Limits...
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.

Show MeSH

Related in: MedlinePlus

Dynamics of the association of Pex1p, Pex6p, and phosphoinositide- and GTP-bp with membranes of P1 and P2 during their priming. (A) L-[35S]methionine–labeled P1 and L-[35S]methionine–labeled P2 were preincubated individually for 5 min at 26°C with or without nystatin (an ergosterol ligand), phosphoinositide-specific antibodies, ATPγS, or nonhydrolyzable GTP analogues. Pretreated P1 and P2 were supplemented with ATP and incubated individually in the presence or absence of unlabeled cytosol, as indicated. After a 10-min incubation at 26°C, peroxisomal vesicles were pelleted. Pex1p and Pex6p were immunoprecipitated under denaturing conditions from the pellet (P) and supernatant (S) fractions. Immunoprecipitates were resolved by SDS-PAGE and visualized by fluorography. (B and C) Intact P1 and P2, either labeled with L-[35S]methionine (B) or unlabeled (C), were primed individually by incubation with unlabeled cytosol and ATP or remained unprimed. Equal aliquots of primed and unprimed peroxisomes (30 μg of protein per aliquot) were treated with 30 μg of trypsin or 1 M NaCl for 30 min on ice. Peroxisomes were then osmotically lysed and subjected to centrifugation. The pellet of membranes recovered after such centrifugation was solubilized with a detergent, n-OG. Detergent-soluble membrane proteins were analyzed by protein-lipid overlay assay using commercial PIP-Strips (B) or by GTP slot-blot with guanosine 5′-α-[32P]triphosphate (C). Lipid- and GTP-bp were visualized by autoradiography.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171827&req=5

fig2: Dynamics of the association of Pex1p, Pex6p, and phosphoinositide- and GTP-bp with membranes of P1 and P2 during their priming. (A) L-[35S]methionine–labeled P1 and L-[35S]methionine–labeled P2 were preincubated individually for 5 min at 26°C with or without nystatin (an ergosterol ligand), phosphoinositide-specific antibodies, ATPγS, or nonhydrolyzable GTP analogues. Pretreated P1 and P2 were supplemented with ATP and incubated individually in the presence or absence of unlabeled cytosol, as indicated. After a 10-min incubation at 26°C, peroxisomal vesicles were pelleted. Pex1p and Pex6p were immunoprecipitated under denaturing conditions from the pellet (P) and supernatant (S) fractions. Immunoprecipitates were resolved by SDS-PAGE and visualized by fluorography. (B and C) Intact P1 and P2, either labeled with L-[35S]methionine (B) or unlabeled (C), were primed individually by incubation with unlabeled cytosol and ATP or remained unprimed. Equal aliquots of primed and unprimed peroxisomes (30 μg of protein per aliquot) were treated with 30 μg of trypsin or 1 M NaCl for 30 min on ice. Peroxisomes were then osmotically lysed and subjected to centrifugation. The pellet of membranes recovered after such centrifugation was solubilized with a detergent, n-OG. Detergent-soluble membrane proteins were analyzed by protein-lipid overlay assay using commercial PIP-Strips (B) or by GTP slot-blot with guanosine 5′-α-[32P]triphosphate (C). Lipid- and GTP-bp were visualized by autoradiography.

Mentions: Fusion of P1 and P2 is a multistep process that includes priming, docking, and fusion events (Titorenko and Rachubinski, 2000). Priming (activation) of P1 and P2 before their physical contact commits both fusion partners to subsequent docking. Priming requires two AAA ATPases, Pex1p and Pex6p (Titorenko et al., 2000). Before priming, Pex1p is associated with the cytosolic surface of P1, whereas Pex1p and Pex6p are bound to the outer surface of P2. During priming, ATP hydrolysis triggers cytosol-dependent release of Pex1p from P1 and of Pex6p from P2, whereas P2-associated Pex1p remains bound to the organelle. We evaluated the requirements for the release of AAA ATPases during peroxisome priming. Using the extent of release of Pex1p from P1 and of Pex6p from P2 as a measure of priming efficiency, we found that, in addition to cytosolic proteins, ATP hydrolysis, and a particular type of AAA ATPase (Pex1p for P1 and Pex6p for P2; Titorenko and Rachubinski, 2000), priming of both fusion partners requires ergosterol, PI(4)P, and PI(4,5)P2 (Fig. 2 A). However, priming does not depend on GTP hydrolysis (Fig. 2 A).


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)

Dynamics of the association of Pex1p, Pex6p, and phosphoinositide- and GTP-bp with membranes of P1 and P2 during their priming. (A) L-[35S]methionine–labeled P1 and L-[35S]methionine–labeled P2 were preincubated individually for 5 min at 26°C with or without nystatin (an ergosterol ligand), phosphoinositide-specific antibodies, ATPγS, or nonhydrolyzable GTP analogues. Pretreated P1 and P2 were supplemented with ATP and incubated individually in the presence or absence of unlabeled cytosol, as indicated. After a 10-min incubation at 26°C, peroxisomal vesicles were pelleted. Pex1p and Pex6p were immunoprecipitated under denaturing conditions from the pellet (P) and supernatant (S) fractions. Immunoprecipitates were resolved by SDS-PAGE and visualized by fluorography. (B and C) Intact P1 and P2, either labeled with L-[35S]methionine (B) or unlabeled (C), were primed individually by incubation with unlabeled cytosol and ATP or remained unprimed. Equal aliquots of primed and unprimed peroxisomes (30 μg of protein per aliquot) were treated with 30 μg of trypsin or 1 M NaCl for 30 min on ice. Peroxisomes were then osmotically lysed and subjected to centrifugation. The pellet of membranes recovered after such centrifugation was solubilized with a detergent, n-OG. Detergent-soluble membrane proteins were analyzed by protein-lipid overlay assay using commercial PIP-Strips (B) or by GTP slot-blot with guanosine 5′-α-[32P]triphosphate (C). Lipid- and GTP-bp were visualized by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171827&req=5

fig2: Dynamics of the association of Pex1p, Pex6p, and phosphoinositide- and GTP-bp with membranes of P1 and P2 during their priming. (A) L-[35S]methionine–labeled P1 and L-[35S]methionine–labeled P2 were preincubated individually for 5 min at 26°C with or without nystatin (an ergosterol ligand), phosphoinositide-specific antibodies, ATPγS, or nonhydrolyzable GTP analogues. Pretreated P1 and P2 were supplemented with ATP and incubated individually in the presence or absence of unlabeled cytosol, as indicated. After a 10-min incubation at 26°C, peroxisomal vesicles were pelleted. Pex1p and Pex6p were immunoprecipitated under denaturing conditions from the pellet (P) and supernatant (S) fractions. Immunoprecipitates were resolved by SDS-PAGE and visualized by fluorography. (B and C) Intact P1 and P2, either labeled with L-[35S]methionine (B) or unlabeled (C), were primed individually by incubation with unlabeled cytosol and ATP or remained unprimed. Equal aliquots of primed and unprimed peroxisomes (30 μg of protein per aliquot) were treated with 30 μg of trypsin or 1 M NaCl for 30 min on ice. Peroxisomes were then osmotically lysed and subjected to centrifugation. The pellet of membranes recovered after such centrifugation was solubilized with a detergent, n-OG. Detergent-soluble membrane proteins were analyzed by protein-lipid overlay assay using commercial PIP-Strips (B) or by GTP slot-blot with guanosine 5′-α-[32P]triphosphate (C). Lipid- and GTP-bp were visualized by autoradiography.
Mentions: Fusion of P1 and P2 is a multistep process that includes priming, docking, and fusion events (Titorenko and Rachubinski, 2000). Priming (activation) of P1 and P2 before their physical contact commits both fusion partners to subsequent docking. Priming requires two AAA ATPases, Pex1p and Pex6p (Titorenko et al., 2000). Before priming, Pex1p is associated with the cytosolic surface of P1, whereas Pex1p and Pex6p are bound to the outer surface of P2. During priming, ATP hydrolysis triggers cytosol-dependent release of Pex1p from P1 and of Pex6p from P2, whereas P2-associated Pex1p remains bound to the organelle. We evaluated the requirements for the release of AAA ATPases during peroxisome priming. Using the extent of release of Pex1p from P1 and of Pex6p from P2 as a measure of priming efficiency, we found that, in addition to cytosolic proteins, ATP hydrolysis, and a particular type of AAA ATPase (Pex1p for P1 and Pex6p for P2; Titorenko and Rachubinski, 2000), priming of both fusion partners requires ergosterol, PI(4)P, and PI(4,5)P2 (Fig. 2 A). However, priming does not depend on GTP hydrolysis (Fig. 2 A).

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