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Dynamic ergosterol- and ceramide-rich domains in the peroxisomal membrane serve as an organizing platform for peroxisome fusion.

Boukh-Viner T, Guo T, Alexandrian A, Cerracchio A, Gregg C, Haile S, Kyskan R, Milijevic S, Oren D, Solomon J, Wong V, Nicaud JM, Rachubinski RA, English AM, Titorenko VI - J. Cell Biol. (2005)

Bottom Line: We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes.Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane.A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Concordia University, Montreal, Quebec H4B 1R6, Canada.

ABSTRACT
We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes. Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane. A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains. All of these proteins are essential for the fusion of the immature peroxisomal vesicles P1 and P2, the earliest intermediates in a multistep pathway leading to the formation of mature, metabolically active peroxisomes. Peroxisome fusion depends on the lateral movement of Pex1p, Pex6p, and phosphatidylinositol-4,5-bisphosphate-binding proteins from ECR domains to a detergent-soluble portion of the membrane, followed by their release to the cytosol. Our data suggest a model for the multistep reorganization of the multicomponent peroxisome fusion machinery that transiently associates with ECR domains.

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Ceramide is distributed symmetrically between the two leaflets of the peroxisomal membrane, whereas phosphatidylserine (PS) associates mostly with the cytosolic leaflet. (A–D) A suspension of purified P1 or P2 was divided into two equal aliquots. One aliquot remained untreated, whereas peroxisomal vesicles in the other aliquot were osmotically lysed. Twofold serial dilutions of intact P1 or P2 (from the first aliquot) and of the membranes recovered after centrifugation of osmotically lysed P1 or P2 (from the second aliquot) in the range of 10–160 μg of protein per milliliter were exposed to anti-ceramide mouse IgG or anti-PS mouse IgM. All samples were then treated with fluorescein-conjugated goat anti–mouse IgG or fluorescein-conjugated goat anti–mouse IgM antibodies. To amplify the signals from fluorescein-labeled secondary antibodies, the samples were first labeled with Alexa Fluor 488 rabbit anti-fluorescein/Oregon green IgG and then treated with Alexa Fluor 488 goat anti–rabbit IgG. The Alexa Fluor 488 fluorescence at 510 nm was monitored in individual samples. Controls were made for the nonspecific binding of mouse IgG, mouse IgM, and/or fluorescein- or Alexa Fluor 488–labeled antibodies to the membrane, and background fluorescence was subtracted. (E and F) The ratio “fluorescence for intact vesicles (Fi)/fluorescence for osmotically lysed vesicles (Fol)” (means ± SD from three experiments) was calculated for each dilution of intact P1 and P2 and of the membranes recovered after osmotic lysis of these peroxisomal vesicles. This ratio is equal to the fraction of the total pool of a monitored lipid that is located in the outer (cytosolic) leaflet of the membrane.
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fig5: Ceramide is distributed symmetrically between the two leaflets of the peroxisomal membrane, whereas phosphatidylserine (PS) associates mostly with the cytosolic leaflet. (A–D) A suspension of purified P1 or P2 was divided into two equal aliquots. One aliquot remained untreated, whereas peroxisomal vesicles in the other aliquot were osmotically lysed. Twofold serial dilutions of intact P1 or P2 (from the first aliquot) and of the membranes recovered after centrifugation of osmotically lysed P1 or P2 (from the second aliquot) in the range of 10–160 μg of protein per milliliter were exposed to anti-ceramide mouse IgG or anti-PS mouse IgM. All samples were then treated with fluorescein-conjugated goat anti–mouse IgG or fluorescein-conjugated goat anti–mouse IgM antibodies. To amplify the signals from fluorescein-labeled secondary antibodies, the samples were first labeled with Alexa Fluor 488 rabbit anti-fluorescein/Oregon green IgG and then treated with Alexa Fluor 488 goat anti–rabbit IgG. The Alexa Fluor 488 fluorescence at 510 nm was monitored in individual samples. Controls were made for the nonspecific binding of mouse IgG, mouse IgM, and/or fluorescein- or Alexa Fluor 488–labeled antibodies to the membrane, and background fluorescence was subtracted. (E and F) The ratio “fluorescence for intact vesicles (Fi)/fluorescence for osmotically lysed vesicles (Fol)” (means ± SD from three experiments) was calculated for each dilution of intact P1 and P2 and of the membranes recovered after osmotic lysis of these peroxisomal vesicles. This ratio is equal to the fraction of the total pool of a monitored lipid that is located in the outer (cytosolic) leaflet of the membrane.

Mentions: Lipids are asymmetrically arranged between the two leaflets of the plasma membrane bilayer in eukaryotic cells. Glycolipids and sphingomyelin, the two major sphingolipid components of lipid rafts in mammals, and the glycerophospholipid PC reside predominantly in the outer (exoplasmic) leaflet of the plasma membrane (Pomorski et al., 2004). In contrast, the glycerophospholipids PE, PI, and PS are restricted to the inner (cytosolic) leaflet of the plasma membrane (Pomorski et al., 2004). Cholesterol, a major sterol constituent of lipid rafts in mammals, is equally distributed across the bilayer (Munro, 2003). Using monoclonal antibodies to ceramide and PS, we evaluated the transbilayer distribution of these two lipids in the membranes of unprimed P1 and P2. In the membranes of osmotically lysed P1 and P2, both leaflets of the bilayer were accessible to anti-ceramide and anti-PS antibodies. In contrast, in the membranes of intact P1 and P2, these monoclonal antibodies could detect only ceramide and PS that resided in the cytosolic leaflet. The levels of ceramide recovered in the membranes of osmotically lysed P1 and P2 exceeded the levels of this sphingolipid detected in intact membranes of both vesicles (Fig. 5, A and C), with about half of the ceramide located in the outer (cytosolic) leaflet of the bilayer (Fig. 5 E). Thus, the sphingolipid component of ECR domains is distributed symmetrically between the two leaflets of the membrane bilayers in P1 and P2. In contrast, the glycerophospholipid PS resides predominantly in the outer (cytosolic) leaflets of the membranes of P1 and P2. In fact, the vast majority of this lipid in intact P1 and P2 was accessible to anti-PS antibodies (Fig. 5, B, D, and F).


Dynamic ergosterol- and ceramide-rich domains in the peroxisomal membrane serve as an organizing platform for peroxisome fusion.

Boukh-Viner T, Guo T, Alexandrian A, Cerracchio A, Gregg C, Haile S, Kyskan R, Milijevic S, Oren D, Solomon J, Wong V, Nicaud JM, Rachubinski RA, English AM, Titorenko VI - J. Cell Biol. (2005)

Ceramide is distributed symmetrically between the two leaflets of the peroxisomal membrane, whereas phosphatidylserine (PS) associates mostly with the cytosolic leaflet. (A–D) A suspension of purified P1 or P2 was divided into two equal aliquots. One aliquot remained untreated, whereas peroxisomal vesicles in the other aliquot were osmotically lysed. Twofold serial dilutions of intact P1 or P2 (from the first aliquot) and of the membranes recovered after centrifugation of osmotically lysed P1 or P2 (from the second aliquot) in the range of 10–160 μg of protein per milliliter were exposed to anti-ceramide mouse IgG or anti-PS mouse IgM. All samples were then treated with fluorescein-conjugated goat anti–mouse IgG or fluorescein-conjugated goat anti–mouse IgM antibodies. To amplify the signals from fluorescein-labeled secondary antibodies, the samples were first labeled with Alexa Fluor 488 rabbit anti-fluorescein/Oregon green IgG and then treated with Alexa Fluor 488 goat anti–rabbit IgG. The Alexa Fluor 488 fluorescence at 510 nm was monitored in individual samples. Controls were made for the nonspecific binding of mouse IgG, mouse IgM, and/or fluorescein- or Alexa Fluor 488–labeled antibodies to the membrane, and background fluorescence was subtracted. (E and F) The ratio “fluorescence for intact vesicles (Fi)/fluorescence for osmotically lysed vesicles (Fol)” (means ± SD from three experiments) was calculated for each dilution of intact P1 and P2 and of the membranes recovered after osmotic lysis of these peroxisomal vesicles. This ratio is equal to the fraction of the total pool of a monitored lipid that is located in the outer (cytosolic) leaflet of the membrane.
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Related In: Results  -  Collection

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fig5: Ceramide is distributed symmetrically between the two leaflets of the peroxisomal membrane, whereas phosphatidylserine (PS) associates mostly with the cytosolic leaflet. (A–D) A suspension of purified P1 or P2 was divided into two equal aliquots. One aliquot remained untreated, whereas peroxisomal vesicles in the other aliquot were osmotically lysed. Twofold serial dilutions of intact P1 or P2 (from the first aliquot) and of the membranes recovered after centrifugation of osmotically lysed P1 or P2 (from the second aliquot) in the range of 10–160 μg of protein per milliliter were exposed to anti-ceramide mouse IgG or anti-PS mouse IgM. All samples were then treated with fluorescein-conjugated goat anti–mouse IgG or fluorescein-conjugated goat anti–mouse IgM antibodies. To amplify the signals from fluorescein-labeled secondary antibodies, the samples were first labeled with Alexa Fluor 488 rabbit anti-fluorescein/Oregon green IgG and then treated with Alexa Fluor 488 goat anti–rabbit IgG. The Alexa Fluor 488 fluorescence at 510 nm was monitored in individual samples. Controls were made for the nonspecific binding of mouse IgG, mouse IgM, and/or fluorescein- or Alexa Fluor 488–labeled antibodies to the membrane, and background fluorescence was subtracted. (E and F) The ratio “fluorescence for intact vesicles (Fi)/fluorescence for osmotically lysed vesicles (Fol)” (means ± SD from three experiments) was calculated for each dilution of intact P1 and P2 and of the membranes recovered after osmotic lysis of these peroxisomal vesicles. This ratio is equal to the fraction of the total pool of a monitored lipid that is located in the outer (cytosolic) leaflet of the membrane.
Mentions: Lipids are asymmetrically arranged between the two leaflets of the plasma membrane bilayer in eukaryotic cells. Glycolipids and sphingomyelin, the two major sphingolipid components of lipid rafts in mammals, and the glycerophospholipid PC reside predominantly in the outer (exoplasmic) leaflet of the plasma membrane (Pomorski et al., 2004). In contrast, the glycerophospholipids PE, PI, and PS are restricted to the inner (cytosolic) leaflet of the plasma membrane (Pomorski et al., 2004). Cholesterol, a major sterol constituent of lipid rafts in mammals, is equally distributed across the bilayer (Munro, 2003). Using monoclonal antibodies to ceramide and PS, we evaluated the transbilayer distribution of these two lipids in the membranes of unprimed P1 and P2. In the membranes of osmotically lysed P1 and P2, both leaflets of the bilayer were accessible to anti-ceramide and anti-PS antibodies. In contrast, in the membranes of intact P1 and P2, these monoclonal antibodies could detect only ceramide and PS that resided in the cytosolic leaflet. The levels of ceramide recovered in the membranes of osmotically lysed P1 and P2 exceeded the levels of this sphingolipid detected in intact membranes of both vesicles (Fig. 5, A and C), with about half of the ceramide located in the outer (cytosolic) leaflet of the bilayer (Fig. 5 E). Thus, the sphingolipid component of ECR domains is distributed symmetrically between the two leaflets of the membrane bilayers in P1 and P2. In contrast, the glycerophospholipid PS resides predominantly in the outer (cytosolic) leaflets of the membranes of P1 and P2. In fact, the vast majority of this lipid in intact P1 and P2 was accessible to anti-PS antibodies (Fig. 5, B, D, and F).

Bottom Line: We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes.Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane.A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Concordia University, Montreal, Quebec H4B 1R6, Canada.

ABSTRACT
We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes. Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane. A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains. All of these proteins are essential for the fusion of the immature peroxisomal vesicles P1 and P2, the earliest intermediates in a multistep pathway leading to the formation of mature, metabolically active peroxisomes. Peroxisome fusion depends on the lateral movement of Pex1p, Pex6p, and phosphatidylinositol-4,5-bisphosphate-binding proteins from ECR domains to a detergent-soluble portion of the membrane, followed by their release to the cytosol. Our data suggest a model for the multistep reorganization of the multicomponent peroxisome fusion machinery that transiently associates with ECR domains.

Show MeSH
Related in: MedlinePlus