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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

Quantitative analysis of peroxisome numbers in WT and HpPex11p phosphorylation mutant strains.(A-D) Quantification of peroxisome numbers from images represented in Fig. 2A–D. For each experiment, at least 600 cells were counted per strain. Error bars represent standard deviation between two separate experiments. (E and F) Western blots to compare protein levels between WT and phosphorylation single mutant strains (E), or phosphorylation double mutant strains (F). In the case of mutants S174D and S161D, S174D, the introduction of an extra negative charge results in a mobility shift. Cells were grown for 16 hours on medium containing methanol. Blots were probed with antibodies raised against Pyruvate carboxylase-1 (Pyc1; loading control) or Pex11p. Equal amounts of protein were loaded per lane.
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f3: Quantitative analysis of peroxisome numbers in WT and HpPex11p phosphorylation mutant strains.(A-D) Quantification of peroxisome numbers from images represented in Fig. 2A–D. For each experiment, at least 600 cells were counted per strain. Error bars represent standard deviation between two separate experiments. (E and F) Western blots to compare protein levels between WT and phosphorylation single mutant strains (E), or phosphorylation double mutant strains (F). In the case of mutants S174D and S161D, S174D, the introduction of an extra negative charge results in a mobility shift. Cells were grown for 16 hours on medium containing methanol. Blots were probed with antibodies raised against Pyruvate carboxylase-1 (Pyc1; loading control) or Pex11p. Equal amounts of protein were loaded per lane.

Mentions: Next, fluorescence microscopy analysis (Fig. 2A–C; Fig. 3A–C) revealed that peroxisome numbers were comparable between strains expressing WT or mutant versions of HpPex11p, independent of whether cells were grown on glucose (Fig. 2A) or methanol (Fig. 2B,C). Western blotting revealed that Pex11p levels were comparable among strains (Fig. 3E). These findings suggest that phosphorylation of Serine 174 has no significant effect on peroxisome proliferation in glucose or methanol grown H. polymorpha.


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)

Quantitative analysis of peroxisome numbers in WT and HpPex11p phosphorylation mutant strains.(A-D) Quantification of peroxisome numbers from images represented in Fig. 2A–D. For each experiment, at least 600 cells were counted per strain. Error bars represent standard deviation between two separate experiments. (E and F) Western blots to compare protein levels between WT and phosphorylation single mutant strains (E), or phosphorylation double mutant strains (F). In the case of mutants S174D and S161D, S174D, the introduction of an extra negative charge results in a mobility shift. Cells were grown for 16 hours on medium containing methanol. Blots were probed with antibodies raised against Pyruvate carboxylase-1 (Pyc1; loading control) or Pex11p. Equal amounts of protein were loaded per lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Quantitative analysis of peroxisome numbers in WT and HpPex11p phosphorylation mutant strains.(A-D) Quantification of peroxisome numbers from images represented in Fig. 2A–D. For each experiment, at least 600 cells were counted per strain. Error bars represent standard deviation between two separate experiments. (E and F) Western blots to compare protein levels between WT and phosphorylation single mutant strains (E), or phosphorylation double mutant strains (F). In the case of mutants S174D and S161D, S174D, the introduction of an extra negative charge results in a mobility shift. Cells were grown for 16 hours on medium containing methanol. Blots were probed with antibodies raised against Pyruvate carboxylase-1 (Pyc1; loading control) or Pex11p. Equal amounts of protein were loaded per lane.
Mentions: Next, fluorescence microscopy analysis (Fig. 2A–C; Fig. 3A–C) revealed that peroxisome numbers were comparable between strains expressing WT or mutant versions of HpPex11p, independent of whether cells were grown on glucose (Fig. 2A) or methanol (Fig. 2B,C). Western blotting revealed that Pex11p levels were comparable among strains (Fig. 3E). These findings suggest that phosphorylation of Serine 174 has no significant effect on peroxisome proliferation in glucose or methanol grown H. polymorpha.

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