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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) Two-hybrid interactions of Pex22p with Pex4p. The bars denote the parts of the genes, which were cloned into the two-hybrid vectors pBTM116 (BD), and pVP16 (AD). Plasmids were: Pex22, pTK12 or 13; Pex22.1, pTK14; Pex22.2, pTK16; Pex22.3, pTK18; Pex4, pKNSD119 or 118; Pex4.1, pTK21; Pex4.2, pTK23; Pex4.3, pTK25; and Pex4.4, pTK27. The specified plasmids were transformed into the yeast two-hybrid strain L40, spotted on nitrocellulose and checked for β-galactosidase activity. TM is the transmembrane domain of Pex22p, INS2 is the Insertion Element 2 of Pex4p and * indicates the active-site cysteine (C133) of Pex4p. (B) Pex22p binds to Pex4p in vivo. A lysate from a strain expressing a 6HIS-Pex4p (STK14 + pTK36) or a wild-type (wt) strain (SMD1163) was incubated with (+) or without (−) the cross-linker DSP. 6HIS-Pex4p and cross-linked proteins were precipitated with Ni2+-NTA agarose beads. Bound proteins were subjected to SDS-PAGE and blotted for Pex22p, Pex4p, and Pex3p. Lane c shows the proteins in a TCA lysate of the 6HIS-Pex4p–expressing strain.
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Figure 8: (A) Two-hybrid interactions of Pex22p with Pex4p. The bars denote the parts of the genes, which were cloned into the two-hybrid vectors pBTM116 (BD), and pVP16 (AD). Plasmids were: Pex22, pTK12 or 13; Pex22.1, pTK14; Pex22.2, pTK16; Pex22.3, pTK18; Pex4, pKNSD119 or 118; Pex4.1, pTK21; Pex4.2, pTK23; Pex4.3, pTK25; and Pex4.4, pTK27. The specified plasmids were transformed into the yeast two-hybrid strain L40, spotted on nitrocellulose and checked for β-galactosidase activity. TM is the transmembrane domain of Pex22p, INS2 is the Insertion Element 2 of Pex4p and * indicates the active-site cysteine (C133) of Pex4p. (B) Pex22p binds to Pex4p in vivo. A lysate from a strain expressing a 6HIS-Pex4p (STK14 + pTK36) or a wild-type (wt) strain (SMD1163) was incubated with (+) or without (−) the cross-linker DSP. 6HIS-Pex4p and cross-linked proteins were precipitated with Ni2+-NTA agarose beads. Bound proteins were subjected to SDS-PAGE and blotted for Pex22p, Pex4p, and Pex3p. Lane c shows the proteins in a TCA lysate of the 6HIS-Pex4p–expressing strain.

Mentions: To determine interactions of Pex22p with other Pex proteins, the yeast two-hybrid system was employed. PEX22 was fused to the DB domain of LexA, or the AD of VP16. All published P. pastoris PEX genes (PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX8, PEX10, PEX12, and PEX13) were also fused to these domains. These plasmids were then transformed in combination into the S. cerevisiae strain L40 and interaction of these proteins was assessed by the production of β-galactosidase activity. Only the combination of Pex22p with Pex4p, a ubiquitin-conjugating enzyme, produced any detectable enzyme activity. Almost the whole Pex22p protein (construct Pex22.1) was needed for interaction with Pex4p, whereas the COOH-terminal 39% of Pex4p (construct Pex4.2) interacted with Pex22p (Fig. 8 A). Control experiments performed by exchanging the backbone vectors confirmed our findings (data not shown). We were also able to show that these two fragments of Pex22p (Pex22.1) and Pex4p (Pex4.2) interacted with each other (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)

(A) Two-hybrid interactions of Pex22p with Pex4p. The bars denote the parts of the genes, which were cloned into the two-hybrid vectors pBTM116 (BD), and pVP16 (AD). Plasmids were: Pex22, pTK12 or 13; Pex22.1, pTK14; Pex22.2, pTK16; Pex22.3, pTK18; Pex4, pKNSD119 or 118; Pex4.1, pTK21; Pex4.2, pTK23; Pex4.3, pTK25; and Pex4.4, pTK27. The specified plasmids were transformed into the yeast two-hybrid strain L40, spotted on nitrocellulose and checked for β-galactosidase activity. TM is the transmembrane domain of Pex22p, INS2 is the Insertion Element 2 of Pex4p and * indicates the active-site cysteine (C133) of Pex4p. (B) Pex22p binds to Pex4p in vivo. A lysate from a strain expressing a 6HIS-Pex4p (STK14 + pTK36) or a wild-type (wt) strain (SMD1163) was incubated with (+) or without (−) the cross-linker DSP. 6HIS-Pex4p and cross-linked proteins were precipitated with Ni2+-NTA agarose beads. Bound proteins were subjected to SDS-PAGE and blotted for Pex22p, Pex4p, and Pex3p. Lane c shows the proteins in a TCA lysate of the 6HIS-Pex4p–expressing strain.
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Figure 8: (A) Two-hybrid interactions of Pex22p with Pex4p. The bars denote the parts of the genes, which were cloned into the two-hybrid vectors pBTM116 (BD), and pVP16 (AD). Plasmids were: Pex22, pTK12 or 13; Pex22.1, pTK14; Pex22.2, pTK16; Pex22.3, pTK18; Pex4, pKNSD119 or 118; Pex4.1, pTK21; Pex4.2, pTK23; Pex4.3, pTK25; and Pex4.4, pTK27. The specified plasmids were transformed into the yeast two-hybrid strain L40, spotted on nitrocellulose and checked for β-galactosidase activity. TM is the transmembrane domain of Pex22p, INS2 is the Insertion Element 2 of Pex4p and * indicates the active-site cysteine (C133) of Pex4p. (B) Pex22p binds to Pex4p in vivo. A lysate from a strain expressing a 6HIS-Pex4p (STK14 + pTK36) or a wild-type (wt) strain (SMD1163) was incubated with (+) or without (−) the cross-linker DSP. 6HIS-Pex4p and cross-linked proteins were precipitated with Ni2+-NTA agarose beads. Bound proteins were subjected to SDS-PAGE and blotted for Pex22p, Pex4p, and Pex3p. Lane c shows the proteins in a TCA lysate of the 6HIS-Pex4p–expressing strain.
Mentions: To determine interactions of Pex22p with other Pex proteins, the yeast two-hybrid system was employed. PEX22 was fused to the DB domain of LexA, or the AD of VP16. All published P. pastoris PEX genes (PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX8, PEX10, PEX12, and PEX13) were also fused to these domains. These plasmids were then transformed in combination into the S. cerevisiae strain L40 and interaction of these proteins was assessed by the production of β-galactosidase activity. Only the combination of Pex22p with Pex4p, a ubiquitin-conjugating enzyme, produced any detectable enzyme activity. Almost the whole Pex22p protein (construct Pex22.1) was needed for interaction with Pex4p, whereas the COOH-terminal 39% of Pex4p (construct Pex4.2) interacted with Pex22p (Fig. 8 A). Control experiments performed by exchanging the backbone vectors confirmed our findings (data not shown). We were also able to show that these two fragments of Pex22p (Pex22.1) and Pex4p (Pex4.2) interacted with each other (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