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Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae.

Nair DM, Purdue PE, Lazarow PB - J. Cell Biol. (2004)

Bottom Line: Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol.The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid.These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export.

View Article: PubMed Central - PubMed

Affiliation: Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7p's COOH terminus shifts Pex7p's intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export.

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Different subcellular distributions of native Pex7p and Pex7p-GFP. (A) Rabbit antibodies against Pex7p recognize the endogenous protein in a total cell lysate of wild-type W303 cells by immunoblotting. (B) Subcellular distribution of endogenous Pex7p and peroxisomal enzymes in wild-type and Δpex7 cells. A postnuclear supernatant (PNS) fraction was centrifuged at 15,000 rpm for 20 min to separate an organelle pellet (P) from the cytosol-containing supernatant (S). Immunoblots of the peroxisomal enzymes thiolase and acyl-CoA oxidase (AOx) that use the Pex7p and Pex5p receptors, respectively, are included as controls. (C) Intracellular distribution of Pex7p-GFP fusion protein as well as native Pex7p in wild-type and Δpex14 cells. (Top) Cell fractionation and anti-Pex7p immunoblots. (Bottom) Fluorescence of the Pex7p-GFP.
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fig1: Different subcellular distributions of native Pex7p and Pex7p-GFP. (A) Rabbit antibodies against Pex7p recognize the endogenous protein in a total cell lysate of wild-type W303 cells by immunoblotting. (B) Subcellular distribution of endogenous Pex7p and peroxisomal enzymes in wild-type and Δpex7 cells. A postnuclear supernatant (PNS) fraction was centrifuged at 15,000 rpm for 20 min to separate an organelle pellet (P) from the cytosol-containing supernatant (S). Immunoblots of the peroxisomal enzymes thiolase and acyl-CoA oxidase (AOx) that use the Pex7p and Pex5p receptors, respectively, are included as controls. (C) Intracellular distribution of Pex7p-GFP fusion protein as well as native Pex7p in wild-type and Δpex14 cells. (Top) Cell fractionation and anti-Pex7p immunoblots. (Bottom) Fluorescence of the Pex7p-GFP.

Mentions: Polyclonal antibodies raised against S. cerevisiae Pex7p specifically recognize the protein by immunoblotting in wild-type W303 cells. No band of corresponding molecular mass is detected in the Δpex7 strain (Fig. 1 A). These antibodies also recognize a protein of ∼60 kD; it probably is in mitochondria (cell fractionation not depicted) and is not depleted in Δpex7 cells.


Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae.

Nair DM, Purdue PE, Lazarow PB - J. Cell Biol. (2004)

Different subcellular distributions of native Pex7p and Pex7p-GFP. (A) Rabbit antibodies against Pex7p recognize the endogenous protein in a total cell lysate of wild-type W303 cells by immunoblotting. (B) Subcellular distribution of endogenous Pex7p and peroxisomal enzymes in wild-type and Δpex7 cells. A postnuclear supernatant (PNS) fraction was centrifuged at 15,000 rpm for 20 min to separate an organelle pellet (P) from the cytosol-containing supernatant (S). Immunoblots of the peroxisomal enzymes thiolase and acyl-CoA oxidase (AOx) that use the Pex7p and Pex5p receptors, respectively, are included as controls. (C) Intracellular distribution of Pex7p-GFP fusion protein as well as native Pex7p in wild-type and Δpex14 cells. (Top) Cell fractionation and anti-Pex7p immunoblots. (Bottom) Fluorescence of the Pex7p-GFP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172567&req=5

fig1: Different subcellular distributions of native Pex7p and Pex7p-GFP. (A) Rabbit antibodies against Pex7p recognize the endogenous protein in a total cell lysate of wild-type W303 cells by immunoblotting. (B) Subcellular distribution of endogenous Pex7p and peroxisomal enzymes in wild-type and Δpex7 cells. A postnuclear supernatant (PNS) fraction was centrifuged at 15,000 rpm for 20 min to separate an organelle pellet (P) from the cytosol-containing supernatant (S). Immunoblots of the peroxisomal enzymes thiolase and acyl-CoA oxidase (AOx) that use the Pex7p and Pex5p receptors, respectively, are included as controls. (C) Intracellular distribution of Pex7p-GFP fusion protein as well as native Pex7p in wild-type and Δpex14 cells. (Top) Cell fractionation and anti-Pex7p immunoblots. (Bottom) Fluorescence of the Pex7p-GFP.
Mentions: Polyclonal antibodies raised against S. cerevisiae Pex7p specifically recognize the protein by immunoblotting in wild-type W303 cells. No band of corresponding molecular mass is detected in the Δpex7 strain (Fig. 1 A). These antibodies also recognize a protein of ∼60 kD; it probably is in mitochondria (cell fractionation not depicted) and is not depleted in Δpex7 cells.

Bottom Line: Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol.The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid.These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export.

View Article: PubMed Central - PubMed

Affiliation: Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7p's COOH terminus shifts Pex7p's intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export.

Show MeSH