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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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Localization of different GFP constructs in methanol-grown, wild-type cells. (A) PPY12 was transformed with constructs expressing GFP-SKL (pTW74), Pex22-GFP (pTK30) and Pex22(1–25)-GFP (pTK32) and grown on methanol for 16 h. Cells were prepared for immunofluorescence and labeled with α-AOX. The localization of AOX and the GFP construct in the same cells is shown. (B) Wild-type cells expressing either GFP-SKL (pTW74) or Pex22(1–25)-GFP (pTK32) were grown on methanol, spheroplasted, and incubated for 10 min at 30°C with increasing amounts of digitonin or 0.2% Triton X-100 in isotonic buffer. The spheroplasts were pelleted and the supernatants were used for immunoblotting with GFP, Pex3p, and G6PDH antibodies.
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Figure 7: Localization of different GFP constructs in methanol-grown, wild-type cells. (A) PPY12 was transformed with constructs expressing GFP-SKL (pTW74), Pex22-GFP (pTK30) and Pex22(1–25)-GFP (pTK32) and grown on methanol for 16 h. Cells were prepared for immunofluorescence and labeled with α-AOX. The localization of AOX and the GFP construct in the same cells is shown. (B) Wild-type cells expressing either GFP-SKL (pTW74) or Pex22(1–25)-GFP (pTK32) were grown on methanol, spheroplasted, and incubated for 10 min at 30°C with increasing amounts of digitonin or 0.2% Triton X-100 in isotonic buffer. The spheroplasts were pelleted and the supernatants were used for immunoblotting with GFP, Pex3p, and G6PDH antibodies.

Mentions: Sequence analysis of Pex22p did not reveal an obvious mPTS. There is, however, a stretch of positively charged amino acids near the extreme NH2 terminus of Pex22p which does not completely fit the consensus sequence for an mPTS (Elgersma et al. 1997). This stretch is at the same location as the putative mPTS of Pex3p (Hp, Sc, Pp). Therefore, we constructed GFP fusions with full-length Pex22 (Pex22-GFP, pTK30, this construct is able to complement a Δpex22 mutant for growth on oleate and methanol), a second fusion with the first 25 amino acids of Pex22 (Pex22(1–25)-GFP; pTK32), containing the transmembrane domain, a third fusion with only the transmembrane domain (Pex22(8–25)-GFP; pTK44), and a fourth fusion with the first seven amino acids of Pex22 (Pex22(1–7)-GFP; pTK34), not containing the transmembrane domain. These constructs were transformed into PPY12 and the resulting strains induced on methanol. The constructs expressing full-length Pex22-GFP and Pex22(1–25)-GFP showed colocalization with alcohol oxidase, a bona fide peroxisomal matrix protein (Fig. 7 A), proving that these constructs get targeted to peroxisomes, whereas the other two constructs (Pex22(1–7)-GFP, Pex22(8–25)-GFP) were localized in the cytosol (data not shown). The Pex22(1–25)-GFP fusion protein could also be shown to colocalize with peroxisomes when an organelle fraction was separated on Nycodenz gradients (data not shown). Furthermore, this fusion was organelle associated since the fusion protein (Pex22(1–25)-GFP) only leaked from cells at digitonin concentrations that released membrane proteins (Fig. 7 B). The cytosolic protein, G6PDH, was released into the supernatant at low concentrations (25 μg/ml), whereas the peroxisomal matrix protein GFP-SKL started to leak at digitonin concentrations of 50–100 μg/ml, and release was not complete until the concentration of digitonin was 500 μg/ml. Pex3p, a peroxisomal membrane protein, was only fully released into the supernatant at digitonin concentrations exceeding 1,000 μg/ml. The Pex22(1–25)-GFP fusion protein was released into the medium at very high concentrations (1,000–1,500 μg/ml), or when the cells were treated with 0.2% Triton X-100 (Fig. 7 B). These results show that the Pex22(1–25)-GFP construct is targeted to peroxisomal membranes.


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)

Localization of different GFP constructs in methanol-grown, wild-type cells. (A) PPY12 was transformed with constructs expressing GFP-SKL (pTW74), Pex22-GFP (pTK30) and Pex22(1–25)-GFP (pTK32) and grown on methanol for 16 h. Cells were prepared for immunofluorescence and labeled with α-AOX. The localization of AOX and the GFP construct in the same cells is shown. (B) Wild-type cells expressing either GFP-SKL (pTW74) or Pex22(1–25)-GFP (pTK32) were grown on methanol, spheroplasted, and incubated for 10 min at 30°C with increasing amounts of digitonin or 0.2% Triton X-100 in isotonic buffer. The spheroplasts were pelleted and the supernatants were used for immunoblotting with GFP, Pex3p, and G6PDH antibodies.
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Related In: Results  -  Collection

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Figure 7: Localization of different GFP constructs in methanol-grown, wild-type cells. (A) PPY12 was transformed with constructs expressing GFP-SKL (pTW74), Pex22-GFP (pTK30) and Pex22(1–25)-GFP (pTK32) and grown on methanol for 16 h. Cells were prepared for immunofluorescence and labeled with α-AOX. The localization of AOX and the GFP construct in the same cells is shown. (B) Wild-type cells expressing either GFP-SKL (pTW74) or Pex22(1–25)-GFP (pTK32) were grown on methanol, spheroplasted, and incubated for 10 min at 30°C with increasing amounts of digitonin or 0.2% Triton X-100 in isotonic buffer. The spheroplasts were pelleted and the supernatants were used for immunoblotting with GFP, Pex3p, and G6PDH antibodies.
Mentions: Sequence analysis of Pex22p did not reveal an obvious mPTS. There is, however, a stretch of positively charged amino acids near the extreme NH2 terminus of Pex22p which does not completely fit the consensus sequence for an mPTS (Elgersma et al. 1997). This stretch is at the same location as the putative mPTS of Pex3p (Hp, Sc, Pp). Therefore, we constructed GFP fusions with full-length Pex22 (Pex22-GFP, pTK30, this construct is able to complement a Δpex22 mutant for growth on oleate and methanol), a second fusion with the first 25 amino acids of Pex22 (Pex22(1–25)-GFP; pTK32), containing the transmembrane domain, a third fusion with only the transmembrane domain (Pex22(8–25)-GFP; pTK44), and a fourth fusion with the first seven amino acids of Pex22 (Pex22(1–7)-GFP; pTK34), not containing the transmembrane domain. These constructs were transformed into PPY12 and the resulting strains induced on methanol. The constructs expressing full-length Pex22-GFP and Pex22(1–25)-GFP showed colocalization with alcohol oxidase, a bona fide peroxisomal matrix protein (Fig. 7 A), proving that these constructs get targeted to peroxisomes, whereas the other two constructs (Pex22(1–7)-GFP, Pex22(8–25)-GFP) were localized in the cytosol (data not shown). The Pex22(1–25)-GFP fusion protein could also be shown to colocalize with peroxisomes when an organelle fraction was separated on Nycodenz gradients (data not shown). Furthermore, this fusion was organelle associated since the fusion protein (Pex22(1–25)-GFP) only leaked from cells at digitonin concentrations that released membrane proteins (Fig. 7 B). The cytosolic protein, G6PDH, was released into the supernatant at low concentrations (25 μg/ml), whereas the peroxisomal matrix protein GFP-SKL started to leak at digitonin concentrations of 50–100 μg/ml, and release was not complete until the concentration of digitonin was 500 μg/ml. Pex3p, a peroxisomal membrane protein, was only fully released into the supernatant at digitonin concentrations exceeding 1,000 μg/ml. The Pex22(1–25)-GFP fusion protein was released into the medium at very high concentrations (1,000–1,500 μg/ml), or when the cells were treated with 0.2% Triton X-100 (Fig. 7 B). These results show that the Pex22(1–25)-GFP construct is targeted to peroxisomal membranes.

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