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, 4 (4), e5090

Pxmp2 Is a Channel-Forming Protein in Mammalian Peroxisomal Membrane

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Pxmp2 Is a Channel-Forming Protein in Mammalian Peroxisomal Membrane

Aare Rokka et al. PLoS One.

Abstract

Background: Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma.

Methods/principal findings: In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2(-/-) mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane.

Conclusions/significance: Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of "bulky" metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of Pxmp2 deficient peroxisomes and detection of metabolites in blood and urine.
(A) Western blot analysis of postnuclear homogenates from livers of Pxmp2+/+, Pxmp2−/−, and Pxmp2+/− mice, respectively, by using antibodies against Pxmp2. The molecular mass markers are indicated on the left. (B) Morphological examination of peroxisomes. (1) Peroxisome visible in liver cross section of wild-type mouse, p = peroxisome; (2) cluster of Pxmp2-deficient peroxisomes; (3,4) peroxisomes from livers of Pxmp2 −/− (3) and wild-type (4) mice isolated using a Nycodenz gradient (see Figure 2B; fractions 3–4 were collected for analysis). Bar: 500 nm (1,2); 1000 nm (3,4). (C) Effect of Pxmp2 deletion on the integrity of peroxisomes in vitro. The postnuclear homogenates of liver samples were centrifuged using Nycodenz gradients. The activity of L-α-hydroxyacid oxidase was determined as a marker for soluble peroxisomal matrix proteins in both wild-type (black bars) and Pxmp2-deficient (gray bars) preparations. The ordinate axis (left) represents relative enzyme activities in each fraction (percentage of the whole activity loaded on the gradient which consisted of 0.22 units and 0.23 units for wild-type and Pxmp2-deficient samples, respectively). Recoveries of the enzyme were 106% (control) and 94% (Pxmp2−/−), respectively. The line connecting empty cycles indicates density of the gradient (shown on the right ordinate axis). Note an excessive leakage of L-α-hydroxyacid oxidase from Pxmp2-deficient peroxisomes. (D) Effect of Pxmp2 deletion on the latency of peroxisomal urate oxidase. Upper panel: absorbance traces at 292 nm indicating oxidation of uric acid. Incubation medium contained: purified peroxisomes (40 µg) only (1); + uric acid (2); + 0.05% (w/v) Triton X-100 (3). Lower panel, left part: ‘free’ activities of urate oxidase in peroxisomes from livers of control (dark gray column) and Pxmp2 −/− (light gray column) mice. *P = 0.0008, n = 12; Lower panel, right part: total activity of urate oxidase in ‘postnuclear’ liver homogenates. (E) Determination of uric acid in blood (left panel, male mice) and urine (right panel, female mice) of wild-type (•) and Pxmp2−/− (○) mice. Similar results were obtained with mice of both gender types. The excretion of uric acid and allantoin (see below) into urine is presented as molar ratios to creatinine (see ‘Methods’ for details). *P = 0.010; **P = 0.008. (F) Allantoin content in blood (left panel) and urine (right panel) of wild-type (•) and Pxmp2−/− (○) mice. *P = 0.067.
Figure 2
Figure 2. Detection of pore-forming activity in membrane preparations.
(A) Traces of the multiple-channel recording of an artificial membrane in the presence of detergent-solubilized liver peroxisomes. The right trace shows a timescale-expanded current recording from the left trace. The current measurements (Figure 2A–2D and Figure 3B and 3C) were made with 1.0 M KCl on both sides of a bilayer and at applied voltage +20 mV. (B) Histogram of multiple-channel recordings registered in a peroxisomal fraction isolated from livers of wild-type (upper panel) or Pxmp2 −/− (lower panel) mice. The total number of insertion events: upper panel - 213; lower panel - 210. Here, and on Figure 2D each experiment was repeated at least three times and typical pictures are presented; the 1.3 nS conductance level is marked by an arrowhead on each panel. (C) Multiple-channel recordings of PPF preparation isolated from mock-transfected (upper trace) insect cells or those expressing recombinant Pxmp2 (lower trace). Insertion events with a conductance of 1.3 nS in 1.0 M KCl are marked by asterisks. (D) Histogram of insertion events observed in the presence of PPF preparations isolated from mock-transfected (left panel) and Pxmp2-containing (right panel) insect cells. The total number of insertion events: left panel - 230; right panel - 225.
Figure 3
Figure 3. Channel-forming activity of the purified Pxmp2.
(A) SDS-PAGE and immunoblot analysis of the purified native Pxmp2. Left panel: fractions obtained after the final purification step (second size-exclusion chromatography step, see Text S1) and which were enriched with Pxmp2, were concentrated and proteins (∼20 ng) were subjected to SDS-PAGE followed by silver staining. Right panel: immunoblot analysis of the purified Pxmp2. The Pxmp2 band is marked by an asterisk. (B) Multiple-channel recording of a bilayer after addition of purified Pxmp2 (∼10 ng/ml, final concentration). (C) Histogram of insertion events of purified Pxmp2. The total number of increments is 312. (D) Current traces from a bilayer containing single Pxmp2 pore-forming protein (the insertion event shown on the left panel, the lower trace) at different membrane potentials (1.0 M KCl on both sides of the membrane). Applied membrane potentials are indicated. The data were filtered at 1.0 kHz and recorded at 2.0 kHz. (E) Current traces of low- (upper panel) and high (lower panel) conductance channels in response to voltage ramp protocol (from −100 to 100 mV, 10 sec). 1 M KCl on both sides of the membrane. (F) Current-voltage relationship of the fully open (25–30 pA at +20 mV, 1.0 M KCl) single channel (averages of six independent bilayers±S.D.). (G) Voltage dependence of the probability (Popen) of the high-conductance Pxmp2 channel being in a fully open state. After application of different voltage steps at t = 0, mean currents were calculated for a time period of 40 sec (from t = 20 sec to t = 60 sec). Data were normalized to the maximal currents of the fully open state at the corresponding holding potentials. Data points are an average of n = 4 independent bilayers.
Figure 4
Figure 4. Sub-conductance states of the Pxmp2 channel.
(A) Effect of anti-Pxmp2 antibodies on the pore-forming activity of Pxmp2. The results presented on panels A–D were collected on Pxmp2 channels at fully open state; the data were filtered at 0.4 kHz (A and B) or at 1.0 kHz (C and D) and recorded at 2.0 kHz. Single Pxmp2 channel was incorporated into lipid bilayer and analyzed before and after addition (marked by arrow) of the IgG fraction (3 µg) isolated from pre-immune (upper trace) or immune (lower trace) serum to both compartments of the chamber. The time gap is 120 sec. Here, and on panel B, experiments were repeated 6 times, typical pictures are presented. (B) Effect of alkalization of the bath solution on the pore-forming activity of Pxmp2. Current traces of a bilayer containing one Pxmp2 pore-forming protein before and after addition (marked by arrow) of 0.2 M Na2CO3, pH 11.2 (150 µl) to both compartments of the chamber are shown. The time gap is 60 sec. Two steps of the channel closure are marked by arrowheads. (C) High holding potential leads to closure of the Pxmp2 channel. The artificial membrane contained a single high-conductance channel (the insertion event not shown). Note stepwise closure of the channel at high holding potential, the amplitude of each step constitutes one third of that one of the fully open channel. (D) Flickering of a single Pxmp2 channel incorporated into lipid bilayer. The lower trace shows a timescale-expanded recording from the part (in the frame) of the upper trace. The direct transitions between three main subconductance states are marked by asterisks.
Figure 5
Figure 5. Multiple-channel recording of purified Pxmp2 using different organic anions as electrolytes.
The measurements were made using as a bath 1.0 M solutions of potassium or sodium salts of the anions at pH 7.2. The pH of solutions was adjusted by corresponding sodium or potassium hydroxides. The solutions were buffered with 10 mM MOPS, pH 7.2. Phosphate was used as 1 M potassium phosphate buffer, pH 7.2. Molecular masses of the corresponding anions are shown in brackets. The total number of insertion events (I.e.) is also shown. Two different batches of purified Pxmp2 were used with similar results obtained; typical pictures are presented. The purified Pxmp2 channel was also active with glycolic, lactic, acetic and allantoic acids (data not shown). We did not analyze in depth the dependence between size of anions and their conductance level since the hydrated radii of most of these anions are not known. However, as can be seen from the data, the conductivity of the Pxmp2 channel is clearly dependent on the size of the anions if their molecular mass exceeds 300 Da (compare, e.g., panels A and B with panels C and F), indicating partial restriction in the diffusion of these anions through the channel.
Figure 6
Figure 6. Transfer of solutes across peroxisomal membrane.
(A) Cooperation of non-selective channels and selective transporters in transfer of small (○) and ‘bulky’ (•) solutes across the peroxisomal membrane. Ps, peroxisomal lumen; Cyt, cytoplasm. The arrow indicates the vectorial transportation of the ‘bulky’ compound leading to formation of a separate pool for this solute in the peroxisomal lumen. (B) An apparent role of peroxisomal channels in the transfer of shuttle molecules participating in regeneration of NADH produced by the β-oxidation of fatty acids in peroxisomes. LDH: lactate dehydrogenase, G3PDH: NADH-dependent glycerol-3-phosphate dehydrogenase; both enzymes are partially localized to peroxisomes. (C) An apparent role for Nudix hydrolases in removal of cofactors out of peroxisomes (see ‘Discussion’ for details).

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