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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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(A) Δpex4 and Δpex22 strains share similar phenotypes. Wt (SMD1163), Δpex4 (STK14), and Δpex22 (STK12) strains were grown in methanol or oleate for 14 h and a TCA lysate was generated. Equal volumes were subjected to SDS-PAGE and blotted for the specified proteins. (B) Pex22p is the anchor protein for Pex4p. Differential centrifugation was performed with strains expressing NH-Pex4p (Δpex4+pTK51, PPY12+pTK50, Δpex22+pTK50) and equivalent fractions were checked for the presence of the specified proteins.
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Figure 9: (A) Δpex4 and Δpex22 strains share similar phenotypes. Wt (SMD1163), Δpex4 (STK14), and Δpex22 (STK12) strains were grown in methanol or oleate for 14 h and a TCA lysate was generated. Equal volumes were subjected to SDS-PAGE and blotted for the specified proteins. (B) Pex22p is the anchor protein for Pex4p. Differential centrifugation was performed with strains expressing NH-Pex4p (Δpex4+pTK51, PPY12+pTK50, Δpex22+pTK50) and equivalent fractions were checked for the presence of the specified proteins.

Mentions: PpPex4p was previously characterized as a ubiquitin-conjugating enzyme, similar to ScPex4p (Crane et al. 1994). A Δpex4 strain (STK14) behaved similarly in differential centrifugation, as did a Δpex22 strain (data not shown). TCA lysates were made from strains (STK12 and STK14) grown in methanol and oleate. Equal amounts of cells were loaded on a gel and blotted for the presence of Pex3p, Pex4p, Pex5p, Pex7p, and Pex22p. As shown in Fig. 9 A, all the strains showed similar amounts of Pex3p, whereas strains deleted for Δpex4 and Δpex22 did not contain any detectable Pex5p. However, Pex7p was present in wild-type amounts in all the strains and was induced by oleate relative to methanol growth. Interestingly, we were unable to detect any Pex4p in a Δpex22 strain.


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)

(A) Δpex4 and Δpex22 strains share similar phenotypes. Wt (SMD1163), Δpex4 (STK14), and Δpex22 (STK12) strains were grown in methanol or oleate for 14 h and a TCA lysate was generated. Equal volumes were subjected to SDS-PAGE and blotted for the specified proteins. (B) Pex22p is the anchor protein for Pex4p. Differential centrifugation was performed with strains expressing NH-Pex4p (Δpex4+pTK51, PPY12+pTK50, Δpex22+pTK50) and equivalent fractions were checked for the presence of the specified proteins.
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Related In: Results  -  Collection

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Figure 9: (A) Δpex4 and Δpex22 strains share similar phenotypes. Wt (SMD1163), Δpex4 (STK14), and Δpex22 (STK12) strains were grown in methanol or oleate for 14 h and a TCA lysate was generated. Equal volumes were subjected to SDS-PAGE and blotted for the specified proteins. (B) Pex22p is the anchor protein for Pex4p. Differential centrifugation was performed with strains expressing NH-Pex4p (Δpex4+pTK51, PPY12+pTK50, Δpex22+pTK50) and equivalent fractions were checked for the presence of the specified proteins.
Mentions: PpPex4p was previously characterized as a ubiquitin-conjugating enzyme, similar to ScPex4p (Crane et al. 1994). A Δpex4 strain (STK14) behaved similarly in differential centrifugation, as did a Δpex22 strain (data not shown). TCA lysates were made from strains (STK12 and STK14) grown in methanol and oleate. Equal amounts of cells were loaded on a gel and blotted for the presence of Pex3p, Pex4p, Pex5p, Pex7p, and Pex22p. As shown in Fig. 9 A, all the strains showed similar amounts of Pex3p, whereas strains deleted for Δpex4 and Δpex22 did not contain any detectable Pex5p. However, Pex7p was present in wild-type amounts in all the strains and was induced by oleate relative to methanol growth. Interestingly, we were unable to detect any Pex4p in a Δpex22 strain.

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