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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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Pex7p generated by fusion protein cleavage rescues growth of Δpex7 cells on oleic acid as sole carbon source. Strains were precultured to mid-log phase in SCD and inoculated in 10 ml SCO liquid medium at a density of 105 cells/ml. Aliquots were withdrawn at the time points indicated and the OD read at 600 nm.
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fig4: Pex7p generated by fusion protein cleavage rescues growth of Δpex7 cells on oleic acid as sole carbon source. Strains were precultured to mid-log phase in SCD and inoculated in 10 ml SCO liquid medium at a density of 105 cells/ml. Aliquots were withdrawn at the time points indicated and the OD read at 600 nm.

Mentions: A functional peroxisomal β-oxidation system is required for S. cerevisiae to grow on oleic acid (for review see Lazarow and Kunau, 1997). The Δpex7 strain lacks this ability because thiolase is not targeted to peroxisomes in the absence of the PTS2 receptor, Pex7p. Consistent with the Western blot data for thiolase (Fig. 3 A), the Δpex7 strain expressing the fusion protein alone showed detectable but slow growth compared with wild-type yeast (Fig. 4). The rate of growth increased with the coexpression of TEVP in the cytosol, and was larger still when TEVP-SKL was expressed in peroxisomes. These growth data correlate with an increase in free Pex7p produced and with the proportion of thiolase in peroxisomes (Fig. 3, compare B with C). They demonstrate that the Pex7p cleaved from the fusion protein is functional. The greater cleavage in the case of TEVP-SKL expression may be attributed to the concentration of substrate and protease together in the small peroxisome compartment.


Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae.

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

Pex7p generated by fusion protein cleavage rescues growth of Δpex7 cells on oleic acid as sole carbon source. Strains were precultured to mid-log phase in SCD and inoculated in 10 ml SCO liquid medium at a density of 105 cells/ml. Aliquots were withdrawn at the time points indicated and the OD read at 600 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Pex7p generated by fusion protein cleavage rescues growth of Δpex7 cells on oleic acid as sole carbon source. Strains were precultured to mid-log phase in SCD and inoculated in 10 ml SCO liquid medium at a density of 105 cells/ml. Aliquots were withdrawn at the time points indicated and the OD read at 600 nm.
Mentions: A functional peroxisomal β-oxidation system is required for S. cerevisiae to grow on oleic acid (for review see Lazarow and Kunau, 1997). The Δpex7 strain lacks this ability because thiolase is not targeted to peroxisomes in the absence of the PTS2 receptor, Pex7p. Consistent with the Western blot data for thiolase (Fig. 3 A), the Δpex7 strain expressing the fusion protein alone showed detectable but slow growth compared with wild-type yeast (Fig. 4). The rate of growth increased with the coexpression of TEVP in the cytosol, and was larger still when TEVP-SKL was expressed in peroxisomes. These growth data correlate with an increase in free Pex7p produced and with the proportion of thiolase in peroxisomes (Fig. 3, compare B with C). They demonstrate that the Pex7p cleaved from the fusion protein is functional. The greater cleavage in the case of TEVP-SKL expression may be attributed to the concentration of substrate and protease together in the small peroxisome compartment.

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