 complex is responsible for the translocation of proteins across the inner membrane and occasionally into the inner membrane. Precursor proteins using the TIM23 complex typically have a presequence that is both cationic and amphipathic in order to target the precursor proteins to the matrix. Tim23 channel activity of yeast mitochondria was first described in 1995 by patch-clamping mitoplasts (mitochondria with the outer membrane stripped away), and this channel was referred to as the multiple conductance channel (MCC) in early work (e.g., Lohret and Kinnally, 1995a). In short order, the first link of this channel activity to protein import across the inner membrane was made as synthetic peptides whose sequences mimic that of the presequences modified this single-channel behavior (Lohret and Kinnally, 1995b). Considerable evidence indicates that this channel activity corresponds to that of the TIM23 complex. Antiserum against Tim23p that inhibits protein import into mitoplasts also blocks the flow of current through the Tim23 channel (Jensen and Kinnally, 1997; Lohret et al., 1997; Ryan and Jensen, 1995). Furthermore, a mutation that renders the strain import deficient displays altered Tim23 channel activity (Lohret et al., 1997). More recently, recombinant Tim23 was found to display channel activity in planar bilayers. This channel activity is triggered and reversibly regulated by synthetic presequence peptides and not by control (nontargeting) peptides (Kinnally, 2002; Kushnareva et al., 1999, 2001; Lohret and Kinnally, 1995a; Lohret et al., 1997). The maximum pore diameter of the Tim23 channel is estimated at 2 nm, which is large enough to accommodate an unfolded protein (Kinnally, 2002; Kinnally et al., 2000). Targeting peptides for mitochondria are positively charged and Tim23 channels are cation selective (Kinnally et al., 1996; Lohret and Kinnally, 1995a). Furthermore, Tim23 channels share characteristics with other protein import channel activities. The predominant transition size (500 pS) of the Tim23 channel is the same as that of channels implicated in protein import in the endoplasmic reticulum and Escherichia coli cell membrane (Beckmann, 1997; Menetret, 2000; Simon and Blobel, 1991, 1992). EVects of peptides on the E. coli and Tim23 channels are voltage dependent, with a similar dose dependence (Lohret and Kinnally, 1995b; Simon and Blobel, 1992). Finally, the singlechannel characteristics of Tim23 are identical to those of Tom, the import channel of the outer membrane that has been studied in reconstituted systems (Henry et al., 1989, 1996; ThieVry et al., 1992). Both channels are high conductance, cation selective, and regulated by presequence peptides.

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2. Tom Channel Activity as the Electrophysiological Manifestation of the Pore of the TOM Complex The TOM complex is responsible for import of the vast majority of mitochondrial proteins encoded in the nucleus. Tom channel behavior was first reported in 1987 by the laboratories of TheiVry and Henry, and 1 year passed before this activity was attributed to mitochondria (ThieVry et al., 1987, 1988). They quickly realized that this activity, previously referred to as a peptide-sensitive channel (PSC) in earlier work, is associated with protein import. The channel is large, as indicated by its conductance, and a peptide resembling a presequence modifies the channel activity. Importantly, immunoprecipitation of mitochondrial extracts (solubilized in 0.75% octylglucoside) with antibodies against Tom40p correlated with loss of Tom channel activity in planar bilayer experiments (Juin et al., 1997). Furthermore, these antibodies modified the conductance of the Tom channel (Ku¨nkele et al., 1998b). Channel activity similar to that of the Tom channel is detected in planar bilayers on incorporation of purified TOM complex (Ku¨nkele et al., 1998a) and of bacterially expressed Tom40p, the general insertion pore of the TOM complex (Hill et al., 1998). As detailed later, single-particle analysis of purified TOM complexes reveals structures usually containing two or three ‘‘holes,’’ which likely correspond to the pores of the Tom channels (Ku¨nkele et al., 1998a). 3. General Single-Channel Characteristics of the Tim23, Tim22, and Tom Channels of Yeast Patch-clamp techniques are used to directly compare the single-channel properties of the Tim23 and Tom channels in proteoliposomes containing either purified inner or outer membranes, respectively (Muro et al., 2003). As shown by the current traces of Fig. 6A and B, the conductance of the open states of the Tim23 and Tom channels are the same, 1000 pS. Interestingly, both channels have a major half-open state of 500 pS. Tim23 and Tom channels have the same function and share many singlechannel characteristics (Table I). Both channels are voltage dependent and occupy lower conductance levels at small positive potentials, as shown in the current traces, current voltage curves, and open probability (Po) versus voltage plots of Fig. 6A–D. The parameters associated with voltage dependence are indistinguishable for both channels, for example, the gating charge and V0 as shown in Fig. 6E–H. These findings may be surprising as the Tim23 channel is expected to operate at high potentials across the inner membrane, whereas the Tom channel would normally be influenced by low potentials across the outer membrane. The kinetics of channel opening and closing are a reflection of channel conformations with diVerent energy states. For example, rapidly flickering channels have a small energy barrier separating the open and closed states.

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FIG. 6 Single-channel properties of Tim23 and Tom channels are identical. (A and B) Current traces of Tim23 and Tom channels are shown at various voltages with 2-kHz filtration in symmetrical 150 mM KCl–5 mM HEPES, pH 7.4. O, open; c, closed; s, substate (half-open). (C and D) Current–voltage curves of Tim23 and Tom channels show asymmetric voltage dependence closing at lower positive than negative potentials. Current is relative to bath at 0 mV. Open probability (Po) is determined from amplitude histograms (not shown) for recordings (30 s in duration) of Tim23 and Tom channels at various voltages and again shows asymmetric voltage dependence for both channels (E and F). Gating charges of Tim and Tom channels are identical as shown in ln[Po /(1  Po)] plots (G and H). Best fit lines are shown with 95% confidence intervals as dashed lines. Parts of Fig. 6 are reprinted from Muro et al. (2003).

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TABLE I Comparison of Single-Channel Behaviors of Yeast Tom, Tim23, and Tim22 Tom channel

Tim23 channel

Tim22 channela

Peak conductance (pS)

1060  170

1160  140

324  11

Predominant transition (pS)

496  63

490  43

40  3

Pore diameter (nm)

1.88  0.09

1.87  0.08

1.8–2.3

Gating charge

4.3  1.6

4.2  0.5

ND

V0 (mV)

23  10

37  8

ND

Permeability, Kþ/Cl

4.7  0.3

5.0  0.3

3.9

Multiple kinetic states

>3

>3

ND

Mean open time (ms)b

14.5  1.4 (n ¼ 5)

10.6  4.3 (n ¼ 13)

ND

Mean closed time (ms)

1.0  0.5 (n ¼ 9)

0.5  0.2 (n ¼ 11)

ND

Mean burst length (s)

0.4  0.2 (n ¼ 4)

0.5  0.31 (n ¼ 4)

ND

a b

Recalculated from Kovermann et al. (2002). 20 mV.

Remarkably, most kinetic measures of the Tim23 and Tom channels are similar. The mean open and closed times, as well as the burst lengths, are indistinguishable as shown in Table I. In agreement with earlier studies of Tim23 channels, further kinetic analysis showed that open and closed time distributions could usually be best fit with one to three exponentials, supporting the existence of multiple kinetic states (Lohret and Kinnally, 1995a). As shown in Fig. 7, the dwell time constants for the open, substate, and closed levels of Tim23 and Tom channels are similar and reflect the presence of multiple conductance and kinetic states for both channels. The ability to allow selective permeation of various ions is a classic characteristic of ion channels. However, in keeping with the enormous pore size necessary to allow permeation of an unfolded protein, the ion selectivity of Tim23 and Tom channels can be described only as poor. Although both Tim23 and Tom channels are slightly cation selective, their ability to distinguish anions from cations is low as the permeability ratio for PK/PCl is approximately 5 [see Eq. (3) in Section II.A]. Interestingly, this selectivity may be related to recognition of the typically positively charged presequences. B. Pores of Tim23 and Tom Channels Electrophysiological studies also allow predictions of channel architecture. Most ion channels have a single pore, but a few are double- and/or triple-pore channels, such as bacterial porin (Phale et al., 1998). The peak

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FIG. 7 Dwell time distributions for Tim23 and Tom channels reveal multiple kinetic states. Open (1-nS conductance) dwell time distributions for Tim23 (A) and Tom (D) channels in proteoliposomes prepared with purified membranes from yeast mitochondria are fit with the sum of two exponentials. Substate (500-pS conductance) time distributions are fit with a single exponential function for both Tim23 (B) and Tom (E) channels. Closed dwell time distributions (0 nS) for Tim23 (C) and Tom (F) channels are fit with the sum of two exponentials. Fits are shown in gray and data are black. Dwell time constants (t) are as indicated in each plot, and the number of events was typically 5000–6000 events for each conductance level. These data indicate Tim23 and Tom channels have multiple kinetic states with at least two open, two closed, and one subconductance level states. Reprinted from Muro et al. (2003).

conductances of the Tim23 and Tom channels are typically 1000 pS, which, using the method of Hille (2001) [Eq. (2) in Section II.A] would correspond to a single-pore diameter of 2.4–2.7 nm, assuming a pore length of 5.5–7 nm. However, both channel activities have a ‘‘half-open’’ substate corresponding to 500 pS in yeast. Hence, two 1.7- to 1.9-nm pores that form a double barrel and that cooperatively gate could also account for the peak conductances and

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predominant transition sizes of the Tim23 and Tom channels. Although these calculations are a simplification, they can provide the basis of discussion. Regardless of the definition of a single- or double-barrel pore, the conductance calculations indicate the pore diameters of the Tim23 and Tom channels are suYcient to accommodate unfolded polypeptides during translocation. The polymer exclusion method can be used to further define the characteristics of the pore of a channel. Briefly, current records are obtained in the presence of an uncharged polymer (5–15%, w/v) of various molecular weights. If the polymer is permeable, the current inside the channel pore decreases as the polymer displaces a fraction of the ions that normally carry the current. If the polymer is not permeable, the current does not decrease any more than can be accounted for by changes in solution conductivity. Although sugars can be used, polyethylene glycols (PEGs) and dextrans of various molecular weights are used most commonly as they are available in a large range of molecular weights and are thought to be spherical, at least below a molecular weight of 10,000. As shown in Fig. 8A–D, the relative conductances and transition sizes of the Tim23 and Tom channels are decreased by lowmolecular-weight PEGs, but are not aVected by MW 1450 or 3350 PEGs after corrections for solution conductivity. The polymer exclusion method can be used to further compare the pores of Tim23 and Tom channels. Although the conductance of the Tom channel is slightly reduced by MW 1000 PEG, the Tim23 channel is not aVected as shown in Fig. 8C and D. Therefore, the pore of the Tom channel is slightly larger than that of the Tim23 channel. Plots of the second derivative of the relative conductance and polymer radius provide further information regarding the vestibule of the pore structure (Fig. 8E and F). Hence, the polymer exclusion method predicts that the diameters of the pores of Tom and Tim23 channels are 2.0 and 1.8 nm, which closely approximates the diameters calculated by the simplified method of Hille (2001), assuming the channels have a double-barrel pore. Similar studies of the Tim22 channel have been undertaken (Kovermann et al., 2002, Rehling et al., 2003) and indicate this channel also has a size similar to that of Tim23 and Tom channels. Total amplitude histograms are compilations of the time spent in various conductance states and are used to determine the open probability, Po. Channels typically have a single open and closed state, which gives rise to a high and a low current level, respectively. For a single channel, the probability of being in the closed state is 1P, if P is the probability of being in the open state. The distributions of the currents can typically be fit by Gaussian distributions between the open and closed states, which can be predicted even if there is more than one channel in the current recordings, using the binomial Eq. (5): f ðxÞ ¼

n!Px ð1  PÞnx x!ðN  xÞ!

ð5Þ

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FIG. 8 Estimation of the pore sizes of Tim23 and Tom channels by the polymer exclusion method. Current traces of Tim23 and Tom channels are shown in the absence and presence of polyethylene glycols (PEGs) of various molecular weights (A and B). The relative conductance of Tim23 (C) and Tom (D) channels in the presence (g) and absence (go) of PEG is shown as a function of estimated PEG radius (in nanometers). The second derivative of the data fit from (C) and (D) reveals the restriction radii for small and large PEGs in the inner and outer parts of Tim23 (E) and Tom (F) channels.

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where N is the number of channels, x is the number of channels in the open state, and P is the open probability. There are three conducting states that correspond to the open (1000 pS), half-open (500 pS), and closed (0 pS) states for both the Tim23 and Tom channels. Two independent 500-pS channels or a double-barrel channel could account for this observation. Because the distribution of the amplitude histograms for both the Tim23 and Tom channels is not always Gaussian, these pores are likely to be double barrels (Fig. 9B and D). The amplitude histograms fit a Gaussian distribution when the open probability is high (Fig. 9A and C). However, distributions that were simulated to fit the conductance level for two open and independent 500-pS channels (O) poorly predicted the distributions for the two closed (C) or one open/one closed (S) amplitude histograms in many experiments at higher voltages (Muro et al., 2003). These data suggest that the gating of these putative double-barrel pores is cooperative. Importantly, single-particle analyses of TOM complexes suggest the Tom channel has a double-barrel, and perhaps a triple-barrel, structure (Ku¨ nkele

FIG. 9 Fit of total amplitude histograms of Tim23 and Tom channels to Gaussian distributions. Total amplitude histograms at þ20 and þ40 mV are shown for Tim23 (A and B) and Tom (C and D) for experimental data (black) and data simulations (gray) fit to the probability of occupying the 1-nS level, assuming two independent channels and a Gaussian distribution. Histograms are not leak subtracted. Simulations were generated by Electrophysiology Data Recorder version 2.2.3 software (Dempster, University of Strathclyde, Glasgow, Scotland). The durations of open (O) substate(s), and closed (C) intervals, were analyzed at a resolution of 200–500 bins. Reprinted from Muro et al. (2003), with permission.

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et al., 1998a; Model et al., 2002; Rehling et al., 2003) (see Fig. 10B). Although similar studies of the Tim23 channel have not as yet been fruitful, it would not be surprising if the structures were comparable to that of the Tom channel. Interestingly, the Tim22 channel also presents current traces with three conductance levels including a half-open major substate. Importantly, single-particle analysis of the TIM22 complex, like the TOM complex, appears to be a double/triple-barrel pore as shown in Fig. 10.

FIG. 10 Purified TIM22 and TOM complexes contain two coupled pores. (A) Current traces of the TIM22 complex in the presence or absence of internal signal peptide P2 (100 nM) are shown at the indicated voltages under symmetric buVer conditions. (B) Single-particle electron micrographs of purified TIM22 and TOM complexes show both complexes have two pores. Scale bar: 10 nm. Reprinted with permission from Rehling et al. (2003).

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C. Activity of Tim23 and Tom Channels is Modified by Presequence Peptides Presequence or targeting peptides are synthetic peptides whose sequences mimic the targeting regions of mitochondrial precursors, for example, cytochrome oxidase subunit IV. Functional mitochondrial presequences contain several basic amino acids and are thought to fold into amphipathic a helices (Roise and Schatz, 1988). Increasing the net positive charge and/or length of presequence peptides increases their ability to competitively inhibit protein import (Glaser and Cumsky, 1990), whereas similar changes in presequences increased the eYciency of protein import (Isaya et al., 1988; Martin et al., 1991). SynB2 is a synthetic peptide that does not support protein import, that is, function as a targeting signal (Allison and Schatz, 1986). SynB2 is cationic and predicted to be a helical, but this peptide has low amphipathicity and is usually used as a negative control in these types of experiments. Presequence peptides modify the activities of Tim23 and Tom channels. These peptides do not modify the behaviors of two other mitochondrial channels, VDAC and mCS (Lohret and Kinnally, 1995b). In the absence of peptide, Tim23 and Tom channels of yeast have predominant transition sizes of 500 pS, peak conductances of 1000 pS, and mean open times of 10–20 ms at 20 mV (see Table I). Transitions to the major subconductance level of 500 pS of Tim23 and Tom channels are visualized as downward deflections in the current traces of Fig. 11 and are relatively infrequent in the absence of peptide or in the presence of the control peptide SynB2. However, current traces reveal large-amplitude, rapid flickering between the open (1000 pS), subconductance (500 pS), and closed states in the presence of the targeting peptide yCOX-IV1–13 (the first 13 amino acids of yeast cytochrome oxidase subunit IV) for both Tim23 and Tom channels. There is a 4- to 8-fold increase in the number of transition events in the presence of yCOX-IV1–13 compared with the absence (control) and the presence of SynB2 as shown in the histograms of Fig. 11C for both channel activities. These eVects can be quantified as an increase in flicker rate (events per second) or a decrease in mean open time. The mean open time decreases from 10–20 ms to below 5 ms in the presence of presequence peptide. Similarly, the flicker rate increases from 50–100 to 250–800 events/ s when presequence peptides are present. As expected, the open probability decreases as more time is spent in the substate and closed state in the presence of the presequence peptides. Table II summarizes the eVects of diVerent presequence and control peptides on Tim23 and Tom channels. Dose dependencies for the eVects of presequence peptides are the same for Tim23 and Tom channels (Fig. 11D). The micromolar concentrations of the precursor peptides needed to induce these eVects on the two channels (Lohret and Kinnally, 1995b; Lohret et al., 1997; Muro et al., 2003) are similar to those

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FIG. 11 Targeting peptides modulate Tim23 and Tom channel activities. (A and B) Typical current traces are shown at þ20 mV from patches excised from proteoliposomes containing yeast mitochondrial inner membranes for Tim23 channels and outer membranes for Tom channels before (Control, no peptide) and after sequential perfusions of the bath with 50 mM control peptide SynB2 and then with 50 mM yCOX-IV1–13. See Table II for peptide details. O, S, and C correspond to the open (1000 pS), half-open substate (500 pS), and closed conductance levels. (C) Histograms of flicker rates (number of transition events per second) in the absence (control) and presence of SynB2 or yCOX-IV1–13 are similar for Tim23 and Tom channels. (D) Plot showing the dependence of the flickering rate of Tim23 and Tom channels on the concentration of targeting peptide yCOX-IV1–13. Reprinted from Muro et al. (2003), with permission.

known to competitively inhibit protein import (Allison and Schatz, 1986; Glaser and Cumsky, 1990). Presequences that are more positively charged or longer are better competitive inhibitors of protein import (Glaser and Cumsky, 1990). The ability of presequence peptides to induce rapid flickering of Tim23 and Tom channels is similarly sensitive, that is, more positively charged or longer peptides have lower eVective concentrations. For example, 2 mM yCOXIV1–22 or yCOX-VI (þ5 net charges) has about the same eVect on Tim23 channels as 20 mM fCOX-IV or yCOX-IV1–13 (þ3 net charges). Peptide sensitivity is a voltage-dependent phenomenon (see current– voltage curves in Fig. 12) in that rapid flickering of Tim23 and Tom channels is observed when the electrochemical gradient favors movement of the targeting peptides across the membrane. Hence, cationic peptides are eVective when placed in either the bath at positive potentials or the microelectrode at negative potentials. This voltage dependence is a rectification consistent with

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TABLE II Presequence Peptides Regulating Tim23 and Tom Channel Activitiesa

Peptide

Sequence

Charge Presequence

Tim23 flicker

Tom flicker Yes

yCOX-IV1–13

1

MLSLRQSIRFFKY13

þ3

Yesb

Yes

yCOX-IV1–22

1

MLSLRQSIRFFKPATRTLCSSR22

þ5

Yesb

Yes

Yes

yCOX-VI

1

MLSRAIFRNPVINRTLLRAR20

þ5

Yesc

Yes

ND

fCOX-IV

3

RAPALRRSIATTVVRCNAET22

þ3

Yesc

Yes

ND

d

SynB2

MLSRQQSQRQSRQQSQRQSR

þ5

No

No

No

iVDAC

109

þ3

Noc

No

ND

nVDAC

1

0

Noc

No

ND

cVDAC

272

0

Noc

No

ND

RGAKFNLHFKQ119

MAVPAFSDIAKSANDLLNKD20 THKVGTSFTFES283

a

Modified from Lohret et al. (1997). Glaser and Cumsky (1990). c Predicted but not determined. d Allison and Schatz (1986). b

‘‘electrophoresis’’ of the peptides through the channel. There are concomitant shifts in amplitude histograms and open probability plots in the presence of the targeting peptides for both Tim23 and Tom channels (Fig. 12G–J). The closures of Tim23 and Tom channels induced by presequence peptides may correspond to an intermittent blockade of the conductance pathway as the peptides are translocated across the membrane. Alternatively, the closures have relatively long durations of microseconds, suggesting the closures represent some interaction of the peptide with the translocation apparatus that results in a destabilization of the open state. It is expected that translocation events for polypeptides of this size would be in the nanosecond time domain, which cannot be resolved with typical patch-clamp amplifiers. Perhaps, fast translocation events are obscured by the long-duration blockade associated with peptide interaction with receptors. The frequency of closing increases with dose and the magnitude of the potential that drives the positively charged peptide across the membrane. The peptides have little eVect if the polarity is reversed. These eVects of presequence peptides are typically reversible (Lohret and Kinnally, 1995b) (data not shown) and are accompanied by an increase in noise in the current traces of both Tim23 and Tom channels. This change in noise is evidenced by an increase in the width of the current traces in the presence of presequence peptides (50 mM yCOX-IV) (Fig. 11A and B). The normalized statistical variance of the ionic current increases 10-fold on introduction of presequence peptide for both Tim23 and Tom channels (Grigoriev and Kinnally, unpublished results). No significant change in noise is observed

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FIG. 12 Peptide sensitivity is a voltage-dependent phenomenon. Current–voltage curves of Tim23 and Tom channels are shown in the absence (A and B) and presence (C–F) of 50 mM yCOX-IV1–13. (G and H) Open probability-versus-voltage plots of Tim23 (G) and Tom (H) channels in the presence and absence of 50 mM yCOX-IV1–13. Total amplitude histograms for Tim23 (I) and Tom (J) channel at þ20 mV are shown in the presence and absence of 50 mM yCOX-IV1–13. Reprinted from Muro et al. (2003), with permission.

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FIG. 13 FITC-labeled yCOX-IV1–13 is imported into mitochondria under metabolizing conditions. Yeast mitochondria were incubated with FITC–yCOX-IV1–13 under metabolizing conditions for a total of 30 min. DiVerential interference contrast (A), fluorescence (B), and partial overlay (C) images are shown at 60 magnification. Fluorescence increased with time, and accumulation was blocked by the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP, 1 mM, not shown). Similar results are obtained with fluorescently labeled yCOX-IV1–22, but the synthetic peptide FITC–SynB2 does not accumulate (not shown).

with the control peptide SynB2. In summary, the presence of presequence peptides induces a modest decrease in mean conductance that is voltage dependent, reversible, and accompanied by an increase in noise. All these eVects are also observed when charged molecules are found to transit a pore by biochemical methods, for example, RNA through a-hemolysin channels and ATP through VDAC (Kasianowicz and Bezrukov, 1995; Kasianowicz et al., 1996, 2001; Rostovtseva and Bezrukov, 1998; Rostovtseva et al., 2002a,b). By inference, these eVects are consistent with a partitioning of these peptides into the pores and suggest that the presequence peptides transit through Tim23 and Tom channels. In keeping with the notion that precursor peptides are translocated through the pores, fluorescently labeled (fluorescein isothiocyanate, FITC) yCOX-IV1–13 is imported into mitochondria (Fig. 13) in a membrane potential–dependent manner, whereas the control peptide SynB2 is not transported (not shown). The eVects of presequence peptides provide a functional assay for the eVects of mutations of the TIM23 and TOM complex components, as well as biochemical modifications, for example, protease treatments and pH dependence. This assay allows for a link to be made between the function of the apparatus and the ramifications of structural modifications.

D. Biochemical and Molecular Modifications of TIM23 and TOM Complexes That Change Their Channel Activities Many of the components of TIM23 and TOM complexes are essential, which has made studies of knockouts and other mutations more diYcult. Several investigators have avoided this problem by using inducible promoters, so that

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loss of a particular component is not lethal for the strain. For example, strains of yeast have been developed in which the genes for several components, for example, Tim17 and Tim23, are dependent on the presence of galactose (Milisav et al., 2001) or some other controller. These strains are presently being characterized for their eVects on Tim23 and Tom channel activities (Martinez-Caballero, Campo, and Kinnally, unpublished results). Others have developed temperature-sensitive strains (Emtage and Jensen, 1993). However, the application of biochemical modification (e.g., pH and protease treatment) has also been fruitful, as detailed later. 1. pH Dependence Mitochondria maintain a pH gradient across their inner membranes as a result of respiratory chain activity. Whereas protein import into mitochondria and mitoplasts is not aVected by increasing the external pH to as high as pH 9.5, accumulation of mature proteins is inhibited by lowering the external pH (Fig. 14). The median eVective concentration (EC50) for proton inhibition of protein import into mitochondria is pH 6.5, and into mitoplasts it is pH 5.9. Whereas the open probabilities for Tim23 and Tom channels in proteoliposomes are high at alkaline pH (pH 9.5), as shown in the current traces and plots of Fig. 14, acidification of the bath reduces the open probabilities in a proton concentration–dependent fashion. There are concomitant changes in the probabilities of occupying the substate and closed state for both Tim23 and Tom channels (Fig. 14D and E). The EC50 of proton-induced half-closure of the Tim23 channel is pH 6.4, whereas that of the Tom channel is pH 5.7. The pH can be used to probe the relationship between protein import and the channels associated with the import apparatus. Importantly, the EC50 for protein import into mitochondria is indistinguishable from that of the fully open state of the Tim23 channel (Fig. 14). Therefore, Tim23 open probability may limit import of preproteins into mitochondria at acidic pH. Furthermore, the parallel decreases in import and open probability of the fully open state of the Tim23 channel (Fig. 14I) suggest that both pores of the Tim23 channel may need to be open in order for import to occur. Such a relationship does not exist between protein import and the open probability of the Tom channel. 2. Essential Components Revealed by Protease Sensitivity The Tim23 channel has an asymmetric sensitivity to trypsin. Trypsin treatment of the matrix face of the inner membrane has no detectable eVect on the peptide sensitivity of the Tim23 channel. When patches excised from proteoliposomes are perfused with medium, the flicker rates increase severalfold in

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FIG. 14 pH dependence of protein import into mitochondria and mitoplasts. Current traces of Tim23 (A) and Tom (B) channels were recorded from excised patches from proteoliposomes containing purified inner and outer membranes of yeast mitochondria. Current traces are shown after perfusion of the bath with medium at the indicated pH, with the microelectrodes filled with 150 mM KCl–5 mM HEPES, pH 7.4. O, S, and C correspond to open state, substate, and closed state, respectively. (C) The open (1-nS conductance state) probability (Po) of Tim23 (d) and Tom (s) channels was calculated from total amplitude histograms (30 s) of current traces at þ20 mV after perfusion of the bath with medium at the indicated pH while the micropipette buVer pH was 7.4. For several of the points, the pH of the bath and micropipette buVer was the same as indicated for both Tim23 (j) and Tom (u) channels. Proton EC50 values are pH 6.5 and 5.7, and Hill coeYcients are 1.1  0.1 and 1.8  0.3, for Tom and Tim23, respectively. Means  SE with a minimum of four determinations are shown. (D) The probability of occupying the substate (Ps) of Tim23 and Tom channels was calculated as described above. Proton EC50 values for increasing Ps are pH 6.4 and 5.9,

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the presence of targeting peptides before and after trypsin treatment, as illustrated in the current traces of Fig. 15A. This finding indicates that the components of the TIM23 complex important in presequence peptide regulation and/or closely associated with the Tim23 channel are not functionally modified by this proteolytic digestion. In contrast, mild exposure of the intermembrane space face to trypsin virtually eliminates the presequence peptide regulation of the Tim23 channel activity. Targeting peptides do not induce rapid flickering of the Tim23 channel after proteoliposomes are treated with trypsin, as shown in the current traces of Fig. 15B. Furthermore, the same loss of Tim23 peptide sensitivity is observed if mitoplasts are treated with trypsin before reconstitution (Fig. 15C). Interestingly, the ability of presequence peptides to induce flickering of the Tim23 channel is disrupted on both faces, as targeting peptides had no eVect when present in the bath or microelectrode (Fig. 15C). Western blots of Fig. 15D show a loss of Tim23p with no change in the levels of Tim44p, Tim17p, or mtHsp70. These findings are consistent with a pivotal role of Tim23p in the peptide regulation of this channel. This role is further supported by studies of the tim23.1 mutant, in which a point mutation results in a loss of peptide sensitivity of the Tim23 channel (see Section IV.D.4). 3. Trypsin Treatment Indicating Tom20p and Tom70p Are Not Essential for Regulation of Tom Channel Activity by Presequence Peptides Protein import is inhibited by pretreatment of mitochondria with trypsin (Lithgow and Schatz, 1995). Protease treatment was used to probe the structure of the TOM complex to evaluate the contribution of various components to the regulation of Tom channel activity by targeting peptides. The model in Fig. 16A illustrates the orientation of the TOM complex with and Hill coeYcients are 1.4  0.2 and 3.7  0.3, for Tim23 and Tom, respectively. (E) The probability of Tim23 and Tom channels occupying the closed state (Pc) was calculated as described previously. Proton EC50 values are pH 5.25 and 3.2, and Hill coeYcients are 1.4  0.1 and 0.3  0.1, for Tom and Tim23 channels, respectively. (F and G) Autoradiographs show inhibition of protein import into mitochondria and mitoplasts at low pH. P and M are preprotein and mature protein, respectively. Mature protein is processed after import and hence has a lower molecular weight. (H) Protein import, as indicated by mature protein band density, was normalized to that at pH 7.4. The EC50 values are pH 6.5 and 5.9 for mitochondria and mitoplasts, respectively. (I) Protein import capability is linked to Tim23 open probability. Media at various pH values were used to modify the open probability of Tim23 and Tom as in (A) and (B). The relationship between relative protein import function and the open probabilities of the Tim23 (d) and Tom (s) channels are shown. Whereas a best fit line for the Po of Tim23 channel and protein import has a correlation coeYcient of 0.975, data for the Po of Tom do not have a linear relationship with protein import. Modified from Grigoriev et al. (2003).

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FIG. 15 Trypsin treatment of the intermembrane space face of the inner membrane eliminates presequence peptide regulation of the Tim23 channel activity. (A) Current traces of the yeast Tim23 channel are shown in the presence and absence of 50 mM yCOX-IV1–13 in the bath before and after trypsin treatment of the matrix face (bath) of the inner membrane. (B) There is no increase in the flicker rate of the Tim23 channel after introduction of yCOX-IV1–13 if proteoliposomes (i.e., the intermembrane face) are trypsin treated. (C) The same loss of Tim23 peptide sensitivity is observed if mitoplasts are treated with trypsin before reconstitution, as shown by histograms of flicker rate. (D) Western blots using various antibodies show this trypsin treatment resulted in loss of Tim23p but not Tim17p, Tim44p, or mtHsp70. These data suggest loss of Tim23p is associated with loss of recognition of targeting signals.

respect to the membrane face treated with trypsin and targeting peptide. Mitochondria were isolated from VDAC-less yeast and treated without or with trypsin at two concentrations, 200 and 400 mg of trypsin per milligram of protein. As shown in the Western blots of Fig. 16B, these treatments generated three preparations containing diVerent intact TOM complex

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FIG. 16 Trypsin treatment of the cytosol side modifies the peptide sensitivity of the Tom channel. (A) Model illustrating the orientation of the TOM complex with respect to the membrane face treated with trypsin (symbolized by indented open circle in cytosol) and the targeting peptide yCOX-IV1–13. MOM, mitochondrial outer membrane; IMS, intermembrane space. (B) Western blots show loss of various components of the TOM complex in mitochondria treated with trypsin (200 or 400 mg/mg of protein). (C) Peptide sensitivity of the Tom channel lacking Tom70p, Tom20p, and Tom22p (membranes treated with trypsin at 400 mg/mg of protein) is lost as indicated by flicker rates with 50 mM yCOX-IV1–13. Current traces (D) and flicker rates (E) of Tom channel in the presence and absence of 50 mM yCOXIV1–13 in excised patches, before and after treatment with trypsin at 200 mg/mg of protein, show that there is no loss of targeting peptide sensitivity if Tom70p and Tom20p are missing. (F) Flicker rates determined in the presence of various concentrations of yCOX-IV1–22 show the shift in dose dependence caused by treatment with trypsin at 400 mg/mg protein.

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components. Untreated preparations contain Tom20p, Tom22p, Tom70p, and Tom40p. Preparations treated with 200 mg of trypsin per milligram of protein lack Tom20p and Tom70p. Preparations treated with 400 mg of trypsin per milligram of protein maintain Tom40p but lack Tom22p, Tom20p, and Tom70p. The Tom channel activity of these three preparations is compared later to evaluate the functional eVects of the structural modifications of the TOM complex. Tom channel activity reconstituted from outer membranes purified from mitochondria lacking intact Tom20p and Tom70p (200 mg of trypsin) has the same sensitivity to signal peptides as the activity from both
20
70 (see Section IV.D.4) and wild-type strains. That is, cleavage of Tom20p and Tom70p by trypsin has no eVect on flicker rates in the presence of yCOXIV1–13 on either membrane face, as shown in the histograms of Fig. 16C. As expected, identical results were obtained when proteoliposomes prepared from control outer membranes are trypsin treated to cleave Tom20p and Tom70p, but not Tom22p, as shown in the current traces and histograms of Fig. 16D and E. Extensive trypsin treatment of mitochondria causes a loss of Tom22p and abolishes regulation of the Tom channel by presequence peptides. Tom22p is another receptor associated with protein import across the mitochondrial outer membrane. It is relatively trypsin insensitive, but Tom22p can be cleaved with high levels that also disrupt protein import (Lithgow and Schatz, 1995). Mitochondria isolated from the single VDAC deletion strain of yeast (M22-2) were treated with trypsin (400 mg) to eliminate the Tom22p signal in Western blots (Fig. 16B), and Tom channel activity was evaluated after reconstitution. In contrast to that seen from membranes that were untreated or treated with 200 mg of trypsin, 50 mM yCOX-IV1–13 does not induce rapid flickering of the Tom channel (Fig. 16C). Furthermore, trypsin treatment causes a shift in the dose dependence of the flickering induced by yCOX-IV1–13 (not shown) and yCOX-IV1–22 (Fig. 16F). Flickering of the Tom channel increases from 30  12 to 88  64 events/s on addition of 20 mM yCOX-IV1–22 in preparations that contain Tom40p but lack Tom22p (Fig. 16F). However, the addition of 20 mM yCOX-IV1–22 increases the flicker rate from 64  35 to 800  31 events/s in preparations that contain both Tom22p and Tom40p but are lacking Tom20p and Tom70p. Loss of detectable Tom channel activity and Tom40p were associated with more extensive protease treatment, for example, with Pronase (not shown). These findings support a role for Tom22p as a receptor required for the sensitivity of the Tom channel to cationic a-helical peptides. The intermembrane space face of the Tom channel is trypsin insensitive. Similar to Tim23 channel activity, Tom channel activity is not aVected by perfusion of the bath with trypsin in patches excised from proteoliposomes, as shown in the current traces and histograms of Fig. 17. A 3- to 5-fold

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261

FIG. 17 The intermembrane space face of the Tom channel is trypsin insensitive. (A) Model of the orientation of the TOM complex with respect to the membrane face treated with trypsin and targeting peptide. Current traces (B) and histograms (C) of the Tom channel in the presence and absence of 50 mM yCOX-IV1–13 added to the bath, before and after treatment with trypsin at 200 mg/mg of protein.

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increase in flicker rate is observed in the presence yCOX-IV1–13 before and after trypsin treatment of the bath face of the Tom channel. Although these findings suggest that essential components of the Tom channel are not aVected by this trypsin treatment, loss of peptide sensitivity is followed by closure of the channel (as indicated by a loss of conductance) after perfusion of the bath with Pronase (not shown). It is not possible to evaluate the structural state of the TOM complex, as single Tom channels were used in these experiments. Closure should be associated with damage or impairment of Tom40p as this protein is essential and comprises the general insertion pore of the TOM complex (Lithgow et al., 1994). 4. Genetic Manipulation Genetic manipulation of the protein import complexes has generated a wealth of information. This approach is illustrated next with mutants of TIM23 and TOM complexes. The tim23-1 mutant was used in the original identification of Tim23 channel activity. The tim23-1 mutation results in a temperature-dependent deficiency in protein import with the substitution of aspartate for glycine at position 186 in Tim23p (Emtage and Jensen, 1993). Mitochondria isolated from tim23-1 mutants are defective in the import of several diVerent precursor proteins, including subunit IV of yeast cytochrome oxidase (Emtage and Jensen, 1993). The electrophysiological properties of the Tim23 channels isolated from wild-type and tim23-1 strains are virtually identical (Lohret et al., 1997). In particular, Tim23 channels from both strains have the same peak conductance, predominant transition size, mean open time, and cation selectivity. Furthermore, permeability ratios for Kþ/Cl are 6 for wildtype and tim23-1 patches with a 150:30 mM KCl gradient. Conductance through Tim23 channels from both strains is voltage dependent, that is, predominantly open at low (e.g., 20 mV) but not at high potentials of either polarity. The V0 (voltage at which the probabilities of opening and closing are the same) at positive potentials is less than the V0 at negative potentials for both wild-type and tim23-1 strains. In the presence of presequence peptides, however, the channel activities of wild-type and tim23-1 strains diVer dramatically. The conductance of Tim23 channel from wild-type strains is transiently blocked by presequence peptides as described previously. The flicker rate from the wild-type strain increases 4- to 8-fold on addition of yCOX-IV1–13 peptide to the bath at positive potentials (e.g., Fig. 11). Similarly, the flicker rate of wild-type Tim23 channel is 5-fold higher at negative potentials if yCOX-IV1–13 is included in the micropipette buVer. The flicker rate of Tim23 channel from wild-type cells increases with yCOX-IV1–13 concentration in the bath and is saturated by about 25 mM (Fig. 11D). This dose-dependent

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263

blockade is similar to that required for the competitive inhibition of protein import by presequence peptides (Glaser and Cumsky, 1990). However, the tim23-1 mutation essentially eliminates the peptide sensitivity of the Tim23 channel (Lohret et al., 1997). This mutation interferes with the ability of the Tim23 channel to recognize presequence peptides. These data agree with the trypsin digestion described in Section IV.D.3. Tom20p and Tom70p are the putative receptors of the TOM complex for some precursors. A mutant,
20
70, lacking both Tom20 and Tom70 (Lithgow et al., 1994) and its corresponding wild-type strain were examined to determine whether these import complex components are required for regulation of the Tom channel by signal peptides. Although Tom20p and Tom70p are completely lacking, no significant diVerences are detected in the amounts of Tom40p or Tom22p relative to the level of another outer membrane channel, VDAC, as shown in the Western blots of Fig. 18A. The singlechannel characteristics (e.g., conductance, voltage dependence, mean open times, and selectivity) and the peptide sensitivity of both Tom and Tim23 channel activities from this double-deletion mutant are the same as for wild type (Fig. 18B). Furthermore, the flicker rates for Tom channel activities from the two strains of yeast are comparable in the absence or presence of presequence peptides in the microelectrode and in the bath. These findings are consistent with the protease digestion experiments of Section IV.D.3 and indicate that Tom20p and Tom70p are not requisite for the Tom channel to recognize the classic cationic a-helical signal sequence found in many mitochondrial precursors. 5. Determining Components by Recombinant Proteins Loss of channel activity on disruption of a specific gene typically links the gene product to the activity. However, generation of native-like channel activity from recombinant protein has also been eVective in sorting out components of the import complexes, as most knockout disruptions are lethal. In eVorts to determine the molecular identity of the pores of Tim23 and Tom channels, several knockout strains were examined for their channel activity. These studies have eliminated several proteins as potential candidates for the pores, including the adenine nucleotide translocator (Lohret et al., 1996), Tom20p, Tom70p (see earlier discussion), phosphate translocator, mitochondrial ABC transporter (our unpublished results), VDAC (Lohret and Kinnally, 1995a; Muro et al., 2003), the F0 portion of ATP synthase, FeS protein, cytochrome c oxidase, cytochrome b, and the membrane-bound portion of NADH dehydrogenase (Murphy et al., 1998). None of these knockouts modify Tim23 channel activity. Alternatively, strains of yeast have been developed in which the genes for several components, for

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FIG. 18 Tom channel activity is not modified by deletion of Tom70p and Tom20p. (A) Western blots show loss of Tom20p and Tom70p with no change in Tom40p or Tom22p relative to VDAC in a
20
70 mutant strain compared with wild type. (B) Peptide sensitivity of the Tom channel is the same in both strains as indicated by histograms of flicker rates with 50 mM yCOX-IV1–13.

example, Tim17 and Tim23, are dependent on the presence of galactose (Milisav et al., 2001). These strains are presently being characterized for their eVects on Tim23 and Tom channel activities (Martinez-Caballero, Campo, and Kinnally, unpublished results). The study of recombinant proteins in planar bilayers has been a successful approach to understanding Tim22, Tim23, and Tom40 channels. As shown in Table III, these activities approach the behavior observed in the native/ proteoliposome model systems with regard to pore size and peptide sensitivity. However, diVerences in conductances from those reported for the native membrane or those reconstituted in proteoliposomes are observed and are likely related to missing components of the complexes.

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PROTEIN IMPORT CHANNEL ACTIVITY TABLE III Single-Channel Behavior of Recombinant Proteins Tim22a

Tim23b

Tom40c

Permeability, Kþ/Cl

3.9

8.5

8

Conductance (pS)

324  11

270  7

216  2

Transition size (pS)

40  3

84  9

90  2

Pore size (nm)

1.8–2.3

1.3–2.4

2.2  1.4

yCOX-IV1–13

þ

þ

þ

P2

þ

ND

ND

SynB2

þ

–/þ

—

Peptide sensitivity

a

Data recalculated from Kovermann et al. (2002) to be 150 mM salt. Data recalculated from Truscott et al. (2001) to be 150 mM salt. c Data recalculated from Hill et al. (1998) to be 150 mM salt. b

6. Antibody Studies Although not always the case, antibodies against channel components can directly block or modify current flow through the channel pore. Typically, these are polyclonal antibodies that recognize the native protein. Evidence such as antibody blockade lends strong support to the involvement of that epitope/protein in the channel structure. Alternatively, but equally compelling, antibodies can be used to immunodeplete detergent-solubilized proteins from membrane lysates. If the protein is part of a channel, one observes a depletion of channel activity from supernatants of the lysates after immunoprecipitation. The immunodepleted supernatants are typically assayed for the detection of channel activity in planar bilayers or after reconstitution into proteoliposomes. Antibodies against Tim23p that inhibit protein import (Emtage and Jensen, 1993) also block conductance through the Tim23 channel (Lohret et al., 1997). Preimmune serum, antibodies against VDAC, and antibodies against an iron–sulfur protein of the inner membrane have no eVect on the frequency of detecting Tim23 channel activity (not shown). Proteoliposomes prepared with mitochondrial inner membranes are preincubated with antibodies against Tim23p, and then the conductance of patches excised from the treated vesicles is examined. Tim23 channel activity is virtually absent. Proteoliposomes prepared with outer membranes are incubated with equivalent amounts of Tim23 IgG or antibody from preimmune serum. Neither IgG had any eVect on the detection level of Tom channel activity.

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The relationship between channel activity (previously referred to as PSC) and the TOM complex was significantly strengthened by immunodepletion of this channel activity by antibodies against Tom40p. In the study by Juin et al. (1997), strong correlations were made between the amount of Tom40p immunoprecipitated and the loss of channel activity. A variety of control antibodies had no eVect. This direct association of channel activity and the TOM complex was again demonstrated when antibodies against the carboxy terminus of Tom40p were found to modify Tom channel activity. Immunodepletion and/or modification of channel activity are gold standards for connecting a particular protein with channel activity. However, negative data are not informative as many antibodies that work well in Western blots fail to immunoprecipitate or modify the behavior of many channels.

E. Comparison of Mammalian and Yeast Tim23 and Tom Channel Activities The activities of Tim23 and Tom channels are conserved among mouse, yeast, and Neurospora crassa mitochondria, which is consistent with both channels having roles in fundamental cellular processes (Fe´ vre et al., 1990; Ku¨ nkele et al., 1998a; Lohret and Kinnally, 1995a,b). As expected from the low permeability of the inner membrane of mitochondria, the Tim23 channel is normally closed under metabolizing conditions unless it is activated, for example, by targeting peptides (Kinnally et al., 1991, 1992, 1996; Kushnareva et al., 1999). However, Tim23 activity is usually detected after its reconstitution into proteoliposomes, suggesting that regulatory components are lost during the fractionation procedure (Ku¨ nkele et al., 1998b; Lohret et al., 1996). Multiple conductance channel (MCC) activity was originally recorded by patch-clamp techniques on mammalian mitoplasts in the late 1980s and characterized in the early 1990s (Kinnally et al., 1989, 1991, 1992, 1996). However, at least two diVerent channels contributed to the mammalian recordings then referred to as the MCC. One channel activity was the permeability transition pore, PTP, and the other was the Tim23 channel. This confusion was not recognized until the studies were expanded to include yeast, which do not exhibit a mitochondrial permeability transition. Tim23 channels from mammalian and yeast mitochondria are high conductance, voltage dependent, and slightly cation selective, in keeping with their functions (Table IV). Both activities have a half-open state and are likely double-barrel pores. Importantly, Tim23 channels from both sources are sensitive to presequence peptides (Lohret and Kinnally, 1995b). Tim23 and Tom channels from mammalian and yeast mitochondria do display some

267

PROTEIN IMPORT CHANNEL ACTIVITY TABLE IV Single-Channel Behavior of Mammalian Tim23 and Tom Channels Characteristic

Mitoplasts

Tim23

Tom 710  60

Conductance (pS)

750

739  12

Transition size (pS)

350

340  12

340  30

3

3.6  0.8

Ion selectivity (PK/PCl)

3–6

Gating charge

5.4  1.0

1.9  0.1

ND

V0 (mV)

23  3

12  3

10

Mean open time (ms)

ND

5.4  0.1

5

Peptide sensitive

Yes

Yes

Yes

diVerences in their conductance and kinetics. The yeast channels are larger and have slower kinetics, with a conductance of 1000 pS (transition size, 500 pS) and mean open time of 15 ms. The conductances of mammalian channels are 750 pS (transition size, 350–400 pS) with mean open times of 5 ms (Table IV). The transition sizes suggest the presence of 1.4- to 1.6-nm diameter pores in the mammalian pores, whereas those of yeast are 1.8–2.0 nm.

F. MAC: A Protein Export Channel in the Outer Membrane Whereas Tom, Tim23, and Tim22 channels are responsible for the import of unfolded proteins into mitochondria, the mitochondrial apoptosis-induced channel (MAC) is the putative export channel for folded cytochrome c from the intermembrane space into the cytoplasm (Guo et al., 2004; Pavlov et al., 2001). A comparison of current traces from MAC, VDAC, and mammalian Tom channels is shown in Fig. 19. Once released, cytochrome c binds other proteins to form the apoptosome, which facilitates activation of the executioner caspases. Although not normally present, MAC forms in the mitochondrial outer membrane early in apoptosis, and overexpression of the antiapoptotic protein Bcl-2 suppresses the appearance of MAC (Pavlov et al., 2001). The molecular composition of MAC is not known. However, the proapoptotic protein Bax is speculated to be responsible for cytochrome c release and forms channels similar to MAC when expressed in yeast mitochondria (Pavlov et al., 2001), and consequently may be a component of some MAC. MAC presents heterogeneous channel activity (conductance, 2–5 nS, peak transition size, variable, i.e., 2.5  0.6 nS) (Guo et al., 2004; Pavlov et al., 2001). The pore diameter inferred from the peak conductance of this channel is approximately 4–6 nm. In agreement with this calculation, the eVects of

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FIG. 19 Cytochrome c alters MAC activity in proteoliposomes containing MOM from apoptotic FL5.12 cells. (A) Current traces of single channels at þ20 mV show that MAC character is distinct from that of Tom channel and VDAC, two other channels located in the mitochondrial outer membrane. O and C, open and closed conductance levels, respectively. Sampling was at 5 kHz, with 2-kHz filtration. Modified from Pavlov et al. (2001). (B) EVects of cytochrome c on the conductance and noise levels of MAC activity. Type 1 eVect is illustrated by a current trace from a patch with MAC activity in the absence (Control) and presence of 100 mM cytochrome c (Cyt c). The mean current levels of Control, Cyt c, and Cyt c ‘‘plug’’ are 56, 54, and 19 pA, respectively (determined by total amplitude histograms, not shown). The current level remained at 54 pA (Cyt c) for 74 s after perfusion with cytochrome c and then spontaneously decreased to 19 pA (Cyt c ‘‘plug’’), illustrating a likely shift from type 1 (high noise level) to type 2 (low noise level) eVects of cytochrome c. Sampling was at 2 kHz with 1-kHz filtration. Modified from Guo et al. (2004). Patching medium was symmetrical 150 mM KCl–5 mM HEPES-KOH, pH 7.4.

various molecular weight dextrans also indicate that the pore diameter of MAC is typically 4–6 nm, which should be suYcient to allow the passage of 3-nm-diameter cytochrome c, and perhaps slightly larger proteins (Guo et al., 2004). Hence, the pore of this channel is considerably larger than that of Tom, Tim23, and Tim22 channels. This observation is understandable, as MAC translocates folded proteins whereas Tom, Tim23, and Tim22

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channels translocate unfolded proteins. Like the other protein import channels, MAC is slightly cation selective, which may be related to the fact that cytochrome c, the intended permeant, is positively charged at neutral pH. Unlike the protein import channels, MAC is not voltage dependent. Physiological (micromolar) levels of cytochrome c alter MAC activity (Fig. 19) (Guo et al., 2004). Some of these eVects, which we refer to as type 1, are consistent with a partitioning of cytochrome c into the pore of MAC, as was found by others for ATP in VDAC and RNA in a-hemolysin (Bezrukov and Kasianowicz 1997; Kasianowicz et al., 1996; Rostovtseva and Bezrukov, 1998; Rostovtseva et al., 2002b). The type 1 eVects include a modest decrease in conductance that is dose and voltage dependent, reversible, and associated with an increase in noise of the current traces. Type 2 eVects may correspond to a ‘‘plugging’’ of the pore or destabilization of the open state. Type 2 eVects include a dose-dependent, voltage-independent, and irreversible decrease in conductance. Further studies indicate that size, rather than charge, is crucial to the eVects of cytochrome c on MAC (Guo et al., 2004). At this early point in time, there is no evidence suggesting that there is a motor to extrude cytochrome c from the intermembrane space. Hence, MAC is more like the ‘‘hole’’ provided by the TOM complex rather than the TIM23 complex with its various chaperones.

V. Perspectives Future electrophysiological studies may include examination of cotranslation/translocation. Although not essential, proteins may be cotranslationally imported (Bauer et al., 2000; Voisine et al., 1999). If import through TIM23 and TOM complexes can be monitored by changes in the conductance of Tim23 and Tom channels, attempts will be made to place the translation reaction (including ribosomes) in the microelectrode. The mechanism(s) by which the Tim44/mtHsp70A complex facilitates translocation across the inner membrane is presently controversial. Neupert’s group supports the notion that preproteins diVuse across the membrane by Brownian motion. Binding of mtHsp70 prevents backsliding and essentially traps the preprotein in the matrix. Others contend that mtHsp70 unfolds preproteins and generates a pull upon ATP hydrolysis, resulting from ratcheting of preproteinbound mtHsp70 on its membrane anchoring site on Tim44 (Bauer et al., 2000; Voisine et al., 1999). Therefore, Tim44/mtHsp70A and other mutants will be examined to determine whether the putative transient blockade of conductance is modified in duration and amplitude at various voltages. Finally, the pharmacology of protein import is in its infancy. EVorts to

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identify agents that facilitate import might modify the outcome of patients genetically deficient in import, whereas those that suppress import may modify many phenomena, including the commitment step in apoptosis.

Acknowledgments This research was supported by NSF grants MCB-0235834 and INT003797, NIH grant GM57249 to K.W.K., and Junta de Extremadura 2PR02B007 to M.L.C. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or NIH. The authors thank Carla Koehler and JeV Schatz (University of Basel, Basel, Switzerland) for the
20
70 mutant, Mike Forte (Oregon Health State University) for the VDAC knockout strains of yeast, and Stan Korsmeyer (HHMI, Harvard) for the FL5.12 mouse leukemia cell line.

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