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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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Peroxisome reintroduction in pex3 cells requires Pex25. Pex3-GFP was reintroduced in pex3 (A), pex3 pex11C (B), pex3 pex11 cells (C), or pex3 pex25 cells (D). All strains contained PEX3-GFP under control of the inducible amine oxidase promoter (PAMO). Cells were pregrown on glucose/ammonium sulfate media and shifted (at t = 0 h) to glycerol/methanol/methylamine to induce Pex3-GFP synthesis and peroxisome proliferation. Bar, 1 µm. All images are presented at the same magnification. The cell walls are indicated in blue. (E) Levels of endogenous Pex3 in WT cells and Pex3-GFP levels in the indicated strains grown for 0, 2, 4, 8, and 20 h on methanol/glycerol/methylamine medium. Equal amounts of protein were loaded per lane. Pyruvate carboxylase (Pyc1) was used as loading control. The blots were decorated with anti-Pex3 or anti-Pyc1 antibodies. The additional Pex3 band at t = 2, 4, 8, and 20 h in the pex3 pex25 samples originates from degradation of Pex3-GFP as is reinforced by the absence of full-length Pex3-GFP at t = 20 h (compare also vacuolar fluorescence in D). (F) Peroxisomes marked by GFP-SKL in the H. polymorpha pex11 strain grown for 20 h on methanol/glycerol/methylamine medium.
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fig4: Peroxisome reintroduction in pex3 cells requires Pex25. Pex3-GFP was reintroduced in pex3 (A), pex3 pex11C (B), pex3 pex11 cells (C), or pex3 pex25 cells (D). All strains contained PEX3-GFP under control of the inducible amine oxidase promoter (PAMO). Cells were pregrown on glucose/ammonium sulfate media and shifted (at t = 0 h) to glycerol/methanol/methylamine to induce Pex3-GFP synthesis and peroxisome proliferation. Bar, 1 µm. All images are presented at the same magnification. The cell walls are indicated in blue. (E) Levels of endogenous Pex3 in WT cells and Pex3-GFP levels in the indicated strains grown for 0, 2, 4, 8, and 20 h on methanol/glycerol/methylamine medium. Equal amounts of protein were loaded per lane. Pyruvate carboxylase (Pyc1) was used as loading control. The blots were decorated with anti-Pex3 or anti-Pyc1 antibodies. The additional Pex3 band at t = 2, 4, 8, and 20 h in the pex3 pex25 samples originates from degradation of Pex3-GFP as is reinforced by the absence of full-length Pex3-GFP at t = 20 h (compare also vacuolar fluorescence in D). (F) Peroxisomes marked by GFP-SKL in the H. polymorpha pex11 strain grown for 20 h on methanol/glycerol/methylamine medium.

Mentions: To this end we constructed a pex3 pex25 strain that contained PEX3-GFP under control of the inducible amine oxidase promoter (PAMO) using a pex3 strain as a control. After precultivation of the strains on glucose/ammonium sulfate, thus repressing PAMO, Pex3-GFP protein was invariably undetectable (not depicted). Upon a shift of pex3 pex25 PAMOPEX3 cells to fresh glycerol/methanol/methylamine-containing media, Pex3-GFP fluorescence was generally first detected after 2 h of cultivation (Fig. 4 D) and frequently observed as a single spot per cell that did not develop into a peroxisome upon further cultivation. Even after 20 h of cultivation on glycerol/methanol/methylamine media peroxisomes were absent and Pex3-GFP was still infrequently observed in spots or had accumulated in the vacuole. Subsequent electron microscopy analyses also failed to resolve any peroxisome structures in these cells at any time of cultivation (not depicted). Under the same conditions peroxisomes were readily formed in pex3 controls (Fig. 4 A). In identical experiments, using pex3 pex11C or pex3 pex11 cells, peroxisomes were reintroduced like in the pex3 control (Fig. 4, B and C), with the exception that in pex3 pex11 cells relatively low numbers of enlarged peroxisomes were formed, as expected for H. polymorpha pex11 cells (Fig. 4 F).


Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1.

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

Peroxisome reintroduction in pex3 cells requires Pex25. Pex3-GFP was reintroduced in pex3 (A), pex3 pex11C (B), pex3 pex11 cells (C), or pex3 pex25 cells (D). All strains contained PEX3-GFP under control of the inducible amine oxidase promoter (PAMO). Cells were pregrown on glucose/ammonium sulfate media and shifted (at t = 0 h) to glycerol/methanol/methylamine to induce Pex3-GFP synthesis and peroxisome proliferation. Bar, 1 µm. All images are presented at the same magnification. The cell walls are indicated in blue. (E) Levels of endogenous Pex3 in WT cells and Pex3-GFP levels in the indicated strains grown for 0, 2, 4, 8, and 20 h on methanol/glycerol/methylamine medium. Equal amounts of protein were loaded per lane. Pyruvate carboxylase (Pyc1) was used as loading control. The blots were decorated with anti-Pex3 or anti-Pyc1 antibodies. The additional Pex3 band at t = 2, 4, 8, and 20 h in the pex3 pex25 samples originates from degradation of Pex3-GFP as is reinforced by the absence of full-length Pex3-GFP at t = 20 h (compare also vacuolar fluorescence in D). (F) Peroxisomes marked by GFP-SKL in the H. polymorpha pex11 strain grown for 20 h on methanol/glycerol/methylamine medium.
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fig4: Peroxisome reintroduction in pex3 cells requires Pex25. Pex3-GFP was reintroduced in pex3 (A), pex3 pex11C (B), pex3 pex11 cells (C), or pex3 pex25 cells (D). All strains contained PEX3-GFP under control of the inducible amine oxidase promoter (PAMO). Cells were pregrown on glucose/ammonium sulfate media and shifted (at t = 0 h) to glycerol/methanol/methylamine to induce Pex3-GFP synthesis and peroxisome proliferation. Bar, 1 µm. All images are presented at the same magnification. The cell walls are indicated in blue. (E) Levels of endogenous Pex3 in WT cells and Pex3-GFP levels in the indicated strains grown for 0, 2, 4, 8, and 20 h on methanol/glycerol/methylamine medium. Equal amounts of protein were loaded per lane. Pyruvate carboxylase (Pyc1) was used as loading control. The blots were decorated with anti-Pex3 or anti-Pyc1 antibodies. The additional Pex3 band at t = 2, 4, 8, and 20 h in the pex3 pex25 samples originates from degradation of Pex3-GFP as is reinforced by the absence of full-length Pex3-GFP at t = 20 h (compare also vacuolar fluorescence in D). (F) Peroxisomes marked by GFP-SKL in the H. polymorpha pex11 strain grown for 20 h on methanol/glycerol/methylamine medium.
Mentions: To this end we constructed a pex3 pex25 strain that contained PEX3-GFP under control of the inducible amine oxidase promoter (PAMO) using a pex3 strain as a control. After precultivation of the strains on glucose/ammonium sulfate, thus repressing PAMO, Pex3-GFP protein was invariably undetectable (not depicted). Upon a shift of pex3 pex25 PAMOPEX3 cells to fresh glycerol/methanol/methylamine-containing media, Pex3-GFP fluorescence was generally first detected after 2 h of cultivation (Fig. 4 D) and frequently observed as a single spot per cell that did not develop into a peroxisome upon further cultivation. Even after 20 h of cultivation on glycerol/methanol/methylamine media peroxisomes were absent and Pex3-GFP was still infrequently observed in spots or had accumulated in the vacuole. Subsequent electron microscopy analyses also failed to resolve any peroxisome structures in these cells at any time of cultivation (not depicted). Under the same conditions peroxisomes were readily formed in pex3 controls (Fig. 4 A). In identical experiments, using pex3 pex11C or pex3 pex11 cells, peroxisomes were reintroduced like in the pex3 control (Fig. 4, B and C), with the exception that in pex3 pex11 cells relatively low numbers of enlarged peroxisomes were formed, as expected for H. polymorpha pex11 cells (Fig. 4 F).

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