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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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Subcellular localization, floatation gradient, Nycodenz gradient, membrane extraction and protease protection assays for Pex22p. (A) Postnuclear supernatant (PNS) was produced from wild-type (SMD1163) and Δpex22 (STK12) cells grown in oleate and subfractionated into a 27,000-g pellet (27 k p), a 100,000-g pellet (100 k p) and a 100,000-g supernatant (100 k s). Equivalent volumes were loaded on gels, transferred to nitrocellulose and blotted for the specified proteins. (B) The 27-k pellet of wild-type and Δpex22 strains grown in oleate were overlaid with sucrose and centrifuged. Fractions were taken from the top and checked for the localization of Pex3p and the β-subunit of the mitochondrial F1-ATPase (F1). (C) PNS from wild-type cells (SMD1163) grown on oleate was loaded on top of Nycodenz gradients. Equal volumes of fractions from the gradient were analyzed by immunoblotting. (D) The 27-k pellet of oleate-grown wild-type cells was subfractionated into an insoluble pellet fraction (p) and a soluble fraction (s) after treatment with 0.1 M carbonate (pH 11.5), 10 mM Tris (pH 8), 10 mM Tris (pH 8), 1 M NaCl, and 0.1% Triton X-100. The distributions of the specified proteins between supernatant and membranous pellet fractions were examined by immunoblotting. (E) A 27-k pellet of oleate-grown, wild-type cells was treated with the specified amount of trypsin in the presence (+) or absence (−) of 0.1% Triton X-100. The disappearance of the specified proteins was examined by immunoblotting.
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Figure 5: Subcellular localization, floatation gradient, Nycodenz gradient, membrane extraction and protease protection assays for Pex22p. (A) Postnuclear supernatant (PNS) was produced from wild-type (SMD1163) and Δpex22 (STK12) cells grown in oleate and subfractionated into a 27,000-g pellet (27 k p), a 100,000-g pellet (100 k p) and a 100,000-g supernatant (100 k s). Equivalent volumes were loaded on gels, transferred to nitrocellulose and blotted for the specified proteins. (B) The 27-k pellet of wild-type and Δpex22 strains grown in oleate were overlaid with sucrose and centrifuged. Fractions were taken from the top and checked for the localization of Pex3p and the β-subunit of the mitochondrial F1-ATPase (F1). (C) PNS from wild-type cells (SMD1163) grown on oleate was loaded on top of Nycodenz gradients. Equal volumes of fractions from the gradient were analyzed by immunoblotting. (D) The 27-k pellet of oleate-grown wild-type cells was subfractionated into an insoluble pellet fraction (p) and a soluble fraction (s) after treatment with 0.1 M carbonate (pH 11.5), 10 mM Tris (pH 8), 10 mM Tris (pH 8), 1 M NaCl, and 0.1% Triton X-100. The distributions of the specified proteins between supernatant and membranous pellet fractions were examined by immunoblotting. (E) A 27-k pellet of oleate-grown, wild-type cells was treated with the specified amount of trypsin in the presence (+) or absence (−) of 0.1% Triton X-100. The disappearance of the specified proteins was examined by immunoblotting.

Mentions: Differential centrifugation experiments confirmed the results obtained with the GFP fusions. Wild-type cells, SMD1163 (for control), and the Δpex22 strain (STK12) were grown in oleate to induce peroxisomes. Post-nuclear supernatants (PNS) from these strains were centrifuged at 27,000 g (27 k). The supernatant was spun further at 100,000 g (100 k). Equal portions of these fractions (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) were analyzed by immunoblotting. Both catalase and thiolase, which are PTS1- and PTS2-containing proteins, respectively, in yeasts and mammals, were localized in the 27-k pellet in the wild-type strain, whereas in the Δpex22 strain these proteins were cytosolic (100-k supernatant) (Fig. 5 A). Pex3p, however, was localized in the 27-k pellet in both strains. To check if the pelletable Pex3p is membrane bound, the 27-k pellet was resuspended in 65% sucrose and overlaid with layers of 50% and 30% sucrose, respectively. After centrifugation, fractions were collected from the top and analyzed. Immunoblots showed that in both strains, Pex3p floated to the middle or top of the gradient, as did a mitochondrial marker (F1β-ATPase), suggesting that Pex3p is membrane-bound in the Δpex22 strain (Fig. 5 B). Together, these data suggest that both PTS1- and PTS2-containing proteins are not properly targeted in a Δpex22 strain, whereas peroxisomal membrane proteins (Pex3p) are targeted to membrane structures, most likely the peroxisome remnants seen by immunofluorescence and electron microscopy.


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)

Subcellular localization, floatation gradient, Nycodenz gradient, membrane extraction and protease protection assays for Pex22p. (A) Postnuclear supernatant (PNS) was produced from wild-type (SMD1163) and Δpex22 (STK12) cells grown in oleate and subfractionated into a 27,000-g pellet (27 k p), a 100,000-g pellet (100 k p) and a 100,000-g supernatant (100 k s). Equivalent volumes were loaded on gels, transferred to nitrocellulose and blotted for the specified proteins. (B) The 27-k pellet of wild-type and Δpex22 strains grown in oleate were overlaid with sucrose and centrifuged. Fractions were taken from the top and checked for the localization of Pex3p and the β-subunit of the mitochondrial F1-ATPase (F1). (C) PNS from wild-type cells (SMD1163) grown on oleate was loaded on top of Nycodenz gradients. Equal volumes of fractions from the gradient were analyzed by immunoblotting. (D) The 27-k pellet of oleate-grown wild-type cells was subfractionated into an insoluble pellet fraction (p) and a soluble fraction (s) after treatment with 0.1 M carbonate (pH 11.5), 10 mM Tris (pH 8), 10 mM Tris (pH 8), 1 M NaCl, and 0.1% Triton X-100. The distributions of the specified proteins between supernatant and membranous pellet fractions were examined by immunoblotting. (E) A 27-k pellet of oleate-grown, wild-type cells was treated with the specified amount of trypsin in the presence (+) or absence (−) of 0.1% Triton X-100. The disappearance of the specified proteins was examined by immunoblotting.
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Figure 5: Subcellular localization, floatation gradient, Nycodenz gradient, membrane extraction and protease protection assays for Pex22p. (A) Postnuclear supernatant (PNS) was produced from wild-type (SMD1163) and Δpex22 (STK12) cells grown in oleate and subfractionated into a 27,000-g pellet (27 k p), a 100,000-g pellet (100 k p) and a 100,000-g supernatant (100 k s). Equivalent volumes were loaded on gels, transferred to nitrocellulose and blotted for the specified proteins. (B) The 27-k pellet of wild-type and Δpex22 strains grown in oleate were overlaid with sucrose and centrifuged. Fractions were taken from the top and checked for the localization of Pex3p and the β-subunit of the mitochondrial F1-ATPase (F1). (C) PNS from wild-type cells (SMD1163) grown on oleate was loaded on top of Nycodenz gradients. Equal volumes of fractions from the gradient were analyzed by immunoblotting. (D) The 27-k pellet of oleate-grown wild-type cells was subfractionated into an insoluble pellet fraction (p) and a soluble fraction (s) after treatment with 0.1 M carbonate (pH 11.5), 10 mM Tris (pH 8), 10 mM Tris (pH 8), 1 M NaCl, and 0.1% Triton X-100. The distributions of the specified proteins between supernatant and membranous pellet fractions were examined by immunoblotting. (E) A 27-k pellet of oleate-grown, wild-type cells was treated with the specified amount of trypsin in the presence (+) or absence (−) of 0.1% Triton X-100. The disappearance of the specified proteins was examined by immunoblotting.
Mentions: Differential centrifugation experiments confirmed the results obtained with the GFP fusions. Wild-type cells, SMD1163 (for control), and the Δpex22 strain (STK12) were grown in oleate to induce peroxisomes. Post-nuclear supernatants (PNS) from these strains were centrifuged at 27,000 g (27 k). The supernatant was spun further at 100,000 g (100 k). Equal portions of these fractions (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) were analyzed by immunoblotting. Both catalase and thiolase, which are PTS1- and PTS2-containing proteins, respectively, in yeasts and mammals, were localized in the 27-k pellet in the wild-type strain, whereas in the Δpex22 strain these proteins were cytosolic (100-k supernatant) (Fig. 5 A). Pex3p, however, was localized in the 27-k pellet in both strains. To check if the pelletable Pex3p is membrane bound, the 27-k pellet was resuspended in 65% sucrose and overlaid with layers of 50% and 30% sucrose, respectively. After centrifugation, fractions were collected from the top and analyzed. Immunoblots showed that in both strains, Pex3p floated to the middle or top of the gradient, as did a mitochondrial marker (F1β-ATPase), suggesting that Pex3p is membrane-bound in the Δpex22 strain (Fig. 5 B). Together, these data suggest that both PTS1- and PTS2-containing proteins are not properly targeted in a Δpex22 strain, whereas peroxisomal membrane proteins (Pex3p) are targeted to membrane structures, most likely the peroxisome remnants seen by immunofluorescence and electron microscopy.

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