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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.

Show MeSH
Pex7p export from peroxisomes. Δpex7 strains coexpressing Pex7p-cs-GFP without protease (A), with TEVP (B), or with TEVP-SKL (C) were analyzed by cell fractionation followed by SDS-PAGE and immunoblotting with anti-Pex7p (a), anti-GFP (b), anti-thiolase (c), and anti-AOx (d) antibodies. (D) MutPex7p-cs-GFP with TEVP-SKL.
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fig3: Pex7p export from peroxisomes. Δpex7 strains coexpressing Pex7p-cs-GFP without protease (A), with TEVP (B), or with TEVP-SKL (C) were analyzed by cell fractionation followed by SDS-PAGE and immunoblotting with anti-Pex7p (a), anti-GFP (b), anti-thiolase (c), and anti-AOx (d) antibodies. (D) MutPex7p-cs-GFP with TEVP-SKL.

Mentions: When Pex7p-cs-GFP is expressed alone in Δpex7 cells (without any viral protease), neither free Pex7p nor free GFP is detected (Fig. 3 A). The fusion protein shows a dual localization, part in peroxisomes and part in the cytosol, as observed previously. Only a little thiolase is found in the organelle pellet, which is consistent with the limited functionality of the fusion protein to catalyze PTS2 import into peroxisomes (Fig. 3 A).


Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae.

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

Pex7p export from peroxisomes. Δpex7 strains coexpressing Pex7p-cs-GFP without protease (A), with TEVP (B), or with TEVP-SKL (C) were analyzed by cell fractionation followed by SDS-PAGE and immunoblotting with anti-Pex7p (a), anti-GFP (b), anti-thiolase (c), and anti-AOx (d) antibodies. (D) MutPex7p-cs-GFP with TEVP-SKL.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172567&req=5

fig3: Pex7p export from peroxisomes. Δpex7 strains coexpressing Pex7p-cs-GFP without protease (A), with TEVP (B), or with TEVP-SKL (C) were analyzed by cell fractionation followed by SDS-PAGE and immunoblotting with anti-Pex7p (a), anti-GFP (b), anti-thiolase (c), and anti-AOx (d) antibodies. (D) MutPex7p-cs-GFP with TEVP-SKL.
Mentions: When Pex7p-cs-GFP is expressed alone in Δpex7 cells (without any viral protease), neither free Pex7p nor free GFP is detected (Fig. 3 A). The fusion protein shows a dual localization, part in peroxisomes and part in the cytosol, as observed previously. Only a little thiolase is found in the organelle pellet, which is consistent with the limited functionality of the fusion protein to catalyze PTS2 import into peroxisomes (Fig. 3 A).

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