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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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Ergosterol ligands, phosphoinositide-specific antibodies, and nonhydrolyzable GTP analogues are reversible inhibitors of peroxisome fusion. (A, C, D, and F) P1 and P2 were preincubated individually for 5 min at 26°C without inhibitor or with the indicated amounts of ergosterol ligands (A), phosphoinositide-specific antibodies (a; C and D), or nonhydrolyzable GTP analogues (F). Pretreated P1 and P2 were mixed and supplemented with cytosol and ATP to yield standard fusion reactions. After a 90-min incubation at 26°C, the percentage of conversion of the precursor form of thiolase (pTHI) to its mature form (mTHI) as a measure of fusion was calculated. (B, E, and G) P1 and P2 were preincubated individually for 5 min at 26°C with 200 μg/ml nystatin (B), 60 μM of antibodies to PI(4)P or 60 μM of antibodies to PI(4,5)P2 (E), or 2 mM GTPγS or 2 mM GppNHp (G). Pretreated P1 and P2 were then mixed. The samples were supplemented with cytosol and ATP to yield standard fusion reactions and incubated at 26°C in the presence or absence of liposomes without or with the indicated amounts of ergosterol (B), PI(4)P or PI(4,5)P2 (E), or GTP (G). After a 90-min incubation, fusion efficiency was measured.
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fig1: Ergosterol ligands, phosphoinositide-specific antibodies, and nonhydrolyzable GTP analogues are reversible inhibitors of peroxisome fusion. (A, C, D, and F) P1 and P2 were preincubated individually for 5 min at 26°C without inhibitor or with the indicated amounts of ergosterol ligands (A), phosphoinositide-specific antibodies (a; C and D), or nonhydrolyzable GTP analogues (F). Pretreated P1 and P2 were mixed and supplemented with cytosol and ATP to yield standard fusion reactions. After a 90-min incubation at 26°C, the percentage of conversion of the precursor form of thiolase (pTHI) to its mature form (mTHI) as a measure of fusion was calculated. (B, E, and G) P1 and P2 were preincubated individually for 5 min at 26°C with 200 μg/ml nystatin (B), 60 μM of antibodies to PI(4)P or 60 μM of antibodies to PI(4,5)P2 (E), or 2 mM GTPγS or 2 mM GppNHp (G). Pretreated P1 and P2 were then mixed. The samples were supplemented with cytosol and ATP to yield standard fusion reactions and incubated at 26°C in the presence or absence of liposomes without or with the indicated amounts of ergosterol (B), PI(4)P or PI(4,5)P2 (E), or GTP (G). After a 90-min incubation, fusion efficiency was measured.

Mentions: Using an in vitro assay for the fusion of the immature peroxisomal vesicles P1 and P2 (Titorenko et al., 2000), we identified several inhibitors of this process. Fusion of P1 and P2 was inhibited by nystatin, filipin III, and amphotericin B (Fig. 1 A), each known for its propensity to sequester sterol lipids (Simons and Toomre, 2000) but not fully remove them from the membrane (Foster et al., 2003). In the yeast Saccharomyces cerevisiae (Zinser et al., 1991) and Y. lipolytica (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200409045/DC1), ergosterol is the major sterol lipid constituent of the peroxisomal membrane. The inhibitory effect of ergosterol ligands on peroxisome fusion can be reversed by phosphatidylcholine (PC)-based liposomes with ergosterol, but not by PC-based liposomes without it (Fig. 1 B). The inhibition of peroxisome fusion by ergosterol ligands was not due to the impairment of overall peroxisome integrity, as neither of these drugs caused the dissipation of the existing gradient of Ca2+ across the peroxisomal membrane (unpublished data). Together, these results suggest that ergosterol plays a specific role in the fusion of P1 and P2.


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)

Ergosterol ligands, phosphoinositide-specific antibodies, and nonhydrolyzable GTP analogues are reversible inhibitors of peroxisome fusion. (A, C, D, and F) P1 and P2 were preincubated individually for 5 min at 26°C without inhibitor or with the indicated amounts of ergosterol ligands (A), phosphoinositide-specific antibodies (a; C and D), or nonhydrolyzable GTP analogues (F). Pretreated P1 and P2 were mixed and supplemented with cytosol and ATP to yield standard fusion reactions. After a 90-min incubation at 26°C, the percentage of conversion of the precursor form of thiolase (pTHI) to its mature form (mTHI) as a measure of fusion was calculated. (B, E, and G) P1 and P2 were preincubated individually for 5 min at 26°C with 200 μg/ml nystatin (B), 60 μM of antibodies to PI(4)P or 60 μM of antibodies to PI(4,5)P2 (E), or 2 mM GTPγS or 2 mM GppNHp (G). Pretreated P1 and P2 were then mixed. The samples were supplemented with cytosol and ATP to yield standard fusion reactions and incubated at 26°C in the presence or absence of liposomes without or with the indicated amounts of ergosterol (B), PI(4)P or PI(4,5)P2 (E), or GTP (G). After a 90-min incubation, fusion efficiency was measured.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171827&req=5

fig1: Ergosterol ligands, phosphoinositide-specific antibodies, and nonhydrolyzable GTP analogues are reversible inhibitors of peroxisome fusion. (A, C, D, and F) P1 and P2 were preincubated individually for 5 min at 26°C without inhibitor or with the indicated amounts of ergosterol ligands (A), phosphoinositide-specific antibodies (a; C and D), or nonhydrolyzable GTP analogues (F). Pretreated P1 and P2 were mixed and supplemented with cytosol and ATP to yield standard fusion reactions. After a 90-min incubation at 26°C, the percentage of conversion of the precursor form of thiolase (pTHI) to its mature form (mTHI) as a measure of fusion was calculated. (B, E, and G) P1 and P2 were preincubated individually for 5 min at 26°C with 200 μg/ml nystatin (B), 60 μM of antibodies to PI(4)P or 60 μM of antibodies to PI(4,5)P2 (E), or 2 mM GTPγS or 2 mM GppNHp (G). Pretreated P1 and P2 were then mixed. The samples were supplemented with cytosol and ATP to yield standard fusion reactions and incubated at 26°C in the presence or absence of liposomes without or with the indicated amounts of ergosterol (B), PI(4)P or PI(4,5)P2 (E), or GTP (G). After a 90-min incubation, fusion efficiency was measured.
Mentions: Using an in vitro assay for the fusion of the immature peroxisomal vesicles P1 and P2 (Titorenko et al., 2000), we identified several inhibitors of this process. Fusion of P1 and P2 was inhibited by nystatin, filipin III, and amphotericin B (Fig. 1 A), each known for its propensity to sequester sterol lipids (Simons and Toomre, 2000) but not fully remove them from the membrane (Foster et al., 2003). In the yeast Saccharomyces cerevisiae (Zinser et al., 1991) and Y. lipolytica (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200409045/DC1), ergosterol is the major sterol lipid constituent of the peroxisomal membrane. The inhibitory effect of ergosterol ligands on peroxisome fusion can be reversed by phosphatidylcholine (PC)-based liposomes with ergosterol, but not by PC-based liposomes without it (Fig. 1 B). The inhibition of peroxisome fusion by ergosterol ligands was not due to the impairment of overall peroxisome integrity, as neither of these drugs caused the dissipation of the existing gradient of Ca2+ across the peroxisomal membrane (unpublished data). Together, these results suggest that ergosterol plays a specific role in the fusion of P1 and P2.

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