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Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1.

Saraya R, Krikken AM, Veenhuis M, van der Klei IJ - J. Cell Biol. (2011)

Bottom Line: Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes.Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11.These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, 9700 CC Groningen, Netherlands.

ABSTRACT
We identified two proteins, Pex25 and Rho1, which are involved in reintroduction of peroxisomes in peroxisome-deficient yeast cells. These are, together with Pex3, the first proteins identified as essential for this process. Of the three members of the Hansenula polymorpha Pex11 protein family-Pex11, Pex25, and Pex11C-only Pex25 was required for reintroduction of peroxisomes into a peroxisome-deficient mutant strain. In peroxisome-deficient pex3 cells, Pex25 localized to structures adjacent to the ER, whereas in wild-type cells it localized to peroxisomes. Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes. Interestingly, pex11 pex25 double deletion cells, in which both peroxisome fission (due to the deletion of PEX11) and reintroduction (due to deletion of PEX25) was blocked, did display a peroxisome-deficient phenotype. Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11. Reintroduction in the presence of Pex25 required the function of the GTPase Rho1. These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.

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Artificial targeting of Pex3 to the ER does not restore peroxisome formation in the absence of Pex25. PAOX BIPN30PEX3-mCherry was introduced in pex3 cells (A), pex3 pex11 cells (B), pex3 pex25 cells (C), or pex3 pex11 pex25 cells (D). Electron microsopy analysis of cells grown for 16 h on glycerol/methanol-containing media failed to resolve peroxisomal structures in cells lacking Pex25. N, nucleus; P, peroxisome; V, vacuole. Bar, 0.5 µm. The asterisk represents a cytosolic alcohol oxidase crystalloid.
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fig5: Artificial targeting of Pex3 to the ER does not restore peroxisome formation in the absence of Pex25. PAOX BIPN30PEX3-mCherry was introduced in pex3 cells (A), pex3 pex11 cells (B), pex3 pex25 cells (C), or pex3 pex11 pex25 cells (D). Electron microsopy analysis of cells grown for 16 h on glycerol/methanol-containing media failed to resolve peroxisomal structures in cells lacking Pex25. N, nucleus; P, peroxisome; V, vacuole. Bar, 0.5 µm. The asterisk represents a cytosolic alcohol oxidase crystalloid.

Mentions: Because Pex25 is required for reintroduction of peroxisomes in pex3 cells, we hypothesized that Pex3 may not properly sort to the ER in the absence of Pex25 to form new peroxisomes. To address this question, we constructed a pex3 pex25 strain, in which Pex3-mCherry was artificially sorted to the ER. To this end we constructed a gene encoding a fusion protein containing the first N-terminal 30 amino acids of the ER protein BIP (BiPN30) and full-length Pex3 (lacking the start codon) fused to mCherry under control of the inducible alcohol oxidase promoter (PAOXBIPN30PEX3-mCherry). A similar construct was previously reported to functionally complement S. cerevisiae pex3 cells (Kragt et al., 2005). Indeed, upon synthesis of this fusion protein in H. polymorpha pex3 control cells peroxisomes were readily formed (Fig. 5 A). Essentially similar results were obtained when the fusion protein was introduced in pex3 pex11 cells (Fig. 5 B). In contrast, however, peroxisomes were not detected when the construct was expressed in pex3 pex25 or pex3 pex11 pex25 cells (Fig. 5, C and D). In these cells large cytosolic alcohol oxidase crystals were formed (Fig. 5, C and D, asterisk), akin to pex3 cells, demonstrating that these cells are indeed peroxisome deficient. These data suggest that the failure of pex3 pex25 cell to form peroxisomes from the ER cannot be restored by artificial targeting of Pex3 to the ER.


Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1.

Saraya R, Krikken AM, Veenhuis M, van der Klei IJ - J. Cell Biol. (2011)

Artificial targeting of Pex3 to the ER does not restore peroxisome formation in the absence of Pex25. PAOX BIPN30PEX3-mCherry was introduced in pex3 cells (A), pex3 pex11 cells (B), pex3 pex25 cells (C), or pex3 pex11 pex25 cells (D). Electron microsopy analysis of cells grown for 16 h on glycerol/methanol-containing media failed to resolve peroxisomal structures in cells lacking Pex25. N, nucleus; P, peroxisome; V, vacuole. Bar, 0.5 µm. The asterisk represents a cytosolic alcohol oxidase crystalloid.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105547&req=5

fig5: Artificial targeting of Pex3 to the ER does not restore peroxisome formation in the absence of Pex25. PAOX BIPN30PEX3-mCherry was introduced in pex3 cells (A), pex3 pex11 cells (B), pex3 pex25 cells (C), or pex3 pex11 pex25 cells (D). Electron microsopy analysis of cells grown for 16 h on glycerol/methanol-containing media failed to resolve peroxisomal structures in cells lacking Pex25. N, nucleus; P, peroxisome; V, vacuole. Bar, 0.5 µm. The asterisk represents a cytosolic alcohol oxidase crystalloid.
Mentions: Because Pex25 is required for reintroduction of peroxisomes in pex3 cells, we hypothesized that Pex3 may not properly sort to the ER in the absence of Pex25 to form new peroxisomes. To address this question, we constructed a pex3 pex25 strain, in which Pex3-mCherry was artificially sorted to the ER. To this end we constructed a gene encoding a fusion protein containing the first N-terminal 30 amino acids of the ER protein BIP (BiPN30) and full-length Pex3 (lacking the start codon) fused to mCherry under control of the inducible alcohol oxidase promoter (PAOXBIPN30PEX3-mCherry). A similar construct was previously reported to functionally complement S. cerevisiae pex3 cells (Kragt et al., 2005). Indeed, upon synthesis of this fusion protein in H. polymorpha pex3 control cells peroxisomes were readily formed (Fig. 5 A). Essentially similar results were obtained when the fusion protein was introduced in pex3 pex11 cells (Fig. 5 B). In contrast, however, peroxisomes were not detected when the construct was expressed in pex3 pex25 or pex3 pex11 pex25 cells (Fig. 5, C and D). In these cells large cytosolic alcohol oxidase crystals were formed (Fig. 5, C and D, asterisk), akin to pex3 cells, demonstrating that these cells are indeed peroxisome deficient. These data suggest that the failure of pex3 pex25 cell to form peroxisomes from the ER cannot be restored by artificial targeting of Pex3 to the ER.

Bottom Line: Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes.Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11.These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, 9700 CC Groningen, Netherlands.

ABSTRACT
We identified two proteins, Pex25 and Rho1, which are involved in reintroduction of peroxisomes in peroxisome-deficient yeast cells. These are, together with Pex3, the first proteins identified as essential for this process. Of the three members of the Hansenula polymorpha Pex11 protein family-Pex11, Pex25, and Pex11C-only Pex25 was required for reintroduction of peroxisomes into a peroxisome-deficient mutant strain. In peroxisome-deficient pex3 cells, Pex25 localized to structures adjacent to the ER, whereas in wild-type cells it localized to peroxisomes. Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes. Interestingly, pex11 pex25 double deletion cells, in which both peroxisome fission (due to the deletion of PEX11) and reintroduction (due to deletion of PEX25) was blocked, did display a peroxisome-deficient phenotype. Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11. Reintroduction in the presence of Pex25 required the function of the GTPase Rho1. These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.

Show MeSH
Related in: MedlinePlus