Limits...
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.

Show MeSH

Related in: MedlinePlus

Electron microscopy of wild-type and Δpex22 cells. Morphological analysis of wild-type cells (PPY12) (A and B) and Δpex22 (STK11) (C and D). Cells were grown in methanol (A and C) or oleate medium (B and D), and prepared for electron microscopy. Arrows point to the peroxisome remnants. N, nucleus; P, peroxisome. Bars, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199742&req=5

Figure 4: Electron microscopy of wild-type and Δpex22 cells. Morphological analysis of wild-type cells (PPY12) (A and B) and Δpex22 (STK11) (C and D). Cells were grown in methanol (A and C) or oleate medium (B and D), and prepared for electron microscopy. Arrows point to the peroxisome remnants. N, nucleus; P, peroxisome. Bars, 0.5 μm.

Mentions: The Δpex22 (STK11) strain was transformed with GFP constructs to determine the ability of this strain to import peroxisomal matrix proteins. The GFP constructs used were shown to be properly localized to peroxisomes in wild-type cells (Wiemer et al. 1996; Fig. 3). A PTS1-GFP (pTW51) introduced into the Δpex22 strain was not targeted into peroxisomes when grown in methanol medium but was localized in the cytosol (Fig. 3). A PTS2-GFP (expressing the first 17 amino acids of S. cerevisiae thiolase fused to GFP; pTW61) was also not targeted to peroxisomes when grown in oleate but was localized in the cytosol (Fig. 3). However, immunofluorescence with Pex3p antibody showed that this peroxisomal membrane protein localized to punctate structures in the cytosol in the mutant strain, suggesting that the Δpex22 strain retains the ability to target peroxisomal membrane proteins to some peroxisome-like structures, so called remnants (Fig. 3). Electron microscopy revealed that in wild-type cells, the peroxisomes were clearly present in both methanol (Fig. 4 A) and oleate (Fig. 4 B) grown cells. In Δpex22 cells, no normal peroxisomes could be observed (Fig. 4C and Fig. D). However, in both growth media, small single-membrane organelles could be observed, suggesting that Δpex22 cells contain peroxisome remnants.


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)

Electron microscopy of wild-type and Δpex22 cells. Morphological analysis of wild-type cells (PPY12) (A and B) and Δpex22 (STK11) (C and D). Cells were grown in methanol (A and C) or oleate medium (B and D), and prepared for electron microscopy. Arrows point to the peroxisome remnants. N, nucleus; P, peroxisome. Bars, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199742&req=5

Figure 4: Electron microscopy of wild-type and Δpex22 cells. Morphological analysis of wild-type cells (PPY12) (A and B) and Δpex22 (STK11) (C and D). Cells were grown in methanol (A and C) or oleate medium (B and D), and prepared for electron microscopy. Arrows point to the peroxisome remnants. N, nucleus; P, peroxisome. Bars, 0.5 μm.
Mentions: The Δpex22 (STK11) strain was transformed with GFP constructs to determine the ability of this strain to import peroxisomal matrix proteins. The GFP constructs used were shown to be properly localized to peroxisomes in wild-type cells (Wiemer et al. 1996; Fig. 3). A PTS1-GFP (pTW51) introduced into the Δpex22 strain was not targeted into peroxisomes when grown in methanol medium but was localized in the cytosol (Fig. 3). A PTS2-GFP (expressing the first 17 amino acids of S. cerevisiae thiolase fused to GFP; pTW61) was also not targeted to peroxisomes when grown in oleate but was localized in the cytosol (Fig. 3). However, immunofluorescence with Pex3p antibody showed that this peroxisomal membrane protein localized to punctate structures in the cytosol in the mutant strain, suggesting that the Δpex22 strain retains the ability to target peroxisomal membrane proteins to some peroxisome-like structures, so called remnants (Fig. 3). Electron microscopy revealed that in wild-type cells, the peroxisomes were clearly present in both methanol (Fig. 4 A) and oleate (Fig. 4 B) grown cells. In Δpex22 cells, no normal peroxisomes could be observed (Fig. 4C and Fig. D). However, in both growth media, small single-membrane organelles could be observed, suggesting that Δpex22 cells contain peroxisome remnants.

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.

Show MeSH
Related in: MedlinePlus