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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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Identification and localization of Rho1. Rho1 was identified through a screen for conditional mutants affected in the formation of peroxisomal structures in H. polymorpha pex3 cells that produce the first 50 amino acids of the PEX3 gene fused to GFP(N50.Pex3-GFP). (A) Fluorescence microscopy images of methanol/methylamine-grown cells of the pex3 PAMON50PEX3-GFP control strain grown at 35°C (A) or 44°C (B) and pex3 RHO1tsPAMO N50PEX3-GFP grown at 35°C (C) or 44°C (D). (E) Localization of GFP-Rho1, produced in WT H. polymorpha cells that also synthesize DsRed-SKL to mark peroxisomes. (F–I) Pex25-mCherry fluorescence in pex11 pex25 PAOX PEX25-mCherry cells grown at at 35°C (F) or 44°C (G) and in pex25 pex11 RHO1ts PAOX PEX25-mCherry cells upon cultivation at 35°C (H) and 44°C (I). Bar, 1 µm.
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fig9: Identification and localization of Rho1. Rho1 was identified through a screen for conditional mutants affected in the formation of peroxisomal structures in H. polymorpha pex3 cells that produce the first 50 amino acids of the PEX3 gene fused to GFP(N50.Pex3-GFP). (A) Fluorescence microscopy images of methanol/methylamine-grown cells of the pex3 PAMON50PEX3-GFP control strain grown at 35°C (A) or 44°C (B) and pex3 RHO1tsPAMO N50PEX3-GFP grown at 35°C (C) or 44°C (D). (E) Localization of GFP-Rho1, produced in WT H. polymorpha cells that also synthesize DsRed-SKL to mark peroxisomes. (F–I) Pex25-mCherry fluorescence in pex11 pex25 PAOX PEX25-mCherry cells grown at at 35°C (F) or 44°C (G) and in pex25 pex11 RHO1ts PAOX PEX25-mCherry cells upon cultivation at 35°C (H) and 44°C (I). Bar, 1 µm.

Mentions: One of these mutants, 3-34-ts, was used for further analysis. This strain showed GFP fluorescence in the cytosol at restrictive temperature (Fig. 9 D), whereas at permissive temperature (35°C) fluorescent spots were observed (Fig. 9 C). As expected, pex3 control cells producing N50.Pex3-GFP formed normal fluorescent spots at both temperatures (Fig. 9, A and B).


Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1.

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

Identification and localization of Rho1. Rho1 was identified through a screen for conditional mutants affected in the formation of peroxisomal structures in H. polymorpha pex3 cells that produce the first 50 amino acids of the PEX3 gene fused to GFP(N50.Pex3-GFP). (A) Fluorescence microscopy images of methanol/methylamine-grown cells of the pex3 PAMON50PEX3-GFP control strain grown at 35°C (A) or 44°C (B) and pex3 RHO1tsPAMO N50PEX3-GFP grown at 35°C (C) or 44°C (D). (E) Localization of GFP-Rho1, produced in WT H. polymorpha cells that also synthesize DsRed-SKL to mark peroxisomes. (F–I) Pex25-mCherry fluorescence in pex11 pex25 PAOX PEX25-mCherry cells grown at at 35°C (F) or 44°C (G) and in pex25 pex11 RHO1ts PAOX PEX25-mCherry cells upon cultivation at 35°C (H) and 44°C (I). Bar, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105547&req=5

fig9: Identification and localization of Rho1. Rho1 was identified through a screen for conditional mutants affected in the formation of peroxisomal structures in H. polymorpha pex3 cells that produce the first 50 amino acids of the PEX3 gene fused to GFP(N50.Pex3-GFP). (A) Fluorescence microscopy images of methanol/methylamine-grown cells of the pex3 PAMON50PEX3-GFP control strain grown at 35°C (A) or 44°C (B) and pex3 RHO1tsPAMO N50PEX3-GFP grown at 35°C (C) or 44°C (D). (E) Localization of GFP-Rho1, produced in WT H. polymorpha cells that also synthesize DsRed-SKL to mark peroxisomes. (F–I) Pex25-mCherry fluorescence in pex11 pex25 PAOX PEX25-mCherry cells grown at at 35°C (F) or 44°C (G) and in pex25 pex11 RHO1ts PAOX PEX25-mCherry cells upon cultivation at 35°C (H) and 44°C (I). Bar, 1 µm.
Mentions: One of these mutants, 3-34-ts, was used for further analysis. This strain showed GFP fluorescence in the cytosol at restrictive temperature (Fig. 9 D), whereas at permissive temperature (35°C) fluorescent spots were observed (Fig. 9 C). As expected, pex3 control cells producing N50.Pex3-GFP formed normal fluorescent spots at both temperatures (Fig. 9, A and B).

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