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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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Yeast two-hybrid analysis reveals interaction of Pex11 with itself and of Pex25 with itself. Analysis of the interaction of different H. polymorpha proteins with Pex11 and Pex25, using yeast two-hybrid assays. Genes were fused to the LEXA binding domain (LexA-BD) in vector pBTM116 and a VP16 activation domain (Vp16-AD) in vector pVP16. The resulting plasmids were cotransformed into S. cerevisiae L-40. As controls, empty pVP16 or pBTM116 was used for transformation. Pex3 Pex19 interaction was added as positive control. Three independent transformants were tested using a β-galactosidase filter lift assay. Colonies were stained for 8 h.
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fig8: Yeast two-hybrid analysis reveals interaction of Pex11 with itself and of Pex25 with itself. Analysis of the interaction of different H. polymorpha proteins with Pex11 and Pex25, using yeast two-hybrid assays. Genes were fused to the LEXA binding domain (LexA-BD) in vector pBTM116 and a VP16 activation domain (Vp16-AD) in vector pVP16. The resulting plasmids were cotransformed into S. cerevisiae L-40. As controls, empty pVP16 or pBTM116 was used for transformation. Pex3 Pex19 interaction was added as positive control. Three independent transformants were tested using a β-galactosidase filter lift assay. Colonies were stained for 8 h.

Mentions: In pex3 cells Pex25 is localized in spots adjacent to the ER. As reintroduction of peroxisomes in these cells requires Pex25 and involves the initial targeting of Pex3 to the ER, we tested whether Pex3 interacts with Pex25 using a yeast two-hybrid assay. As shown in Fig. 8, no interaction between Pex25 and Pex3 was detected. Also, no interaction between Pex11 and Pex3 was observed. As reported previously for Pex11 proteins from other species, H. polymorpha Pex11 interacts with itself and hence most likely forms oligomers (Rottensteiner et al., 2003; Tam et al., 2003). The same was observed for H. polymorpha Pex25. We could not detect an interaction between H. polymorpha Pex11 and Pex25. These data indicate that Pex25 and Pex3, which are both involved in reintroduction of peroxisomes at the ER, most likely do not show a direct physical interaction.


Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1.

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

Yeast two-hybrid analysis reveals interaction of Pex11 with itself and of Pex25 with itself. Analysis of the interaction of different H. polymorpha proteins with Pex11 and Pex25, using yeast two-hybrid assays. Genes were fused to the LEXA binding domain (LexA-BD) in vector pBTM116 and a VP16 activation domain (Vp16-AD) in vector pVP16. The resulting plasmids were cotransformed into S. cerevisiae L-40. As controls, empty pVP16 or pBTM116 was used for transformation. Pex3 Pex19 interaction was added as positive control. Three independent transformants were tested using a β-galactosidase filter lift assay. Colonies were stained for 8 h.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Yeast two-hybrid analysis reveals interaction of Pex11 with itself and of Pex25 with itself. Analysis of the interaction of different H. polymorpha proteins with Pex11 and Pex25, using yeast two-hybrid assays. Genes were fused to the LEXA binding domain (LexA-BD) in vector pBTM116 and a VP16 activation domain (Vp16-AD) in vector pVP16. The resulting plasmids were cotransformed into S. cerevisiae L-40. As controls, empty pVP16 or pBTM116 was used for transformation. Pex3 Pex19 interaction was added as positive control. Three independent transformants were tested using a β-galactosidase filter lift assay. Colonies were stained for 8 h.
Mentions: In pex3 cells Pex25 is localized in spots adjacent to the ER. As reintroduction of peroxisomes in these cells requires Pex25 and involves the initial targeting of Pex3 to the ER, we tested whether Pex3 interacts with Pex25 using a yeast two-hybrid assay. As shown in Fig. 8, no interaction between Pex25 and Pex3 was detected. Also, no interaction between Pex11 and Pex3 was observed. As reported previously for Pex11 proteins from other species, H. polymorpha Pex11 interacts with itself and hence most likely forms oligomers (Rottensteiner et al., 2003; Tam et al., 2003). The same was observed for H. polymorpha Pex25. We could not detect an interaction between H. polymorpha Pex11 and Pex25. These data indicate that Pex25 and Pex3, which are both involved in reintroduction of peroxisomes at the ER, most likely do not show a direct physical interaction.

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