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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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ScYaf5p is the homologue of Pex22p. (A) The two proteins were aligned using the SSEARCH Pairwise Sequence Alignment program (Smith and Waterman 1981). The colons represent identical amino acids, the dots are similar amino acids and the bars represent gaps introduced to maximize similarity. (B) BJ1991 (wild-type) and ΔScyaf5 (STK16) were transformed with a GFP-SKL construct. Cells were grown on oleate and checked for the localization of GFP under the fluorescence microscope. (C) Full-length ScYAF5 and ScPEX4 were cloned into the two-hybrid vector pBTM116 (BD) or pVP16 (AD) and transformed into the two-hybrid strain L40. The transformants were grown on a nitrocellulose filter and β-galactosidase activity was tested.
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Figure 10: ScYaf5p is the homologue of Pex22p. (A) The two proteins were aligned using the SSEARCH Pairwise Sequence Alignment program (Smith and Waterman 1981). The colons represent identical amino acids, the dots are similar amino acids and the bars represent gaps introduced to maximize similarity. (B) BJ1991 (wild-type) and ΔScyaf5 (STK16) were transformed with a GFP-SKL construct. Cells were grown on oleate and checked for the localization of GFP under the fluorescence microscope. (C) Full-length ScYAF5 and ScPEX4 were cloned into the two-hybrid vector pBTM116 (BD) or pVP16 (AD) and transformed into the two-hybrid strain L40. The transformants were grown on a nitrocellulose filter and β-galactosidase activity was tested.

Mentions: PpPex22p was run against protein databases (SwissProt, SGD) with Blast and Fasta searches. No high-scoring homologue could be found. Only several low-scoring proteins could be found in the Saccharomyces Genome Database (SGD) database. Out of these, only ScYaf5p (open reading frame YAL055w) is of about similar size and exhibits a transmembrane region at the NH2 terminus similar to Pex22p, although it starts at amino acid 14–32 (Fig. 10 A). To determine if ScYaf5p is the real Pex22p homologue, the entire open reading frame of ScYAF5 was replaced by a PCR-generated kanMX2 cassette (Wach et al. 1994). Strains deleted for ScYAF5 were streaked on oleate and glucose plates. ΔScyaf5 strains grew on glucose like wild-type cells, whereas they did not grow on oleate. A ΔScyaf5 strain transformed with a plasmid expressing ScYAF5 from a catalase promoter complemented the growth defect on oleate (data not shown).


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)

ScYaf5p is the homologue of Pex22p. (A) The two proteins were aligned using the SSEARCH Pairwise Sequence Alignment program (Smith and Waterman 1981). The colons represent identical amino acids, the dots are similar amino acids and the bars represent gaps introduced to maximize similarity. (B) BJ1991 (wild-type) and ΔScyaf5 (STK16) were transformed with a GFP-SKL construct. Cells were grown on oleate and checked for the localization of GFP under the fluorescence microscope. (C) Full-length ScYAF5 and ScPEX4 were cloned into the two-hybrid vector pBTM116 (BD) or pVP16 (AD) and transformed into the two-hybrid strain L40. The transformants were grown on a nitrocellulose filter and β-galactosidase activity was tested.
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Related In: Results  -  Collection

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

Figure 10: ScYaf5p is the homologue of Pex22p. (A) The two proteins were aligned using the SSEARCH Pairwise Sequence Alignment program (Smith and Waterman 1981). The colons represent identical amino acids, the dots are similar amino acids and the bars represent gaps introduced to maximize similarity. (B) BJ1991 (wild-type) and ΔScyaf5 (STK16) were transformed with a GFP-SKL construct. Cells were grown on oleate and checked for the localization of GFP under the fluorescence microscope. (C) Full-length ScYAF5 and ScPEX4 were cloned into the two-hybrid vector pBTM116 (BD) or pVP16 (AD) and transformed into the two-hybrid strain L40. The transformants were grown on a nitrocellulose filter and β-galactosidase activity was tested.
Mentions: PpPex22p was run against protein databases (SwissProt, SGD) with Blast and Fasta searches. No high-scoring homologue could be found. Only several low-scoring proteins could be found in the Saccharomyces Genome Database (SGD) database. Out of these, only ScYaf5p (open reading frame YAL055w) is of about similar size and exhibits a transmembrane region at the NH2 terminus similar to Pex22p, although it starts at amino acid 14–32 (Fig. 10 A). To determine if ScYaf5p is the real Pex22p homologue, the entire open reading frame of ScYAF5 was replaced by a PCR-generated kanMX2 cassette (Wach et al. 1994). Strains deleted for ScYAF5 were streaked on oleate and glucose plates. ΔScyaf5 strains grew on glucose like wild-type cells, whereas they did not grow on oleate. A ΔScyaf5 strain transformed with a plasmid expressing ScYAF5 from a catalase promoter complemented the growth defect on oleate (data not shown).

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