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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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Pex1p, Pex6p, and phosphoinositide- and GTP-bp associate with ECR membrane domains that can float to low density during centrifugation in a sucrose density gradient. (A and B) The pellet of membranes recovered after centrifugation of osmotically lysed unprimed P1 (A) or P2 (B) was resuspended in ice-cold MBS buffer and supplemented with a nonionic detergent, Brij 35. After incubation on ice for 30 min, the Brij 35–treated membranes were subjected to centrifugation by flotation in a discontinuous sucrose density gradient. Proteins from equal volumes of gradient fractions were immunoblotted with the indicated antibodies. Equal volumes of gradient fractions were also subjected to protein-lipid overlay assays using nitrocellulose membrane arrays spotted with PI(4)P or PI(4,5)P2, GTP slot-blot, and lipid extraction, which was followed by TLC and visualization of lipids. The positions of Pex1p (arrows) and Pex6p (arrowhead), which were identified by mass spectrometric peptide mapping, are indicated. (C and D) Electron micrographs of ECR domains recovered in the low-density fraction 7 of the flotation sucrose density gradients presented in A and B, respectively. Bars, 100 nm.
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fig4: Pex1p, Pex6p, and phosphoinositide- and GTP-bp associate with ECR membrane domains that can float to low density during centrifugation in a sucrose density gradient. (A and B) The pellet of membranes recovered after centrifugation of osmotically lysed unprimed P1 (A) or P2 (B) was resuspended in ice-cold MBS buffer and supplemented with a nonionic detergent, Brij 35. After incubation on ice for 30 min, the Brij 35–treated membranes were subjected to centrifugation by flotation in a discontinuous sucrose density gradient. Proteins from equal volumes of gradient fractions were immunoblotted with the indicated antibodies. Equal volumes of gradient fractions were also subjected to protein-lipid overlay assays using nitrocellulose membrane arrays spotted with PI(4)P or PI(4,5)P2, GTP slot-blot, and lipid extraction, which was followed by TLC and visualization of lipids. The positions of Pex1p (arrows) and Pex6p (arrowhead), which were identified by mass spectrometric peptide mapping, are indicated. (C and D) Electron micrographs of ECR domains recovered in the low-density fraction 7 of the flotation sucrose density gradients presented in A and B, respectively. Bars, 100 nm.

Mentions: When exposed to cold nonionic detergents, detergent-insoluble protein and lipid components of lipid rafts can float to low density, away from detergent-soluble proteins and lipids, during centrifugation in sucrose density gradients (Brown and Rose, 1992). Our data on the insolubility of a distinct set of membrane proteins and lipids in various detergents (Figs. S2 and S3) suggested that these constituents of the membranes of P1 and P2 reside in ECR domains, perhaps lipid raft(s), that house several essential components of the peroxisome fusion machinery. To confirm the existence of such domains and to purify them for further characterization, Brij 35 extracts of the membranes of unprimed P1 and P2 were subjected to centrifugation by flotation in a discontinuous sucrose density gradient. A discrete group of detergent-insoluble membrane proteins, including the P1-associated forms of Pex1p, PI(4)P-bp, PI(4,5)P2-bp, and GTP-bp (Fig. 4 A) and the P2-bound forms of Pex1p, Pex6p, PI(4)P-bp, and PI(4,5)P2-bp (Fig. 4 B), floated to the low-density fractions 5–9 and peaked in fraction 7 of the gradient. The identity of Pex1p and Pex6p was confirmed by mass spectrometric peptide mapping. Furthermore, many detergent-soluble membrane proteins, including Pex2p and Pex16p, were recovered in the bottom fractions 1, 2, and 3 of the gradient (Fig. 4), with all three fractions corresponding to the load.


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)

Pex1p, Pex6p, and phosphoinositide- and GTP-bp associate with ECR membrane domains that can float to low density during centrifugation in a sucrose density gradient. (A and B) The pellet of membranes recovered after centrifugation of osmotically lysed unprimed P1 (A) or P2 (B) was resuspended in ice-cold MBS buffer and supplemented with a nonionic detergent, Brij 35. After incubation on ice for 30 min, the Brij 35–treated membranes were subjected to centrifugation by flotation in a discontinuous sucrose density gradient. Proteins from equal volumes of gradient fractions were immunoblotted with the indicated antibodies. Equal volumes of gradient fractions were also subjected to protein-lipid overlay assays using nitrocellulose membrane arrays spotted with PI(4)P or PI(4,5)P2, GTP slot-blot, and lipid extraction, which was followed by TLC and visualization of lipids. The positions of Pex1p (arrows) and Pex6p (arrowhead), which were identified by mass spectrometric peptide mapping, are indicated. (C and D) Electron micrographs of ECR domains recovered in the low-density fraction 7 of the flotation sucrose density gradients presented in A and B, respectively. Bars, 100 nm.
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fig4: Pex1p, Pex6p, and phosphoinositide- and GTP-bp associate with ECR membrane domains that can float to low density during centrifugation in a sucrose density gradient. (A and B) The pellet of membranes recovered after centrifugation of osmotically lysed unprimed P1 (A) or P2 (B) was resuspended in ice-cold MBS buffer and supplemented with a nonionic detergent, Brij 35. After incubation on ice for 30 min, the Brij 35–treated membranes were subjected to centrifugation by flotation in a discontinuous sucrose density gradient. Proteins from equal volumes of gradient fractions were immunoblotted with the indicated antibodies. Equal volumes of gradient fractions were also subjected to protein-lipid overlay assays using nitrocellulose membrane arrays spotted with PI(4)P or PI(4,5)P2, GTP slot-blot, and lipid extraction, which was followed by TLC and visualization of lipids. The positions of Pex1p (arrows) and Pex6p (arrowhead), which were identified by mass spectrometric peptide mapping, are indicated. (C and D) Electron micrographs of ECR domains recovered in the low-density fraction 7 of the flotation sucrose density gradients presented in A and B, respectively. Bars, 100 nm.
Mentions: When exposed to cold nonionic detergents, detergent-insoluble protein and lipid components of lipid rafts can float to low density, away from detergent-soluble proteins and lipids, during centrifugation in sucrose density gradients (Brown and Rose, 1992). Our data on the insolubility of a distinct set of membrane proteins and lipids in various detergents (Figs. S2 and S3) suggested that these constituents of the membranes of P1 and P2 reside in ECR domains, perhaps lipid raft(s), that house several essential components of the peroxisome fusion machinery. To confirm the existence of such domains and to purify them for further characterization, Brij 35 extracts of the membranes of unprimed P1 and P2 were subjected to centrifugation by flotation in a discontinuous sucrose density gradient. A discrete group of detergent-insoluble membrane proteins, including the P1-associated forms of Pex1p, PI(4)P-bp, PI(4,5)P2-bp, and GTP-bp (Fig. 4 A) and the P2-bound forms of Pex1p, Pex6p, PI(4)P-bp, and PI(4,5)P2-bp (Fig. 4 B), floated to the low-density fractions 5–9 and peaked in fraction 7 of the gradient. The identity of Pex1p and Pex6p was confirmed by mass spectrometric peptide mapping. Furthermore, many detergent-soluble membrane proteins, including Pex2p and Pex16p, were recovered in the bottom fractions 1, 2, and 3 of the gradient (Fig. 4), with all three fractions corresponding to the load.

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