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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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PI(4,5)P2-bp and P2-bound Pex1p segregate from ECR domains during peroxisome docking. P1 and P2 were incubated individually with cytosol and ATP at 26°C. After a 10-min incubation, P1 and P2 were pelleted, resuspended in a buffer, and mixed. Samples were incubated at 26°C with or without cytosol, ATP, nystatin, GTPγS, ATPγS, antibodies to Pex1p, or antibodies to PI(4)P. Equal aliquots of peroxisomal vesicles were taken at the times indicated. Samples were subjected to osmotic lysis followed by centrifugation. The pellets of membranes recovered after such centrifugation were resuspended in ice-cold MBS buffer and supplemented with a 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. Equal volumes of gradient fractions were analyzed as described in Fig. 4.
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fig7: PI(4,5)P2-bp and P2-bound Pex1p segregate from ECR domains during peroxisome docking. P1 and P2 were incubated individually with cytosol and ATP at 26°C. After a 10-min incubation, P1 and P2 were pelleted, resuspended in a buffer, and mixed. Samples were incubated at 26°C with or without cytosol, ATP, nystatin, GTPγS, ATPγS, antibodies to Pex1p, or antibodies to PI(4)P. Equal aliquots of peroxisomal vesicles were taken at the times indicated. Samples were subjected to osmotic lysis followed by centrifugation. The pellets of membranes recovered after such centrifugation were resuspended in ice-cold MBS buffer and supplemented with a 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. Equal volumes of gradient fractions were analyzed as described in Fig. 4.

Mentions: Docking of preprimed P1 and P2 results in further remodeling of the protein repertoire of their ECR domains. By 5 min of docking, P2-associated Pex1p and proteins that bind PI(4,5)P2 on the cytosolic faces of both fusion partners moved from these floating membrane domains to detergent-soluble, ergosterol- and ceramide-poor domains recovered in the high-density bottom fractions of the flotation gradient (Fig. 7). The movement of Pex1p and PI(4,5)P2-bp to a detergent-soluble portion of the membranes was followed by the release of these proteins to the cytosol, which was evident after 10 min of docking (Fig. 7). Not all proteins moved away from ECR domains during peroxisome docking. The group of ECR resident proteins included P1-associated GTP-bp and proteins that bind PI(4)P on the cytosolic faces of both fusion partners (Fig. 7). Furthermore, no dramatic changes in lipid composition of ECR domains were observed during docking of separately primed P1 and P2 (compare Figs. 6 and 7).


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)

PI(4,5)P2-bp and P2-bound Pex1p segregate from ECR domains during peroxisome docking. P1 and P2 were incubated individually with cytosol and ATP at 26°C. After a 10-min incubation, P1 and P2 were pelleted, resuspended in a buffer, and mixed. Samples were incubated at 26°C with or without cytosol, ATP, nystatin, GTPγS, ATPγS, antibodies to Pex1p, or antibodies to PI(4)P. Equal aliquots of peroxisomal vesicles were taken at the times indicated. Samples were subjected to osmotic lysis followed by centrifugation. The pellets of membranes recovered after such centrifugation were resuspended in ice-cold MBS buffer and supplemented with a 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. Equal volumes of gradient fractions were analyzed as described in Fig. 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171827&req=5

fig7: PI(4,5)P2-bp and P2-bound Pex1p segregate from ECR domains during peroxisome docking. P1 and P2 were incubated individually with cytosol and ATP at 26°C. After a 10-min incubation, P1 and P2 were pelleted, resuspended in a buffer, and mixed. Samples were incubated at 26°C with or without cytosol, ATP, nystatin, GTPγS, ATPγS, antibodies to Pex1p, or antibodies to PI(4)P. Equal aliquots of peroxisomal vesicles were taken at the times indicated. Samples were subjected to osmotic lysis followed by centrifugation. The pellets of membranes recovered after such centrifugation were resuspended in ice-cold MBS buffer and supplemented with a 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. Equal volumes of gradient fractions were analyzed as described in Fig. 4.
Mentions: Docking of preprimed P1 and P2 results in further remodeling of the protein repertoire of their ECR domains. By 5 min of docking, P2-associated Pex1p and proteins that bind PI(4,5)P2 on the cytosolic faces of both fusion partners moved from these floating membrane domains to detergent-soluble, ergosterol- and ceramide-poor domains recovered in the high-density bottom fractions of the flotation gradient (Fig. 7). The movement of Pex1p and PI(4,5)P2-bp to a detergent-soluble portion of the membranes was followed by the release of these proteins to the cytosol, which was evident after 10 min of docking (Fig. 7). Not all proteins moved away from ECR domains during peroxisome docking. The group of ECR resident proteins included P1-associated GTP-bp and proteins that bind PI(4)P on the cytosolic faces of both fusion partners (Fig. 7). Furthermore, no dramatic changes in lipid composition of ECR domains were observed during docking of separately primed P1 and P2 (compare Figs. 6 and 7).

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