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Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane.

Koller A, Snyder WB, Faber KN, Wenzel TJ, Rangell L, Keller GA, Subramani S - J. Cell Biol. (1999)

Bottom Line: Therefore, Pex22p anchors Pex4p at the peroxisomal membrane.Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis.The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California San Diego, La Jolla, California 92093-0322, USA.

ABSTRACT
We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

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Immunoelectron microscopy of wild-type and Δpex22 cells. Cells (Δpex22 [A] and wild-type (PPY12) [B–D]) were grown in methanol (A and D) or oleate (B and C) and labeled with Pex22p antibody. Arrows in C show the patches of Pex22p on peroxisomes. P, peroxisome. Bars, 0.5 μm.
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Figure 6: Immunoelectron microscopy of wild-type and Δpex22 cells. Cells (Δpex22 [A] and wild-type (PPY12) [B–D]) were grown in methanol (A and D) or oleate (B and C) and labeled with Pex22p antibody. Arrows in C show the patches of Pex22p on peroxisomes. P, peroxisome. Bars, 0.5 μm.

Mentions: Antibodies raised against Pex22p (see Materials and Methods) specifically detected a protein of ∼23 kD in cells grown on oleate and methanol (Fig. 5 A). Cells grown in glucose only showed a faint band corresponding to Pex22p (data not shown). No band was apparent in Δpex22 strains as expected (Fig. 5 A). The same fractions as above (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) taken from the wild-type strain were checked for the presence of Pex22p by immunoblotting. Pex22p was localized to the 27-k pellet, suggesting an organellar localization for this protein (Fig. 5 A). The PNS of the wild-type strain was fractionated on a linear Nycodenz gradient and analyzed by immunoblotting. Catalase and thiolase migrated, although with some trailing most likely due to rupture of some peroxisomes, near the bottom of the gradient, as did Pex3p (Fig. 5 C). Pex22p colocalized with the peroxisomal markers catalase, thiolase, and Pex3p. Further evidence that Pex22p is a peroxisomal protein was obtained by immunoelectron microscopy. Sections of methanol- and oleate-grown cells were decorated with Pex22p antibodies followed by incubation with gold-conjugated protein A. The gold particles almost exclusively decorated the peroxisomal membrane in the wild-type (Fig. 6B and Fig. D), but not the Δpex22 strain (Fig. 6 A). Sometimes, Pex22p was localized to patches on peroxisomes (Fig. 6 C).


Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane.

Koller A, Snyder WB, Faber KN, Wenzel TJ, Rangell L, Keller GA, Subramani S - J. Cell Biol. (1999)

Immunoelectron microscopy of wild-type and Δpex22 cells. Cells (Δpex22 [A] and wild-type (PPY12) [B–D]) were grown in methanol (A and D) or oleate (B and C) and labeled with Pex22p antibody. Arrows in C show the patches of Pex22p on peroxisomes. P, peroxisome. Bars, 0.5 μm.
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Figure 6: Immunoelectron microscopy of wild-type and Δpex22 cells. Cells (Δpex22 [A] and wild-type (PPY12) [B–D]) were grown in methanol (A and D) or oleate (B and C) and labeled with Pex22p antibody. Arrows in C show the patches of Pex22p on peroxisomes. P, peroxisome. Bars, 0.5 μm.
Mentions: Antibodies raised against Pex22p (see Materials and Methods) specifically detected a protein of ∼23 kD in cells grown on oleate and methanol (Fig. 5 A). Cells grown in glucose only showed a faint band corresponding to Pex22p (data not shown). No band was apparent in Δpex22 strains as expected (Fig. 5 A). The same fractions as above (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) taken from the wild-type strain were checked for the presence of Pex22p by immunoblotting. Pex22p was localized to the 27-k pellet, suggesting an organellar localization for this protein (Fig. 5 A). The PNS of the wild-type strain was fractionated on a linear Nycodenz gradient and analyzed by immunoblotting. Catalase and thiolase migrated, although with some trailing most likely due to rupture of some peroxisomes, near the bottom of the gradient, as did Pex3p (Fig. 5 C). Pex22p colocalized with the peroxisomal markers catalase, thiolase, and Pex3p. Further evidence that Pex22p is a peroxisomal protein was obtained by immunoelectron microscopy. Sections of methanol- and oleate-grown cells were decorated with Pex22p antibodies followed by incubation with gold-conjugated protein A. The gold particles almost exclusively decorated the peroxisomal membrane in the wild-type (Fig. 6B and Fig. D), but not the Δpex22 strain (Fig. 6 A). Sometimes, Pex22p was localized to patches on peroxisomes (Fig. 6 C).

Bottom Line: Therefore, Pex22p anchors Pex4p at the peroxisomal membrane.Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis.The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California San Diego, La Jolla, California 92093-0322, USA.

ABSTRACT
We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

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Related in: MedlinePlus