Peroxisomen zijn belangrijke organellen in eukaryote cellen. Ze bestaan uit een enkele membraan met een eiwitrijke matrix. Belangrijke peroxisomale processen zijn de β-oxidatie van vetzuren en de afbraak van waterstofperoxide. Momenteel is er veel discussie over de wijze waarop peroxisomen worden gevormd. In wild-type gisten worden ze voornamelijk aangemaakt door deling van al aanwezige organellen. Er bestaan echter mutanten waarin peroxisomen volledig afwezig zijn (b.v. in pex3 of pex19 mutanten). Tot nu toe werd gedacht dat na het herintroduceren van het PEX3 of PEX19 gen in deze mutanten nieuwe peroxisomen werden gevormd vanuit het endoplasmatisch reticulum (ER). Experimenten die beschreven zijn in dit proefschrift wezen uit dat in pex3 en pex19 mutanten wel degelijk peroxisomale membraan structuren aanwezig zijn. Deze structuren zijn de basis voor de vorming van nieuwe peroxisomen als het PEX3 of PEX19 gen wordt geherintroduceerd. Onder deze omstandigheden worden peroxisomen dus niet uit het ER gevormd. Peroxisoom homeostase omvat een nauwkeurige balans tussen de vorming en afbraak (pexofagie) van peroxisomen. We hebben laten zien dat de aanwezigheid van eiwitaggregaten slecht is voor cellen en leidt tot verhoogde niveaus van zuurstof radicalen. Deze eiwitaggregaten stimuleerden asymmetrische deling van peroxisomen en afbraak van de aggregaat bevattende organellen. Tenslotte hebben we een nieuwe functie gevonden voor het peroxisomale membraan eiwit Wsc. Dit eiwit speelt een rol in de deling en overerving van peroxisomen in gisten die groeien op glucose

Manivannan, Selvambigai

Peroxisomen zijn belangrijke organellen in eukaryote cellen. Ze bestaan uit een enkele membraan met een eiwitrijke matrix. Belangrijke peroxisomale processen zijn de β-oxidatie van vetzuren en de afbraak van waterstofperoxide. Momenteel is er veel discussie over de wijze waarop peroxisomen worden gevormd. In wild-type gisten worden ze voornamelijk aangemaakt door deling van al aanwezige organellen. Er bestaan echter mutanten waarin peroxisomen volledig afwezig zijn (b.v. in pex3 of pex19 mutanten). Tot nu toe werd gedacht dat na het herintroduceren van het PEX3 of PEX19 gen in deze mutanten nieuwe peroxisomen werden gevormd vanuit het endoplasmatisch reticulum (ER).Experimenten die beschreven zijn in dit proefschrift wezen uit dat in pex3 en pex19 mutanten wel degelijk peroxisomale membraan structuren aanwezig zijn. Deze structuren zijn de basis voor de vorming van nieuwe peroxisomen als het PEX3 of PEX19 gen wordt geherintroduceerd. Onder deze omstandigheden worden peroxisomen dus niet uit het ER gevormd.Peroxisoom homeostase omvat een nauwkeurige balans tussen de vorming en afbraak (pexofagie) van peroxisomen. We hebben laten zien dat de aanwezigheid van eiwitaggregaten slecht is voor cellen en leidt tot verhoogde niveaus van zuurstof radicalen. Deze eiwitaggregaten stimuleerden asymmetrische deling van peroxisomen en afbraak van de aggregaat bevattende organellen. Tenslotte hebben we een nieuwe functie gevonden voor het peroxisomale membraan eiwit Wsc. Dit eiwit speelt een rol in de deling en overerving van peroxisomen in gisten die groeien op glucose.Peroxisomes are important eukaryotic organelles, consisting of a protein rich matrix, surrounded by a single membrane. Generalized functions include β-oxidation of fatty acids and detoxification of hydrogen peroxide. Currently, it is highly debated how peroxisomes are formed. In wild-type yeast cells the organelles primarily develop by fission of pre-existing ones. However, it has been proposed that they can also form de novo from the endoplasmic reticulum (ER) in mutant cells fully lacking peroxisomal membrane structures (e.g. pex3 or pex19), upon reintroduction of the corresponding genes. Studies described in this thesis revealed, however, in these mutants, pre-peroxisomal structures are present, which serve as template for re-introduction of peroxisomes. Hence, under these conditions peroxisomes do not derive from the ER.Peroxisome homeostasis involves a delicate balance between peroxisome biogenesis and degradation (pexophagy). We demonstrate that the presence of protein aggregates in the peroxisomal matrix had physiological disadvantages as it affected growth of the cells and caused enhanced levels of reactive oxygen species. These protein aggregates promoted asymmetric peroxisome fission and degradation of peroxisomes containing protein aggregates via autophagy Finally, this thesis describes a novel function for the peroxisomal membrane protein Wsc, which is important for peroxisome fission and segregation in glucose-grown yeast cells

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Yeast peroxisomes:de novo formation and maintenance

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Yeast peroxisomes: de novo formation and maintenance

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 University of Groningen
Yeast peroxisomes
Manivannan, Selvambigai
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1 
 
 
 
 
 
 
 
 
 
 
 
Yeast peroxisomes: de novo formation and 
maintenance 
 
 
 
 
 
2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                                                  
 
 
The studies presented in this thesis were performed in the research group Molecular Cell Biology 
of the Groningen Biomolecular Sciences and Biotechnology Institute (GBB) of the University of 
Groningen, The Netherlands.  
 
 
3 
 
 
    
 
Yeast peroxisomes  
De novo formation and maintenance 
 
 
PhD thesis 
 
 
to obtain the degree of PhD at the 
University of Groningen 
on the authority of the 
Rector Magnificus Prof. E. Sterken 
and in accordance with 
the decision by the College of Deans. 
 
This thesis will be defended in public on  
 
Friday 2 May 2014 at 11.00 hours 
 
 
 
by 
 
Selvambigai Manivannan 
born on 18 January 1986 
in Velur, India 
4 
 
 
 
 
  Supervisor 
  Prof.  I. J. van der Klei  
 
 
 Assessment committee 
 Prof. M. Schrader 
 Prof. B. M. Bakker 
 Prof. D. J. Slotboom  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5 
 
 
Contents 
 
 
Aim and Outline                                    13 
 
Chapter 1: Introduction                                   17 
 
Chapter 2:  Lumenal peroxisomal protein aggregates are removed                                57 
by concerted fission and autophagy events      
         
Chapter 3:  Preperoxisomal vesicles can form in the absence                                91 
of Pex3               
 
Chapter 4: Pre-peroxisome formation in Hansenula polymorpha                                129 
 pex19 cells does not require the ER  
            
Chapter 5:  The Hansenula polymorpha WSC homolog is involved                              157 
in peroxisomal membrane shaping and organelle  
partitioning            
 
Summary                                      189 
 
Samenvatting                                      197 
 
 
 
 
6 
 
Aim and Outline 
 Hallmark of eukaryotic cells is the presence of different membrane bound components 
termed organelles, each comprised of a unique structure and function. Of these, peroxisomes are 
important organelles that characteristically contain at least one hydrogen peroxide producing 
oxidase together with catalase to remove the hydrogen peroxide by-product. Peroxisomes are 
inducible in nature and dynamic which facilitates a rapid adaptation of their number and function 
in relation to the metabolic needs of the cell. Indeed, the number of peroxisomes per cell is 
carefully regulated. When required, they are induced but when redundant for growth, they may 
be rapidly removed via selective autophagy (also termed pexophagy). Peroxisomes are the 
primary sites of lipid metabolism of the cell. Presently, the major contentious issues in 
peroxisome field are the role of the ER in peroxisome formation and sorting of peroxisomal 
membrane proteins (PMPs). In yeast, peroxisomes are pre-dominantly formed by division of pre-
existing organelle. However, peroxisomes can also be formed from ER by de novo particularly in 
PEX mutants which lacks peroxisomes such as Δpex3 and Δpex19 upon complementation with 
their corresponding gene. Hitherto, the sorting and insertion pathway of PMPs is a conundrum. 
Various models have been proposed ranging from the view that all PMPs travel via the ER to the 
opposite that PMPs are directly post-translationally sorted. Also intermediate have been 
proposed in that only few PMPs travel via the ER whereas others insert directly. This Thesis 
project aims at further elucidating the molecular mechanisms behind peroxisome biosynthesis 
and maintenance. 
Chapter 1 contains a synopsis of the contemporary views on peroxisome formation, homeostasis 
(biogenesis and degradation), and their implications in cellular aging, apoptosis and cell death. 
7 
 
Chapter 2 describes the relation that exists between peroxisome fission and degradation. Using 
mutants of Saccharomyces cerevisiae and Hansenula polymorpha that are defective in 
peroxisome fission (∆dnm1 and ∆pex11) we show that peroxisome degradation is inhibited, like 
in the ∆atg1 control strain. Moreover, we demonstrated that, upon introduction of catalase 
protein aggregates into peroxisomal lumen, these aberrant structures are rapidly removed from 
the organelles by a concerted action of peroxisome fission and selective autophagy. Removal of 
these aggregates was important to sustain cell health as their presence was associated with 
distinct physiological disadvantages as it affected growth and caused enhanced levels of reactive 
oxygen species. Removal required the normal peroxisome fission machinery as it was inhibited 
in peroxisome fission mutants (∆dnm1 and ∆pex11). Taken together, these data demonstrate that 
peroxisomal housekeeping mechanisms exist to remove unwanted or harmful components from 
the organelles, thus keeping them vital and functional. 
 Chapter 3 deals with de novo peroxisome formation in a Hansenula polymorpha pex3 mutant 
which occurs when the PEX3 gene is reintroduced in this mutant. The current view was that pex3 
cell lack peroxisome membrane remnants and new peroxisomes were created using the ER as 
membrane template. Our current data indicate that this view in not generally valid and requires 
adaptation. This is based on our finding that pex3 cell do have peroxisomal membrane remnants 
which are very unstable and subject to rapid degradation. However, subsequent deletion of 
ATG1, a gene essential for autophagy, resulted in stabilization of these peroxisomal membrane 
vesicles. Biochemical and microscopy studies revealed that these vesicles contained Pex8 
together with the docking site proteins Pex13 and Pex14. Other PMPs (i.e. Pex10, Pex11, and 
Pmp47) were unstable and localized to the cytosol. Finally, we showed that the Pex14-containing 
structures were the target for peroxisome development after reintroduction of Pex3 in pex3 atg1 
8 
 
cells. Together, these findings suggest that i) peroxisomal membrane structures can be formed in 
the absence of Pex3, and ii) alternative sorting mechanisms exist for Pex13 and Pex14, which 
reach their target membrane independent of the Pex3/Pex19 docking machinery. 
Chapter 4 is a continuation of chapter 3, as it aims to study de novo formation of peroxisomes in 
a second mutant described to lack peroxisomal membranes, namely pex19. We show that also H. 
polymorpha pex19 cells do contains cluster of peroxisomal vesicles, which are unstable but could 
be stabilized by blocking autophagy (ATG1 deletion). Our data further showed that the 
peroxisomal membrane proteins such as Pex3, Pex13, Pex14 and Pex8 are targeted to the 
vesicular structures of pex19 atg1 cells, whereas the Pex10, Pex11, Pex26 and Pmp47 are not. 
Finally our data revealed that upon complementation with PEX19, the vesicles present in pex19 
cells acts as template for peroxisome formation and not the ER. Therefore, our findings suggest 
that Pex19 is not essential for peroxisomal vesicles formation from ER and also for sorting of 
Pex3. 
The work in Chapter 5 is aimed to investigate the function of putative H. polymorpha orthologs 
of the PMP22/MPV17 family of proteins (WSC, SYM1, and YOR292C). In silico analyses 
revealed the presence of homologs in H. polymorpha. Fluorescence microscopy (FM), using 
GFP fusions of these proteins revealed that under peroxisome inducing conditions, the woronin 
sorting complex (Wsc) protein is localized to peroxisomal membrane whereas the Sym1 and 
Yor292c levels were below the limit of detection by FM. WSC deletion cells (Δwsc) grew 
normally on methanol and showed no alteration in  peroxisome abundance. However on glucose, 
a partial peroxisome segregation defect was observed during vegetative cell reproduction in 
conjunction with aberrations in peroxisome morphology. Therefore, Wsc might be essential for 
9 
 
segregation of peroxisomes and shaping of the organelle membrane.


Yeast peroxisomes:de novo formation and maintenance

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