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Pxmp2 is a channel-forming protein in Mammalian peroxisomal membrane.

Rokka A, Antonenkov VD, Soininen R, Immonen HL, Pirilä PL, Bergmann U, Sormunen RT, Weckström M, Benz R, Hiltunen JK - PLoS ONE (2009)

Bottom Line: Concerning small solutes, the molecular nature of their traffic has remained an enigma.The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter Oulu, University of Oulu, Oulu, Finland.

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.

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Related in: MedlinePlus

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.
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pone-0005090-g005: 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.

Mentions: To further analyze the function of the Pxmp2 channel we made direct measurements of the pore-forming activity of purified protein using various organic anions as electrolytes (Figure 5A–5G). A variety of small mono- and divalent anions known to be peroxisomal metabolites, such as glycolate, pyruvate, 2-ketoglutarate, and others, can be transferred through the Pxmp2 channel. As expected, if the size of the anion is over 300 Da, e.g, lactobionic acid (358 Da) or AMP (347 Da), the single-channel conductance of Pxmp2 is significantly decreased, indicating that the movement of these compounds inside the channel is partially restricted. The only traces of channel-forming activity with a conductance well below 20 pS were detected in the presence of NAD (663 Da). No channel-forming activities were observed with ATP (507 Da), probably due to the high net negative charge of this molecule, which prevents diffusion of ATP through the cation-selective Pxmp2 channel.


Pxmp2 is a channel-forming protein in Mammalian peroxisomal membrane.

Rokka A, Antonenkov VD, Soininen R, Immonen HL, Pirilä PL, Bergmann U, Sormunen RT, Weckström M, Benz R, Hiltunen JK - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662417&req=5

pone-0005090-g005: 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.
Mentions: To further analyze the function of the Pxmp2 channel we made direct measurements of the pore-forming activity of purified protein using various organic anions as electrolytes (Figure 5A–5G). A variety of small mono- and divalent anions known to be peroxisomal metabolites, such as glycolate, pyruvate, 2-ketoglutarate, and others, can be transferred through the Pxmp2 channel. As expected, if the size of the anion is over 300 Da, e.g, lactobionic acid (358 Da) or AMP (347 Da), the single-channel conductance of Pxmp2 is significantly decreased, indicating that the movement of these compounds inside the channel is partially restricted. The only traces of channel-forming activity with a conductance well below 20 pS were detected in the presence of NAD (663 Da). No channel-forming activities were observed with ATP (507 Da), probably due to the high net negative charge of this molecule, which prevents diffusion of ATP through the cation-selective Pxmp2 channel.

Bottom Line: Concerning small solutes, the molecular nature of their traffic has remained an enigma.The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter Oulu, University of Oulu, Oulu, Finland.

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.

Show MeSH
Related in: MedlinePlus