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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 and Pex6p move from detergent-resistant ECR domains to a detergent-soluble portion of the membrane during peroxisome priming. P1 (A) or P2 (B) vesicles were incubated individually with cytosol and ATP at 26°C. Equal aliquots of peroxisomal vesicles were taken at the times indicated. P1 and P2 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. The positions of Pex1p (arrows) and Pex6p (arrowheads), which were identified by mass spectrometric peptide mapping, are indicated.
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fig6: Pex1p and Pex6p move from detergent-resistant ECR domains to a detergent-soluble portion of the membrane during peroxisome priming. P1 (A) or P2 (B) vesicles were incubated individually with cytosol and ATP at 26°C. Equal aliquots of peroxisomal vesicles were taken at the times indicated. P1 and P2 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. The positions of Pex1p (arrows) and Pex6p (arrowheads), which were identified by mass spectrometric peptide mapping, are indicated.

Mentions: The release of P1-bound Pex1p and of P2-associated Pex6p from the organelle surface to the cytosol is a hallmark event of the priming of P1 and P2 (Titorenko and Rachubinski, 2000). It seems that this event includes two consecutive steps. Specifically, both AAA ATPases initially relocate from ECR domains to an ergosterol- and ceramide-poor portion of the membrane, from which they are then released to the cytosol. In fact, when Brij 35 extracts of the membranes of unprimed P1 and P2 were subjected to centrifugation by flotation in a sucrose density gradient, the majority of P1-bound Pex1p and of P2-associated Pex6p was recovered in detergent-resistant ECR domains (Fig. 6). However, already by 3 min of priming, both proteins were seen only in detergent-soluble, ergosterol- and ceramide-poor membrane domains that were recovered in the high-density bottom fractions 1–3 of the gradient (Fig. 6). By 6 min of priming, both AAA ATPases were released from these detergent-soluble domains to the cytosol (Fig. 6). The two consecutive steps in the priming-specific release of Pex1p and Pex6p from the organelle surface to the cytosol have different requirements. Whereas the relocation of both proteins from ECR domains to an ergosterol- and ceramide-poor portion of the membrane depends on ergosterol, PI(4)P, and PI(4,5)P2, their subsequent release to the cytosol requires cytosolic proteins and ATP hydrolysis (Fig. 6).


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 and Pex6p move from detergent-resistant ECR domains to a detergent-soluble portion of the membrane during peroxisome priming. P1 (A) or P2 (B) vesicles were incubated individually with cytosol and ATP at 26°C. Equal aliquots of peroxisomal vesicles were taken at the times indicated. P1 and P2 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. The positions of Pex1p (arrows) and Pex6p (arrowheads), which were identified by mass spectrometric peptide mapping, are indicated.
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Related In: Results  -  Collection

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fig6: Pex1p and Pex6p move from detergent-resistant ECR domains to a detergent-soluble portion of the membrane during peroxisome priming. P1 (A) or P2 (B) vesicles were incubated individually with cytosol and ATP at 26°C. Equal aliquots of peroxisomal vesicles were taken at the times indicated. P1 and P2 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. The positions of Pex1p (arrows) and Pex6p (arrowheads), which were identified by mass spectrometric peptide mapping, are indicated.
Mentions: The release of P1-bound Pex1p and of P2-associated Pex6p from the organelle surface to the cytosol is a hallmark event of the priming of P1 and P2 (Titorenko and Rachubinski, 2000). It seems that this event includes two consecutive steps. Specifically, both AAA ATPases initially relocate from ECR domains to an ergosterol- and ceramide-poor portion of the membrane, from which they are then released to the cytosol. In fact, when Brij 35 extracts of the membranes of unprimed P1 and P2 were subjected to centrifugation by flotation in a sucrose density gradient, the majority of P1-bound Pex1p and of P2-associated Pex6p was recovered in detergent-resistant ECR domains (Fig. 6). However, already by 3 min of priming, both proteins were seen only in detergent-soluble, ergosterol- and ceramide-poor membrane domains that were recovered in the high-density bottom fractions 1–3 of the gradient (Fig. 6). By 6 min of priming, both AAA ATPases were released from these detergent-soluble domains to the cytosol (Fig. 6). The two consecutive steps in the priming-specific release of Pex1p and Pex6p from the organelle surface to the cytosol have different requirements. Whereas the relocation of both proteins from ECR domains to an ergosterol- and ceramide-poor portion of the membrane depends on ergosterol, PI(4)P, and PI(4,5)P2, their subsequent release to the cytosol requires cytosolic proteins and ATP hydrolysis (Fig. 6).

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