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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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Map of PEX22, PEX22 disruption construct and complementation assay of PEX22 constructs. (A) Plasmid p82.09 and derivatives (p82-20–p82-25 and pTK10) containing the DNA indicated by a line were tested for ability to complement the pex22.1 mutant (STK20). Plus (+) and minus (−) signs indicate ability and inability, respectively, to complement the mutant. For the Δpex22 disruption, the whole open reading frame of PEX22 was replaced by the Zeocin-resistance gene (see Materials and Methods). (B) Δpex22::Zeocin (STK11) was transformed with an empty plasmid (pPIC3K) or plasmid pTK10 and streaked on minimal methanol medium (SMethanol). Wt is PPY12.
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Figure 1: Map of PEX22, PEX22 disruption construct and complementation assay of PEX22 constructs. (A) Plasmid p82.09 and derivatives (p82-20–p82-25 and pTK10) containing the DNA indicated by a line were tested for ability to complement the pex22.1 mutant (STK20). Plus (+) and minus (−) signs indicate ability and inability, respectively, to complement the mutant. For the Δpex22 disruption, the whole open reading frame of PEX22 was replaced by the Zeocin-resistance gene (see Materials and Methods). (B) Δpex22::Zeocin (STK11) was transformed with an empty plasmid (pPIC3K) or plasmid pTK10 and streaked on minimal methanol medium (SMethanol). Wt is PPY12.

Mentions: The pex22.1 mutant (STK10) was transformed with a wild-type genomic library and plasmids (p82.2, p82.3, p82.9, p82.13, and p82.15) from colonies that grew on methanol medium were isolated and rechecked for their ability to restore growth on methanol and oleate. The five inserts contained an overlapping fragment of 1.1 kb which was isolated from p82.13 as a BamHI fragment and subcloned into the pSG560 vector (Gould et al. 1992) to check for complementation (p82.20; Fig. 1 A). The smallest, complementing fragment was the 0.9-kb EcoRV-BamHI fragment (p82.25). The whole 1.1-kb fragment was sequenced to obtain the PEX22 gene which is 564 bp long, encoding a protein of 187 amino acid (calculated molecular mass of 20,984 D and pI of 5.76; Fig. 2). The protein contains a putative membrane-spanning region between amino acids 7 or 8 and 24 or 25. Otherwise the protein does not contain any known motifs. The whole PEX22 gene was replaced in wild-type cells with the Zeocin-resistance gene (see Materials and Methods). The resulting Δpex22 strain grew normally on glucose, but not on methanol and oleate, for which growth was complemented upon reintroduction of PEX22 (pTK10; Fig. 1 B).


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)

Map of PEX22, PEX22 disruption construct and complementation assay of PEX22 constructs. (A) Plasmid p82.09 and derivatives (p82-20–p82-25 and pTK10) containing the DNA indicated by a line were tested for ability to complement the pex22.1 mutant (STK20). Plus (+) and minus (−) signs indicate ability and inability, respectively, to complement the mutant. For the Δpex22 disruption, the whole open reading frame of PEX22 was replaced by the Zeocin-resistance gene (see Materials and Methods). (B) Δpex22::Zeocin (STK11) was transformed with an empty plasmid (pPIC3K) or plasmid pTK10 and streaked on minimal methanol medium (SMethanol). Wt is PPY12.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199742&req=5

Figure 1: Map of PEX22, PEX22 disruption construct and complementation assay of PEX22 constructs. (A) Plasmid p82.09 and derivatives (p82-20–p82-25 and pTK10) containing the DNA indicated by a line were tested for ability to complement the pex22.1 mutant (STK20). Plus (+) and minus (−) signs indicate ability and inability, respectively, to complement the mutant. For the Δpex22 disruption, the whole open reading frame of PEX22 was replaced by the Zeocin-resistance gene (see Materials and Methods). (B) Δpex22::Zeocin (STK11) was transformed with an empty plasmid (pPIC3K) or plasmid pTK10 and streaked on minimal methanol medium (SMethanol). Wt is PPY12.
Mentions: The pex22.1 mutant (STK10) was transformed with a wild-type genomic library and plasmids (p82.2, p82.3, p82.9, p82.13, and p82.15) from colonies that grew on methanol medium were isolated and rechecked for their ability to restore growth on methanol and oleate. The five inserts contained an overlapping fragment of 1.1 kb which was isolated from p82.13 as a BamHI fragment and subcloned into the pSG560 vector (Gould et al. 1992) to check for complementation (p82.20; Fig. 1 A). The smallest, complementing fragment was the 0.9-kb EcoRV-BamHI fragment (p82.25). The whole 1.1-kb fragment was sequenced to obtain the PEX22 gene which is 564 bp long, encoding a protein of 187 amino acid (calculated molecular mass of 20,984 D and pI of 5.76; Fig. 2). The protein contains a putative membrane-spanning region between amino acids 7 or 8 and 24 or 25. Otherwise the protein does not contain any known motifs. The whole PEX22 gene was replaced in wild-type cells with the Zeocin-resistance gene (see Materials and Methods). The resulting Δpex22 strain grew normally on glucose, but not on methanol and oleate, for which growth was complemented upon reintroduction of PEX22 (pTK10; Fig. 1 B).

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