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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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Intracellular distribution and cleavability of Pex7p-cs-GFP. (A) Cell fractionation of Δpex7 cells expressing Pex7p-cs-GFP (right) or MutPex7p-cs-GFP (middle) or no fusion protein (left). Immunoblots with anti-Pex7p, anti-thiolase, and anti-AOx antibodies. (B) In vitro cleavage of the fusion protein. Total cell lysates of Δpex7 cells, expressing either Pex7p-cs-GFP or Pex7p-GFP, were treated with or without purified TEVP (Invitrogen) for 30 min at 30°C. Pex7p cleavage product is seen only in the second lane that has Pex7p-cs-GFP treated with TEVP. (C) Intracellular distribution of TEVP and TEVP-SKL in Δpex7 cells. Immunoblot with anti-TEVP.
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fig2: Intracellular distribution and cleavability of Pex7p-cs-GFP. (A) Cell fractionation of Δpex7 cells expressing Pex7p-cs-GFP (right) or MutPex7p-cs-GFP (middle) or no fusion protein (left). Immunoblots with anti-Pex7p, anti-thiolase, and anti-AOx antibodies. (B) In vitro cleavage of the fusion protein. Total cell lysates of Δpex7 cells, expressing either Pex7p-cs-GFP or Pex7p-GFP, were treated with or without purified TEVP (Invitrogen) for 30 min at 30°C. Pex7p cleavage product is seen only in the second lane that has Pex7p-cs-GFP treated with TEVP. (C) Intracellular distribution of TEVP and TEVP-SKL in Δpex7 cells. Immunoblot with anti-TEVP.

Mentions: Pex7p-cs-GFP was partly cytosolic and partly peroxisomal when expressed in the Δpex7 strain, as expected (Fig. 2 A, right). The peroxisomal localization of Pex7p-cs-GFP was confirmed by colocalization of GFP fluorescence with thiolase immunofluorescence (not depicted). The fusion protein demonstrated limited function in catalyzing PTS2 protein import; a little thiolase was found in peroxisomes but most of it was cytosolic (Fig. 2 A, right).


Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae.

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

Intracellular distribution and cleavability of Pex7p-cs-GFP. (A) Cell fractionation of Δpex7 cells expressing Pex7p-cs-GFP (right) or MutPex7p-cs-GFP (middle) or no fusion protein (left). Immunoblots with anti-Pex7p, anti-thiolase, and anti-AOx antibodies. (B) In vitro cleavage of the fusion protein. Total cell lysates of Δpex7 cells, expressing either Pex7p-cs-GFP or Pex7p-GFP, were treated with or without purified TEVP (Invitrogen) for 30 min at 30°C. Pex7p cleavage product is seen only in the second lane that has Pex7p-cs-GFP treated with TEVP. (C) Intracellular distribution of TEVP and TEVP-SKL in Δpex7 cells. Immunoblot with anti-TEVP.
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Related In: Results  -  Collection

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fig2: Intracellular distribution and cleavability of Pex7p-cs-GFP. (A) Cell fractionation of Δpex7 cells expressing Pex7p-cs-GFP (right) or MutPex7p-cs-GFP (middle) or no fusion protein (left). Immunoblots with anti-Pex7p, anti-thiolase, and anti-AOx antibodies. (B) In vitro cleavage of the fusion protein. Total cell lysates of Δpex7 cells, expressing either Pex7p-cs-GFP or Pex7p-GFP, were treated with or without purified TEVP (Invitrogen) for 30 min at 30°C. Pex7p cleavage product is seen only in the second lane that has Pex7p-cs-GFP treated with TEVP. (C) Intracellular distribution of TEVP and TEVP-SKL in Δpex7 cells. Immunoblot with anti-TEVP.
Mentions: Pex7p-cs-GFP was partly cytosolic and partly peroxisomal when expressed in the Δpex7 strain, as expected (Fig. 2 A, right). The peroxisomal localization of Pex7p-cs-GFP was confirmed by colocalization of GFP fluorescence with thiolase immunofluorescence (not depicted). The fusion protein demonstrated limited function in catalyzing PTS2 protein import; a little thiolase was found in peroxisomes but most of it was cytosolic (Fig. 2 A, right).

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