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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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Peroxisome docking requires ergosterol, PI(4,5)P2, cytosolic proteins, and the hydrolysis of ATP and GTP. P1 and P2 were individually preprimed by incubation with cytosol and ATP for 10 min at 26°C. Peroxisomal vesicles were reisolated by centrifugation, washed, and resuspended in buffer. Reisolated P1 and P2 were incubated individually for 5 min at 26°C with or without nystatin, phosphoinositide-specific mAbs, GTPγS, or ATPγS in the presence or absence of cytosol, as indicated. P1 and P2 were mixed and supplemented with ATP. After a 10-min incubation at 26°C, peroxisomal vesicles were subjected to fractionation by flotation on a multistep sucrose gradient. The percentage of recovery of loaded protein is presented. Equal volumes of gradient fractions were analyzed by immunoblotting with anti-Pex16p and anti-thiolase antibodies. The peak fractions for P1 and P2, their docking complex (P1/P2), and the product of their fusion (P3) are indicated. The positions of the precursor form of thiolase (pTHI), which was found in P2 and P1/P2, and of the mature form of thiolase (mTHI), which was associated with P3, are shown.
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fig3: Peroxisome docking requires ergosterol, PI(4,5)P2, cytosolic proteins, and the hydrolysis of ATP and GTP. P1 and P2 were individually preprimed by incubation with cytosol and ATP for 10 min at 26°C. Peroxisomal vesicles were reisolated by centrifugation, washed, and resuspended in buffer. Reisolated P1 and P2 were incubated individually for 5 min at 26°C with or without nystatin, phosphoinositide-specific mAbs, GTPγS, or ATPγS in the presence or absence of cytosol, as indicated. P1 and P2 were mixed and supplemented with ATP. After a 10-min incubation at 26°C, peroxisomal vesicles were subjected to fractionation by flotation on a multistep sucrose gradient. The percentage of recovery of loaded protein is presented. Equal volumes of gradient fractions were analyzed by immunoblotting with anti-Pex16p and anti-thiolase antibodies. The peak fractions for P1 and P2, their docking complex (P1/P2), and the product of their fusion (P3) are indicated. The positions of the precursor form of thiolase (pTHI), which was found in P2 and P1/P2, and of the mature form of thiolase (mTHI), which was associated with P3, are shown.

Mentions: The efficiency of peroxisome docking for fusion can be evaluated by monitoring the extent of in vitro formation of the docking complex P1/P2 (Titorenko et al., 2000). In this docking assay, P1 and P2 are first individually primed with cytosol and ATP, and then mixed and incubated with cytosol and ATP. Peroxisomes are finally subjected to fractionation by flotation on a multistep sucrose gradient. Under these conditions, the P1/P2 docking complex can be separated from undocked P1 and P2 and from P3, the product of fusion between P1 and P2 (Fig. 3; Titorenko et al., 2000). Using the in vitro assay for peroxisome docking, we found that, in addition to P2-bound Pex1p, cytosolic proteins, and ATP hydrolysis (Titorenko and Rachubinski, 2000), the docking of primed P1 and P2 depends on ergosterol, PI(4,5)P2, and GTP hydrolysis by GTPase(s) (Fig. 3). However, docking does not require PI(4)P (Fig. 3).


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)

Peroxisome docking requires ergosterol, PI(4,5)P2, cytosolic proteins, and the hydrolysis of ATP and GTP. P1 and P2 were individually preprimed by incubation with cytosol and ATP for 10 min at 26°C. Peroxisomal vesicles were reisolated by centrifugation, washed, and resuspended in buffer. Reisolated P1 and P2 were incubated individually for 5 min at 26°C with or without nystatin, phosphoinositide-specific mAbs, GTPγS, or ATPγS in the presence or absence of cytosol, as indicated. P1 and P2 were mixed and supplemented with ATP. After a 10-min incubation at 26°C, peroxisomal vesicles were subjected to fractionation by flotation on a multistep sucrose gradient. The percentage of recovery of loaded protein is presented. Equal volumes of gradient fractions were analyzed by immunoblotting with anti-Pex16p and anti-thiolase antibodies. The peak fractions for P1 and P2, their docking complex (P1/P2), and the product of their fusion (P3) are indicated. The positions of the precursor form of thiolase (pTHI), which was found in P2 and P1/P2, and of the mature form of thiolase (mTHI), which was associated with P3, are shown.
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Related In: Results  -  Collection

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fig3: Peroxisome docking requires ergosterol, PI(4,5)P2, cytosolic proteins, and the hydrolysis of ATP and GTP. P1 and P2 were individually preprimed by incubation with cytosol and ATP for 10 min at 26°C. Peroxisomal vesicles were reisolated by centrifugation, washed, and resuspended in buffer. Reisolated P1 and P2 were incubated individually for 5 min at 26°C with or without nystatin, phosphoinositide-specific mAbs, GTPγS, or ATPγS in the presence or absence of cytosol, as indicated. P1 and P2 were mixed and supplemented with ATP. After a 10-min incubation at 26°C, peroxisomal vesicles were subjected to fractionation by flotation on a multistep sucrose gradient. The percentage of recovery of loaded protein is presented. Equal volumes of gradient fractions were analyzed by immunoblotting with anti-Pex16p and anti-thiolase antibodies. The peak fractions for P1 and P2, their docking complex (P1/P2), and the product of their fusion (P3) are indicated. The positions of the precursor form of thiolase (pTHI), which was found in P2 and P1/P2, and of the mature form of thiolase (mTHI), which was associated with P3, are shown.
Mentions: The efficiency of peroxisome docking for fusion can be evaluated by monitoring the extent of in vitro formation of the docking complex P1/P2 (Titorenko et al., 2000). In this docking assay, P1 and P2 are first individually primed with cytosol and ATP, and then mixed and incubated with cytosol and ATP. Peroxisomes are finally subjected to fractionation by flotation on a multistep sucrose gradient. Under these conditions, the P1/P2 docking complex can be separated from undocked P1 and P2 and from P3, the product of fusion between P1 and P2 (Fig. 3; Titorenko et al., 2000). Using the in vitro assay for peroxisome docking, we found that, in addition to P2-bound Pex1p, cytosolic proteins, and ATP hydrolysis (Titorenko and Rachubinski, 2000), the docking of primed P1 and P2 depends on ergosterol, PI(4,5)P2, and GTP hydrolysis by GTPase(s) (Fig. 3). However, docking does not require PI(4)P (Fig. 3).

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