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Phosphorylation of Pex11p does not regulate peroxisomal fission in the yeast Hansenula polymorpha.

Thomas AS, Krikken AM, van der Klei IJ, Williams CP - Sci Rep (2015)

Bottom Line: Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p.Also, no effect on peroxisome inheritance was observed.Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands.

ABSTRACT
Pex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein's trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

No MeSH data available.


Related in: MedlinePlus

Phosphorylation of HpPex11p does not affect localization of the protein.(A–C) Wide field fluorescence microscopy images of pex11Δ cells grown on methanol for 4h (A), 8h (B) or 16 h (C). Besides DsRed-SKL, cells also produced GFP fusions of the Pex11 proteins. All scale bars represent 1 μm. (D) Western blot to compare protein levels of Pex11 S174A-GFP or Pex11 S174D-GFP with WT Pex11-GFP. Cells were grown for 16 hours on methanol. Blots were probed with antibodies raised against Pyc1 or GFP.
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f4: Phosphorylation of HpPex11p does not affect localization of the protein.(A–C) Wide field fluorescence microscopy images of pex11Δ cells grown on methanol for 4h (A), 8h (B) or 16 h (C). Besides DsRed-SKL, cells also produced GFP fusions of the Pex11 proteins. All scale bars represent 1 μm. (D) Western blot to compare protein levels of Pex11 S174A-GFP or Pex11 S174D-GFP with WT Pex11-GFP. Cells were grown for 16 hours on methanol. Blots were probed with antibodies raised against Pyc1 or GFP.

Mentions: To analyse if Pex11p phosphorylation influences the subcellular location of Pex11p, strains were constructed producing WT and mutant versions of Pex11p C-terminally tagged with GFP. Cells were pre-cultivated on glucose and subsequently shifted to medium containing methanol, to induced peroxisome proliferation. At 4 and 8 hours after the shift, we observed that like WT Pex11-GFP, both mutant forms co-localise with DsRed-SKL, introduced into the strains to mark the peroxisomal matrix (Fig. 4A–C). Western blotting revealed that the protein levels were comparable among strains (Fig. 4D). These data suggest that phosphorylation does not regulate HpPex11p localisation.


Phosphorylation of Pex11p does not regulate peroxisomal fission in the yeast Hansenula polymorpha.

Thomas AS, Krikken AM, van der Klei IJ, Williams CP - Sci Rep (2015)

Phosphorylation of HpPex11p does not affect localization of the protein.(A–C) Wide field fluorescence microscopy images of pex11Δ cells grown on methanol for 4h (A), 8h (B) or 16 h (C). Besides DsRed-SKL, cells also produced GFP fusions of the Pex11 proteins. All scale bars represent 1 μm. (D) Western blot to compare protein levels of Pex11 S174A-GFP or Pex11 S174D-GFP with WT Pex11-GFP. Cells were grown for 16 hours on methanol. Blots were probed with antibodies raised against Pyc1 or GFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4477233&req=5

f4: Phosphorylation of HpPex11p does not affect localization of the protein.(A–C) Wide field fluorescence microscopy images of pex11Δ cells grown on methanol for 4h (A), 8h (B) or 16 h (C). Besides DsRed-SKL, cells also produced GFP fusions of the Pex11 proteins. All scale bars represent 1 μm. (D) Western blot to compare protein levels of Pex11 S174A-GFP or Pex11 S174D-GFP with WT Pex11-GFP. Cells were grown for 16 hours on methanol. Blots were probed with antibodies raised against Pyc1 or GFP.
Mentions: To analyse if Pex11p phosphorylation influences the subcellular location of Pex11p, strains were constructed producing WT and mutant versions of Pex11p C-terminally tagged with GFP. Cells were pre-cultivated on glucose and subsequently shifted to medium containing methanol, to induced peroxisome proliferation. At 4 and 8 hours after the shift, we observed that like WT Pex11-GFP, both mutant forms co-localise with DsRed-SKL, introduced into the strains to mark the peroxisomal matrix (Fig. 4A–C). Western blotting revealed that the protein levels were comparable among strains (Fig. 4D). These data suggest that phosphorylation does not regulate HpPex11p localisation.

Bottom Line: Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p.Also, no effect on peroxisome inheritance was observed.Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands.

ABSTRACT
Pex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein's trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

No MeSH data available.


Related in: MedlinePlus