Channel-forming activities of peroxisomal membrane proteins from the yeast Saccharomyces cerevisiae Silke Grunau1,2,*, Sabrina Mindthoff1,*, Hanspeter Rottensteiner1, Raija T Sormunen3, J Kalervo Hiltunen2, Ralf Erdmann1 and Vasily D Antonenkov2 Institut fur Physiologische Chemie, Abt Systembiochemie, Bochum, Germany ă Department of Biochemistry and Biocenter Oulu, University of Oulu, Finland Department of Pathology, University of Oulu, Finland Keywords channels; membranes; peroxisomes; Saccharomyces cerevisiae; yeast Correspondence V D Antonenkov, Department of Biochemistry and Biocenter Oulu, University of Oulu, Linnanmaa, PO Box 3000, FI-90014 Oulu, Finland Fax: +358 5531141 Tel: +358 5531201 E-mail: vasily.antonenkov@oulu.fi *These authors contributed equally to this work (Received 21 October 2008, revised January 2009, accepted 12 January 2009) Highly-purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)-forming proteins in the membrane of these organelles Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore-forming activity was studied by means of multiple-channel monitoring or singlechannel analysis Two abundant pore-forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively The high-conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (PK+ ⁄ PCl)  1.3) and showed an unusual flickering at elevated (> ±40 mV) holding potentials directed upward relative to the open state of the channel The data obtained for the properties of the low-conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high-conductance channel represents a cluster of two low-conductance pores The results lead to conclusion that the yeast peroxisomes contain membrane pore-forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm doi:10.1111/j.1742-4658.2009.06903.x Peroxisomes are ubiquitous subcellular organelles involved in diverse metabolic activities, ranging from the oxidation of fatty acids, purines, hydroxyacids, alcohols and polyamines to the synthesis of plasmalogens, ketone bodies and bile acids [1,2] The protein composition of peroxisomes depends on both the species and environmental conditions For example, the peroxisomes from fungi and plants, but not from mammals, contain enzymes of the glyoxylate cycle that allow the conversion of acetyl-CoA molecules generated mainly by peroxisomal b-oxidation of fatty acids into succinate, which can be used in a variety of reactions, including the biosynthesis of amino acids or carbohydrates [3] A role for peroxisomal membrane as a permeability barrier to solutes has been a matter of debate for more than 40 years Only recently was a ‘consensus’ reached on the idea that this membrane is impermeable to bulky solutes such as ATP and the cofactors, NAD ⁄ H ⁄ , NADP ⁄ H ⁄ , CoA and its acyl derivatives [1,4,5] By contrast, the permeability of the membrane to small solutes, including inorganic ions and organic metabolites, is still a matter of controversy [1,4,5] For example, contradictory results were obtained concerning the existence of pH [6,7] or Ca2+ [8,9] gradients across the peroxisomal membrane Moreover, the assumption that the presence of such gradients confirms the impermeability of the peroxisomal membrane has recently been challenged [5] Our previous studies on mammalian peroxisomes showed that the membrane of these particles is permeable to small solutes [10] and contains pore-forming proteins [11] Likewise, Abbreviation VDAC, voltage-dependent anion channel 1698 FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS S Grunau et al channel-forming activity with properties of substratespecific pores has been reported for plant peroxisomes [12,13] A study on yeast is beneficial due to its simplicity with respect to genetic manipulations Yeast Saccharomyces cerevisiae grown on oleic acid contains well-developed peroxisomes involved mainly in the b-oxidation of fatty acids [14] In the present study, we report on the discovery and characterization of poreforming proteins in the peroxisomal membrane from baker’s yeast, which were detected by an electrophysiological study of peroxisomal proteins reconstituted into artificial membranes Results Characterization of yeast peroxisomal fraction Peroxisomes were isolated from oleic acid-grown S cerevisiae by Optiprep density centrifugation of freshly prepared post-nuclear supernatants The purity of peroxisomes collected from the bottom of the gradient (fractions 3–4; Fig 1A,B) was estimated by analysis of the activity of marker enzymes for different organelles: peroxisomes (catalase), mitochondria (cytochrome c oxidase) and lysosomes (acid phosphatase) (Fig 1A), as well as by immunoblotting using antibodies generated against marker proteins for peroxisomal membrane (Pex11p), mitochondrial outer membrane (voltage-dependent anion channel; VDAC), endoplasmic reticulum (Kar 2p) (Fig 1B) and membranes of vacuoles (alkaline phosphatase) (data not shown) The peroxisomes were well separated from the other cellular organelles, including mitochondria that may be a main potential source of contaminating channel-forming activities [15], and the purity was confirmed by electron microscopic examination (Fig 1C, panels and 2) The images obtained demonstrate that the fraction almost exclusively consists of peroxisomes Most of them were filled with matrix of variable electron density, whereas, in some particles, only the membrane was visible, indicating damage of peroxisomes during isolation On some occasions, an electron dense material that was not surrounded by a membrane was detected (Fig 1C, panel 1) Apparently, this material represents aggregates of peroxisomal matrix proteins escaping from the particles To study the pore-forming activity, only peroxisomal fractions were used that contained less than 0.3% of the total cytochrome c oxidase activity loaded on the gradient and that showed no traces of the marker proteins for different organelles on immunoblots under standard assay conditions (Fig 1B) For control Channels in yeast peroxisomal membrane experiments (see below), the mitochondrial fraction was selected from the same gradients (fractions 14–16; Fig 1A) As judged from the analysis of the distribution of markers for different organelles (Fig 1A,B) and data from electron microscopy (Fig 1C, panel 3), this fraction not only contained mitochondria, but also membranes of other organelles, including endoplasmic reticulum, vacuoles and peroxisomes Latency of yeast peroxisomal enzymes To determine whether or not the yeast peroxisomal membrane is permeable to solutes, we measured the latency of some enzymes confined to the particles (Fig 2A) The activity of these enzymes was detected in the isolated peroxisomal fraction before (‘free’ activity) and then after disruption of the membrane by detergent (‘total’ activity; for details, see Experimental procedures) Only 35% of the ‘total’ catalase activity was registered in the absence of detergent The latency of catalase is a well known phenomenon that has been attributed to the very high concentration in peroxisomes of this extremely active enzyme [16] Similar to catalase, the activities of cofactor-dependent peroxisomal enzymes (i.e NAD-dependent malate dehydrogenase, citrate synthase and malate synthase) were found to be latent, indicating that at least one of the cosubstrates ⁄ cofactors involved in the enzymatic reactions is unable to freely traverse the peroxisomal membrane By contrast, detection under similar conditions of an aspartate aminotransferase reaction showed that the ‘total’ and ‘free’ activities of the enzyme were almost equal (Fig 2A) Importantly, the latter enzyme does not require addition into the reaction medium of any cofactor or other bulky solute The data of the latency determination support the notion that, similar to the mammalian peroxisomal membrane [10], the membrane of yeast peroxisomes provides free access to the particles for small solutes but prevents diffusion of bulky solutes such as cofactors (NAD ⁄ H ⁄ , NADP ⁄ H ⁄ , CoA and its acylated derivatives) and ATP [5] Such an arrangement of the barrier function of the yeast peroxisomal membrane predicts that it contains poreforming proteins that allow the free diffusion of small solutes The channel-forming activity in yeast peroxisomes To detect the predicted channel-forming activity of purified peroxisomal preparations, we applied a multiple-channel recording procedure that allows estimation of the number and conductance of the FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS 1699 Channels in yeast peroxisomal membrane S Grunau et al Fig Purification of peroxisomes from oleate-grown yeast S cerevisiae Organelles from a post-nuclear supernatant were separated by Optiprep density gradient centrifugation and organelle segregation was monitored by (A) enzyme measurements and (B) immunoblot analysis of marker proteins (A) Fractions from the linear Optiprep gradient were analyzed for marker enzyme activities: catalase (a, filled bars, peroxisomes), cytochrome c oxidase (a, gray bars, mitochondria), acid phosphatase (b, filled bars, lysosomes) or protein content (b, gray bars) The results obtained are expressed as the relative activity Enzyme (protein) recoveries varied in the range 88–110% The line connecting small open squares (b) marks the density of the gradient (B) Proteins from equal volumes (20 lL) of each fraction (fractions 1–8 and 22–28) or every second fraction (fractions 8–22) (Fig 1A) were separated by SDS ⁄ PAGE and analyzed by an immunoblot technique using antibodies against the organelle markers: Pex11p (peroxisomes), VDAC (mitochondria) and Kar2p (endoplasmic reticulum) (C) Electron micrographs of subcellular organelles isolated by means of Optiprep gradient centrifugation Gradient fractions enriched in peroxisomes (fractions 3–4; Fig 1A,B) or mitochondria and other organelles (fractions 14–16) were combined and mixed with an equal volume of 2% (w ⁄ v) glutaraldehyde prepared on 40% (w ⁄ v) Optiprep solution to avoid osmotic damage of peroxisomes (for further details, see Experimental procedures) After overnight fixation, the organelles were sedimented and processed for electron microscopy Panels and 2: isolated peroxisomes at lower (1) and higher (2) magnifications, scale bars = 1000 and 200 nm, respectively; panel 3: mitochondrial fraction, scale bar = 2000 nm Amorphous electron-dense material that might indicate aggregates of escaped peroxisomal matrix proteins is marked by an asterisk in (C, panel 1) single-channel events registered Indeed, the proteins solubilized from purified yeast peroxisomes were able to form membrane pores (Fig 2B) displaying two predominant types of the channel-forming activity, with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl (Fig 2C, upper panel) The pattern of the 1700 conductance distribution differed significantly from that detected in the mitochondrial fraction (compare the upper and lower panels in Fig 2C), indicating that the pore-forming activity is determined by peroxisomal proteins but not by contaminating proteins from other organelles FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS S Grunau et al Channels in yeast peroxisomal membrane Fig Latency of peroxisomal enzymes and multiple-channel monitoring of the poreforming activity in yeast peroxisomes (A) ‘Free’ activity of yeast peroxisomal enzymes presented as a percentage of the ‘total’ activity detected after incubation of purified peroxisomes with 0.1% (w ⁄ v, final concentration) Triton X-100 (B) Traces of the multiple-channel monitoring of an artificial membrane in the presence of detergentsolubilized peroxisomes (upper panel) or mitochondria (lower panel) The trace in the frame (upper panel) shows a timescaleexpanded current recording of the upper trace The bath solution contained M KCl on the both sides of the membrane The temperature was +20 °C and the applied voltage was +20 mV (C) Histograms of insertion events observed during multiplechannel monitoring (B) in the presence of purified detergent-solubilized peroxisomes (upper panel; combined gradient fractions and 4; see Fig 1A) or fraction-enriched in mitochondria and other organelles (lower panel; combined gradient fractions 14–16; see Fig 1A) The total number of insertion events was 180–200 for each membrane preparation analyzed Each experiment was repeated three times; typical histograms are presented (D) Channel conductance as a function of KCl concentration The data for high-conductance activity ( 0.6 nS in 1.0 M KCl; see Fig 2C) are shown The mean ± SD conductance for 30–40 single insertion events was calculated Next, we investigated the subperoxisomal localization of the channel-forming activities Peroxisomal matrix proteins were separated from membrane fragments by centrifugation in sucrose gradients after organelle disruption by sonication When proteins solubilized from the isolated membrane fragments were analysed for channel-forming activity, a conductance pattern analogous to that of the whole peroxisomal preparations was obtained (data not shown) By contrast, the matrix proteins did not show any poreforming activity The conductance pattern of the peroxisomal channels was not affected by a shift in pH of the bath solution from pH 6.0 (unbuffered 1.0 m KCl) to pH 7.6 or pH 8.4 by adding 20 mm (final concentration) Mops or Tris buffers, respectively, or by preincubation of protein preparations with dithiothreitol (5 mm, final concentration, data not shown) The later finding may indicate that the redox state of SH groups in molecules of the channel-forming protein(s) does not affect the activity of these proteins significantly As expected for large water-filled channels, the singlechannel conductance registered by multiple-channel recording increased almost linearly with an increasing KCl concentration (Fig 2D) According to our data, the peroxisomal pore-forming proteins were active not only with KCl as an electrolyte, but also activity was detected with other small ions that were tested at a concentration of 1.0 m (pH 6.8): NH4Cl, LiCl, potassium acetate and sodium phosphate However, we were unable to register any activity with some larger electrolytes [e.g tetraethylammonium chloride (1.0 m) or AMP (0.25 m potassium salt, pH 6.8)], although the activity with these ions was evidently present in fractions enriched with mitochondria Electrophysiological properties of a high-conductance channel We used a single-channel analysis to describe properties of the peroxisomal membrane channel with an FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS 1701 Channels in yeast peroxisomal membrane S Grunau et al average conductance of 0.6 nS in 1.0 m KCl (see above) Figure 3A displays the current recordings at holding potentials +20, +60 and )60 mV and an equal concentration of electrolyte (1.0 m KCl) on both sides of an artificial membrane, being characteristic of an ion channel with a slope conductance of K = 0.68 ± 0.4 nS (n = 6) The channel inserted in the membrane was mainly fully open during the whole period of activity registration (minutes) at a wide range of holding potentials (from )80 to +80 mV) The channel showed strong flickering that was directed upwards (at positive voltages), especially at higher holding potentials This flickering was detected in all twenty measurements The direct transition of some spikes down to the fully-closed state of the channel was observed (Fig 3A, lower trace in the right panel), indicating that the current transitions are substates of a single channel rather than amplitudes of several independent channels inserted in the membrane The flickering of the channel resembles the behaviour of the CLC-0 chloride channel consisting of two pore-forming protein molecules [17] (for details, see Discussion) This may indicate that, similar to the CLC-0 channel, the peroxisomal channel represents a cluster of poreforming proteins in which only one is in a permanently open state, whereas others display a fast, transient gating The nature of this gating deserves further investigation The reversal potential of a single channel (Fig 3B,C) under asymmetric salt conditions (1.0 m Fig Single-channel analysis of the highconductance channel (A) Current traces of a bilayer containing a single high-conductance pore-forming protein (the insertion event is marked by asterisk) at different membrane potentials (1.0 M KCl on both sides of the membrane) Applied membrane potentials are indicated Note that the channel displayed an intensive flickering at +60 and )60 mV, respectively The largest current amplitude of this flickering is approximately threefold higher than the amplitude of the channel itself The lower trace in the right panel shows the direct transition of one of the spikes down to the fully-closed state of the channel (marked by arrowhead) (B) Current traces of a bilayer containing one high-conductance channel (the insertion event is marked by asterisk) under asymmetric salt conditions: 1.0 M KCl cis ⁄ 0.5 M KCl trans compartment Control experiments demonstrated that, after adjustment of the electrolyte concentration on both sides of the membrane to 1.0 M KCl, the channel displayed a current amplitude of 12–15 pA at +20 mV, which reflects the conductance detected in the multiple-channel recording experiments (Fig 2C) (C) Current–voltage relationship of the high-conductance channel under asymmetric salt concentrations (see Fig 3B); data points are mean ± SD of at least six independent measurements 1702 FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS S Grunau et al KCl cis ⁄ 0.5 m KCl trans compartment) was Erev = +2.0 mV, indicating that the channel has a slight preference for cations over anions (PK+ ⁄ PCl) = 1.3) The slope conductance of the channel was K = 0.95 nS (in 1.0 m ⁄ 0.5 m KCl) As with symmetric salt conditions, the channel displayed high-conductance flickering, especially at negative holding potentials (Fig 3B) Channels in yeast peroxisomal membrane Single-channel analysis of a low-conductance channel More than 80 insertion events were detected in the single-channel analysis experiments Approximately 60% of the inserted channels showed properties of the high-conductance channel (0.6 nS in 1.0 m KCl; see previous section) The second largest group of inser- Fig Single-channel analysis of the low-conductance channel (A) Upper panel: current trace of the channel (the insertion event is marked by asterisk) The bath solution (A–D) comprised 1.0 M KCl on both sides of the membrane Lower panel: current trace of the low-conductance channel in response to voltage ramp protocol (from )100 to 100 mV, 10 s) (B) Current trace of the low-conductance channel undergoing transition to the high-conductance channel Upper panel: direct transition of the channel-forming activity between the closed state and the fully-open state (marked by asterisk) and between the fully-open state and the intermediate state (marked by an arrowhead) The intermediate state corresponds to the low-conductance channel activity Lower panel: current trace of the same channel as that shown in the upper panel but at a higher holding potential The fully-closed state of the channel (marked by an asterisk) side by side with the spikes of the flickering (marked by arrowheads) is visible (C) Upper panel: current trace showing two sub-conductance states with almost equal amplitude; each of these states represents the amplitude of the low-conductance channel Lower panel: count rate histogram of the upper current trace (D) Upper panel: current trace of the super-large conductance channel with current amplitude in the fully open state  130 pA (1.0 M KCl, +20 mV) Lower panel: a timescale-expanded current recording of the part shown in the frame in the upper trace Note the direct transition from the closed to the fully-open state and the short lifetime of the open state The partial closure of the channel leads to the appearance of a stable substate with a current amplitude comprising one-third of the amplitude characteristic of the fully-open channel FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS 1703 Channels in yeast peroxisomal membrane S Grunau et al tion events (Fig 4A) displayed a conductance similar to that of the low-conductance channel activity detected by multiple-channel monitoring (i.e 0.2– 0.3 nS in 1.0 m KCl; see above) The current–voltage relationship measured for these channels revealed a slope conductance of K = 0.28 nS (1.0 m KCl on both sides of the membrane; data not shown) Recording of the activity at elevated holding potentials showed an upward flickering (at positive potentials) with different open channel amplitudes (Fig 4A,B, lower panels) The flickering that was upward relative to the open state of the main channel (at positive potentials) resembled the similar behaviour of the high-conductance channel (for comparison, see Fig 3A,B) The reversal potential of the low-conductance channel in asymmetric KCl solutions (1.0 m KCl cis ⁄ 0.5 m KCl trans compartment) was Erev = +2.4 mV (data not shown), which is close to the result obtained for the high-conductance channel (Erev = 2.0 mV) Therefore, it appears that the ion selectivity of low- and high-conductance channels is almost identical Approximately half of the low-conductance traces displayed multiple transitions to the level with higher current amplitudes (Fig 4B,C), which approximately corresponded to the amplitude of the high-conductance channel under the same experimental conditions The smaller current amplitude reached approximately one half of the full conductance (Fig 4C) Direct transitions from the closed to the fully-open state (largest current amplitude), and from the closed state to the intermediate state (smaller amplitude), were observed (Fig 4B) The partially open state was also approached from the fully-open state (Fig 4B,C) These observations indicate that the intermediate amplitude is a substate of a single-channel molecule inserted in the membrane, rather than amplitudes of two independently active channel molecules Taken together, the data obtained in the single-channel analysis lead to the suggestion that the high-conductance channel may represent a cluster of two low-conductance channels (for details, see Discussion) The selectivity of the channel-forming activities towards cations (see above), which is opposite to ionselectivity of the VDAC at low holding potentials [18,19], provided an additional opportunity to assess whether the activities described in the present study are truly peroxisomal or determined by mitochondrial contamination Therefore, using single-channel analysis, we compared ion-selectivity of the channel-forming proteins from peroxisomal and mitochondrial fractions, respectively At asymmetric salt conditions (1.0 m KCl cis ⁄ 0.5 m KCl trans compartment) and zero holding potential, all 56 inserted channels dis1704 played selectivity towards cations when the peroxisomal fraction was used in the experiments However, when measurements were made on the mitochondrial fraction, only 19 out of 48 inserted channels showed cation selectivity, whereas the other channels were selective towards anions These results strongly support our conclusion that peroxisomes from baker’s yeast contain cation-selective channel-forming protein On several occasions, the channel activities registered in the peroxisomal fraction showed very large current amplitudes (Fig 4D) The channels with current amplitudes in a fully-open state of  30, 60 and 130 pA (1.0 m KCl, +20 mV) were registered They displayed strong downward (relative to the open states) flickering, indicating closure of the channels The average mean lifetime of the fully-open channels was relatively low (sopen < 100 ms) Properties of the ‘super-large’ conductance channels (Fig 4D) were not analysed further because of their low abundance Discussion The results of the present study demonstrate that the membrane of peroxisomes from the yeast S cerevisiae contains channel ⁄ pore-forming proteins, as concluded from the following data: (a) proteins solubilized from highly-purified peroxisomal preparations showed an abundant channel-forming activity in the multiplechannel recording experiments, the pattern of which is completely different from that of the mitochondrial fraction; (b) the channel-forming activity was shown to associate with membrane proteins of peroxisomes; and (c) the results of the single-channel analysis experiments revealed that, in regard to single-channel conductance, ion-selectivity and voltage-dependence, the investigated peroxisomal channels was found to be distinctly different from VDAC of the outer mitochondrial membrane [18,19], which can be expected as a major source of the channel-forming activity contamination in the peroxisomal preparations Our finding regarding channel-forming activity in the yeast peroxisomal membrane is consistent with previous observations describing such activity in plants [12,13] and mammals [11] In addition, the 31 kDa protein isolated from peroxisomal preparations of the yeast Hansenula polymorpha showed channel-forming properties [20] However, further characterization of this protein revealed properties similar to mitochondrial VDAC [20] An electrophysiological analysis of the channel-forming activity related to the 31 kDa protein has not been described Therefore, it is unclear whether this protein represents a peroxisomal constituent or a mitochondrial contamination FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS S Grunau et al The peroxisomal channels from different sources share features characteristic of bacterial and mammalian porins monitored by the planar lipid bilayer technique: (a) the pores remain open for a prolonged period of time (seconds to minutes); (b) the conductance of the channels is relatively large (> 0.2 nS in 1.0 m KCl); and (c) there is a total absence or weak voltage-dependence of channel gating Interestingly, according to our preliminary data (unpublished results), the diameter of the peroxisomal channel is rather small (< 0.6 nm) and, in this sense, the channel shows similarity to the plant peroxisomal porin [12,13] However, in contrast to the plant protein, which displays strong anion selectivity and weak voltage dependence, the yeast peroxisomal channel is slightly cation-selective and not voltage-gated The plant peroxisomal channel, similar to some porins from the outer bacterial membranes [21], can be considered as a specific porin that preferably allows passing through of mono- and dicarboxylic acids [13] Whether the yeast peroxisomal channels show similar properties is currently under investigation The conductance pattern of the plant peroxisomal channel detected by the multiple channel recording at +20 mV using 1.0 m KCl as an electrolyte reveals two main conductance levels: 0.3 and 0.6 nS, respectively [12], which is very close to the pattern described in the present study (Fig 2B,C) These results can be explained by both plant and yeast high-conductance channels functioning as dimers consisting of two low-conductance channels This notion was substantiated by the single-channel analysis experiments The cluster organization is not a unique feature of the peroxisomal pore-forming proteins Some other examples are trimeric organization of bacterial porins [21] and the dimeric structure of the CLC chloride channels [17] The latter shares an unusual behavior of the yeast peroxisomal channels: an upward flickering relative to the main open state (at positive holding potentials) representing fast-gating transitions The lifetime in the fully-open state of the dimer of the monomeric subunits of the chloride channel is not the same; usually, it is much longer than that for one of the monomers [17] The fast gating of one monomer produces an upward flickering relative to the baseline, which is determined by the fully-open state of the counterpart channel This might comprise one of the mechanistic explanations for the ‘upward’ flickering of the yeast peroxisomal channels The proposed mechanism leads to the prediction that the channel is organized in complex clusters containing a dozen, or even more, monomeric subunits However, at present, we cannot exclude the Channels in yeast peroxisomal membrane possibility of other mechanisms that might lead to the ‘upward’ flickering of the channels The functional role of the yeast peroxisomal channels remains to be established Presumably, the channels form a general diffusion pore in the membrane and function as a size-selective filter that allows crossing of the membrane by a wide variety of small solutes, but prevents transfer of ‘bulky’ compounds, including ATP and cofactors (NAD ⁄ H, NADP ⁄ H and CoA) The channels may provide a route to transfer metabolites such as carnitine or di- and tricarboxilic acids, which participate in the peroxisomal b-oxidation of fatty acids and the glyoxylate cycle, respectively They are also apparently involved in shuttle mechanisms required for metabolic conversion of peroxisomal cofactors [22] Our preliminary data suggest that some peroxisomal metabolites may be transferred by a so-called ‘specific’ channel This type of channel is characteristic of the outer membrane of Gram-negative bacteria [21] Therefore, the overall functional organization of the yeast peroxisomal membrane transport system may be similar to that of the outer membrane of bacteria Experimental procedures Strains, media and culture conditions The yeast strain used in the study was S cerevisiae UTL7-A (MATa leu2-3, 112 ura3-52 trp1) [23] Yeast cells were grown under aerobic conditions on YNDO [0.1% (w ⁄ v) yeast extract, 0.67% (w ⁄ v) yeast nitrogen base without amino acids, 0.1% (w ⁄ v) oleic acid, 0.1% (w ⁄ v) glucose, 0.05% (v ⁄ v) Tween 40] medium at pH 6.0 [23] Isolation of peroxisomes Preparation of yeast spheroplasts, cell homogenization and isolation of a postnuclear supernatant were performed as described previously [23] Peroxisomes (10 mg of protein) were isolated using a preformed linear 2.25–24.0% (w ⁄ v) Optiprep (Iodixanol; Axis-shield PoC AS, Oslo, Norway) density gradient The gradients were centrifuged in a vertical rotor (TV860; Sorvall; Thermo Fisher Scientific Inc., Waltham, MA, USA) at 48 000 g (maximum) for 1.5 h The fractions were collected from the bottom of the tubes and used immediately for analysis of the activities of marker enzymes for subcellular organelles Measurement of enzyme activities and latency determination Catalase and cytochrome c oxidase, marker enzymes for peroxisomes and mitochondria, respectively, were detected FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS 1705 Channels in yeast peroxisomal membrane S Grunau et al as described previously [24] Acid phosphatase was measured as marker for lysosomes [25] Activities of NADdependent malate dehydrogenase, citrate synthase and malate synthase [26], and the activity of aspartate aminotransferase [26,27], were measured in the purified peroxisomal fraction Protein content was determined according to the method of Bradford [27a] The latency of peroxisomal enzymes was detected after resedimentation of the particles in a two-step [20% (w ⁄ v) and 50% (w ⁄ v) sucrose] gradient in a vertical rotor at 100 000 g (maximum) for 45 ‘Free’ enzyme activity was measured prior to disruption of the peroxisomal membrane by Triton X-100 [0.1% (w ⁄ v) final concentration] to reveal the ‘total’ enzyme activity [10] Antibodies and immunoblot analysis Polyclonal rabbit antibodies were raised against Pex11p, VDAC [28], Kar2p [29] and alkaline phosphatase (Molecular Probes, Leiden, The Netherlands) to detect the corresponding marker proteins for different cellular organelles SDS ⁄ PAGE and immunoblotting were performed according to standard procedures and blots were developed using the ECL system (GE Healthcare, Chalfont, St Giles, UK) Electron microscopy For transmission electron microscopy, the isolated peroxisomes were fixed in 1% (w ⁄ v) glutaraldehyde overnight at °C The organelles were sedimented at 20 000 g (maximum) for 30 and processed as described previously [30] Detection of the pore-forming activity To study pore-forming activities of yeast peroxisomal proteins, multiple-channel recording and single-channel analysis of solubilized membrane proteins reconstituted into an artificial lipid bilayer were applied The peroxisomal fraction collected from Optiprep gradients was diluted 10-fold with 20 mm Mops buffer, pH 7.2 (0.02–0.04 mgỈmL)1 protein) and treated with 0.5% (w ⁄ v, final concentration) Genapol X-080 (Fluka, Buchs, Switzerland) by rotating for h at °C After sedimentation of insoluble material at 100 000 g (maximum) for 45 min, the resulting supernatant was immediately used for detection of the pore-forming activity Multiple-channel recordings were performed as described previously [31] in a Teflon chamber separated into two compartments (5 mL each) by the wall containing a circular hole (0.2 mm2) The lipid bilayer was formed across this hole by the painting technique using 1% (w ⁄ v) diphytanoyl phosphatidylcholine (Avanti Polar Lipids Inc., Alabaster, AL, USA), dissolved in n-decane ⁄ butanol (9 : 1, v ⁄ v) After formation of a stable membrane with a typical capacitance 1706 of 400–700 pF, the solubilized membrane proteins (4 lL) were added to both compartments of the chamber, which were equipped with magnetic stirrers One molar bath solutions of KCl were used, unless otherwise stated The temperature was 20 °C A Planar Lipid Bilayer Workstation equipped with a BC-535 amplifier and an pole lowpass Bessel filter (Warner Instruments, Hamden, CT, USA) was used for the current detection Acquisition and analysis were performed using pCLAMP software (Axon Instruments, Foster City, CA, USA) Membrane currents were measured at a holding potential of +20 mV (unless otherwise stated) with a pair of Ag ⁄ AgCl electrodes The data were filtered at 30 Hz and collected at 2.0 kHz Multiplechannel recording allows measurements of a large number of insertion events of reconstituted pore-forming proteins, as well as quantitative analysis of these activities by using histograms of insertion frequency relative to current amplitudes For each histogram, the absolute number of insertion events with a certain current amplitude (bin size = 2.0 pA) was calculated For a more detailed investigation of the electrophysiological properties of the pore-forming proteins, we used a single-channel analysis, which allows characterization at the high-time resolution of a single channel inserted in the artificial membrane Commercial chambers (Warner Instruments) with two compartments (4 mL each) separated by wall with a circular hole (0.04 mm2) were used The Ag ⁄ AgCl electrodes were connected to the compartments via m KCl-agar bridges As in the case of multiple-channel recordings, the electrode of the trans compartment was directly connected to the headstage of a current amplifier Reported membrane potentials are referred to the trans compartment The capacitance of the bilayer was in the range 70–90 pF The data were filtered at 1.0 kHz and collected at 2.0 kHz Measurements of reversal potentials were performed by establishing a twofold (1.0 m KCl cis ⁄ 0.5 m KCl trans compartment) salt gradient after formation a stable lipid bilayer After insertion of a single channel, the current was initially recorded at mV and, subsequently, at different membrane potentials In a separate set of experiments, the number of cation-selective versus anion-selective channels was calculated in peroxisomal and mitochondrial fractions, respectively Asymmetric salt conditions (1.0 m KCl cis ⁄ 0.5 m KCl trans compartment) were used and each insertion event was initially detected at zero holding potential followed by application of a voltage ramp protocol (from )40 to +40 mV, 10 s) Acknowledgements This work was supported by grants from the Academy of Finland, Sigrid Juselius Foundation; the Deutsche Forschungsgemeinschaft (ER 178 ⁄ 2-4); and the FEBS Journal 276 (2009) 1698–1708 ª 2009 The Authors Journal compilation ª 2009 FEBS S Grunau et al European Union Project ‘Peroxisomes’ (LSHG-CT2004-512018) We are grateful to Professor R Benz from the Lehrstuhl fur Biotechnologie, Universitat ă Wurzburg, Germany, and Professor M Weckstrom ă from the 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yeast peroxisomal channels... bulky solute The data of the latency determination support the notion that, similar to the mammalian peroxisomal membrane [10], the membrane of yeast peroxisomes provides free access to the particles... threefold higher than the amplitude of the channel itself The lower trace in the right panel shows the direct transition of one of the spikes down to the fully-closed state of the channel (marked