Peroxisomal Fission Research Articles

De novo DNM1L variant presenting with severe muscular atrophy, dystonia and sensory neuropathy

DNM1L encodes dynamin-related protein 1 (DRP1), a multi-domain GTPase essential for mitochondrial and peroxisomal division. Autosomal dominant and recessive variants in DNM1L cause encephalopathy due to defective mitochondrial and peroxisomal fission 1 (EMPF1), which presents as a complex and clinically heterogeneous neurological disorder of variable severity, often accompanied by seizures. Clinical features are diverse, and no clear phenotype-genotype correlations were drawn to date. DNM1L-related sensory neuropathy has recently been reported as a predominant feature in one case with a de novo variant in the GTPase domain. Herein we present a second case with DNM1L-related sensory neuropathy as the predominant underlying feature without motor neuron involvement, which resulted in severe muscular atrophy and generalized dystonia.

DNM1, a Dynamin-Related Protein That Contributes to Endocytosis and Peroxisome Fission, Is Required for the Vegetative Growth, Sporulation, and Virulence of Metarhizium robertsii.

Although dynamins and dynamin-related proteins (DRPs), a large GTPase superfamily, are involved in the budding of transport vesicles and division of organelles in eukaryotic cells, the function of these proteins in entomopathogenic fungi has not been reported to date. Here, DNM1, a DRP in Metarhizium robertsii, was characterized using gene disruption and complementation strategies. Mutant phenotype assays showed that the ΔDnm1 strain displayed increased defects in radial growth (∼24%) and conidial production (∼42%) compared to those of the wild type (WT), and reduced conidiation levels were accompanied by the repression of several key conidiation-related genes, including flbA, wetA, and flbD Additionally, mutant bioassays revealed that disruption of Dnm1 impaired the virulence (both topical inoculation and injection) of M. robertsii in the insect Galleria mellonella Further analysis demonstrated that deleting Dnm1 in fungi suppressed the transcriptional levels of several virulence genes in the insect hemocoel. Moreover, we found that DNM1 colocalized with peroxisomes and mitochondria. Importantly, disruption of Dnm1 abolished normal fungal endocytosis, resulting in significantly decreased numbers of, as well as morphological changes in, peroxisomes. These findings indicate that deletion of Dnm1 causes significant changes in the vegetative growth, sporulation, and virulence of M. robertsii due to changes in cell function and peroxisomes.IMPORTANCEDnm1 was found to be involved in fungal development and virulence, mediated peroxisomal fission, and normal endocytosis. This finding provides new insights into the cellular processes and pathogenicity in entomopathogenic fungi.

DnmA and FisA Mediate Mitochondria and Peroxisome Fission, and Regulate Mitochondrial Function, ROS Production and Development in Aspergillus nidulans.

The dynamin-like protein Drp1 and its receptor Fis-1 are required for mitochondria and peroxisome fission in animal and yeast cells. Here, we show that in the fungus Aspergillus nidulans the lack of Drp1 and Fis-1 homologs DnmA and FisA has strong developmental defects, leading to a notable decrease in hyphal growth and asexual and sexual sporulation, with some of these defects being aggravated or partially remediated by different carbon sources. Although both DnmA and FisA, are essential for mitochondrial fission, participate in peroxisomal division and are fully required for H2O2-induced mitochondrial division, they also appear to play differential functions. Despite their lack of mitochondrial division, ΔdnmA and ΔfisA mutants segregate mitochondria to conidiogenic cells and produce viable conidia that inherit a single mitochondrion. During sexual differentiation, ΔdnmA and ΔfisA mutants develop fruiting bodies (cleistothecia) that differentiate excessive ascogenous tissue and a reduced number of viable ascospores. ΔdnmA and ΔfisA mutants show decreased respiration and notably high levels of mitochondrial reactive oxygen species (ROS), which likely correspond to superoxide. Regardless of this, ΔdnmA mutants can respond to an external H2O2 challenge by re-localizing the MAP kinase-activated protein kinase (MAPKAP) SrkA from the cytoplasm to the nuclei. Our results show that ROS levels regulate mitochondrial dynamics while a lack of mitochondrial fission results in lower respiration, increased mitochondrial ROS and developmental defects, indicating that ROS, mitochondrial division and development are critically interrelated processes.

Recent insights into peroxisome biogenesis and associated diseases.

Peroxisomes are single-membrane organelles present in eukaryotes. The functional importance of peroxisomes in humans is represented by peroxisome-deficient peroxisome biogenesis disorders (PBDs), including Zellweger syndrome. Defects in the genes that encode the 14 peroxins that are required for peroxisomal membrane assembly, matrix protein import and division have been identified in PBDs. A number of recent findings have advanced our understanding of the biology, physiology and consequences of functional defects in peroxisomes. In this Review, we discuss a cooperative cell defense mechanisms against oxidative stress that involves the localization of BAK (also known as BAK1) to peroxisomes, which alters peroxisomal membrane permeability, resulting in the export of catalase, a peroxisomal enzyme. Another important recent finding is the discovery of a nucleoside diphosphate kinase-like protein that has been shown to be essential for how the energy GTP is generated and provided for the fission of peroxisomes. With regard to PBDs, we newly identified a mild mutation, Pex26-F51L that causes only hearing loss. We will also discuss findings from a new PBD model mouse defective in Pex14, which manifested dysregulation of the BDNF-TrkB pathway, an essential signaling pathway in cerebellar morphogenesis. Here, we thus aim to provide a current view of peroxisome biogenesis and the molecular pathogenesis of PBDs.

Muscle Disuse Atrophy Caused by Discord of Intracellular Signaling.

Significance: Regular contractile activity plays a critical role in maintaining skeletal muscle morphological integrity and physiological function. If the muscle is forced to stop contraction, such as during limb immobilization (IM), the IGF/Akt/mTOR signaling pathway that normally stimulates protein synthesis and inhibits proteolysis will be suppressed, whereas the FoxO-controlled catabolic pathways such as ubiquitin-proteolysis and autophagy/mitophagy will be activated and dominate, resulting in muscle fiber atrophy. Recent Advances: Mitochondria occupy a central position in the regulation of both protein synthesis and degradation through several redox-sensitive pathways, including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial fusion and fission proteins, mitophagy, and sirtuins. Prolonged IM downregulates PGC-1α due to AMPK (5'-AMP-activated protein kinase) and FoxO activation, thus decreasing mitochondrial biogenesis and causing oxidative damage. Decrease of mitochondrial inner membrane potential and increase of mitochondrial fission can trigger cascades of mitophagy leading to loss of mitochondrial homeostasis (mitostasis), inflammation, and apoptosis. The phenotypic outcomes of these disorders are compromised muscle function and fiber atrophy. Critical Issues: Given the molecular mechanism of the pathogenesis, it is imperative that the integrity of intracellular signaling be restored to prevent the deterioration. So far, overexpression of PGC-1α via transgene and in vivo DNA transfection has been found to be effective in ameliorating mitostasis and reduces IM-induced muscle atrophy. Nutritional supplementation of select amino acids and phytochemicals also provides mechanistic and practical insights into the prevention of muscle disuse atrophy. Future Directions: In light of the importance of mitochondria in regulating the various critical signaling pathways, future work should focus on exploring new epigenetic strategies to restore mitostasis and redox balance.

Encephalopathy due to mutation in mitochondrial fission factor gene

Mitochondrial fission factor (MFF) is a part of the protein complex that promotes mitochondrial and peroxisome fission. Mitochondria and peroxisome fission are complex processes of vital importance for cell growth and survival. Here, we are presenting a case of 5-year-old boy with spastic diplegic cerebral palsy, global developmental delay, and intellectual disability with a history of perinatal asphyxia and encephalopathy. MRI brain was suggestive of symmetrical hyper-intensities in bilateral thalami. Whole-genome sequencing indicates homozygous MFF gene mutation at exon 3 of c.19_20delAGinsTT; p.Ser7Phe variant.

Novel role of dynamin-related-protein 1 in dynamics of ER-lipid droplets in adipose tissue.

Dynamin-Related-Protein 1 (DRP1) critically regulates mitochondrial and peroxisomal fission in multicellular organisms. However, the impact of DRP1 on other organelles, especially its direct influence on ER functions remains largely unclear. Here, we report that DRP1 translocates to endoplasmic reticulum (ER) in response to β-adrenergic stimulation. To further investigate the function of DRP1 on ER-lipid droplet (LD) dynamics and the metabolic subsequences, we generated an adipose tissue-specific DRP1 knockout model (Adipo-Drp1flx/flx ). We found that the LDs in adipose tissues of Adipo-Drp1flx/flx mice exhibited more unilocular morphology with larger sizes, and formed less multilocular structures upon cold exposure. Mechanistically, we discovered that abnormal LD morphology occurs because newly generated micro-LDs fail to dissociate from the ER due to DRP1 ablation. Conversely, the ER retention of LDs can be rescued by the overexpressed DRP1 in the adipocytes. The alteration of LD dynamics, combined with abnormal mitochondrial and autophagy functions in adipose tissue, ultimately lead to abnormalities in lipid metabolism in Adipo-Drp1flx/flx mice.

Distinct Contributions of the Peroxisome-Mitochondria Fission Machinery During Sexual Development of the Fungus Podospora anserina.

Mitochondria and peroxisomes are organelles whose activity is intimately associated and that play fundamental roles in development. In the model fungus Podospora anserina, peroxisomes and mitochondria are required for different stages of sexual development, and evidence indicates that their activity in this process is interrelated. Additionally, sexual development involves precise regulation of peroxisome assembly and dynamics. Peroxisomes and mitochondria share the proteins mediating their division. The dynamin-related protein Dnm1 (Drp1) along with its membrane receptors, like Fis1, drives this process. Here we demonstrate that peroxisome and mitochondrial fission in P. anserina depends on FIS1 and DNM1. We show that FIS1 and DNM1 elimination affects the dynamics of both organelles throughout sexual development in a developmental stage-dependent manner. Moreover, we discovered that the segregation of peroxisomes, but not mitochondria, is affected upon elimination of FIS1 or DNM1 during the division of somatic hyphae and at two central stages of sexual development—the differentiation of meiocytes (asci) and of meiotic-derived spores (ascospores). Furthermore, we found that FIS1 and DNM1 elimination results in delayed karyogamy and defective ascospore differentiation. Our findings reveal that sexual development relies on complex remodeling of peroxisomes and mitochondria, which is driven by their common fission machinery.

Peroxisomal fission is modulated by the mitochondrial Rho‐GTPases, Miro1 and Miro2

Peroxisomes are essential for a number of cellular functions, including reactive oxygen species metabolism, fatty acid β‐oxidation and lipid synthesis. To ensure optimal functionality, peroxisomal size, shape and number must be dynamically maintained; however, many aspects of how this is regulated remain poorly characterised. Here, we show that the localisation of Miro1 and Miro2—outer mitochondrial membrane proteins essential for mitochondrial trafficking—to peroxisomes is not required for basal peroxisomal distribution and long‐range trafficking, but rather for the maintenance of peroxisomal size and morphology through peroxisomal fission. Mechanistically, this is achieved by Miro negatively regulating Drp1‐dependent fission, a function that is shared with the mitochondria. We further find that the peroxisomal localisation of Miro is regulated by its first GTPase domain and is mediated by an interaction through its transmembrane domain with the peroxisomal‐membrane protein chaperone, Pex19. Our work highlights a shared regulatory role of Miro in maintaining the morphology of both peroxisomes and mitochondria, supporting a crosstalk between peroxisomal and mitochondrial biology.

The novel peroxin Pex37: the Pxmp2 family joins the peroxisomal fission machinery.

Peroxisomes can undergo fission during cell division, followed by their segregation between mother and daughter cells. Despite species-specific variations in the molecular composition of the fission machinery, the central mechanistic factors can be assigned to two groups: the Pex11 family and the dynamin-related protein family. In a recent study, Singh et al. describe the involvement of a member of the Pxmp2-related protein family in peroxisome fission: the novel peroxin Pex37.

Hansenulapolymorpha Pex37 is a peroxisomal membrane protein required for organelle fission and segregation.

Here, we describe a novel peroxin, Pex37, in the yeast Hansenula polymorpha. H. polymorpha Pex37 is a peroxisomal membrane protein, which belongs to a protein family that includes, among others, the Neurospora crassa Woronin body protein Wsc, the human peroxisomal membrane protein PXMP2, the Saccharomyces cerevisiae mitochondrial inner membrane protein Sym1, and its mammalian homologue MPV17. We show that deletion of H. polymorpha PEX37 does not appear to have a significant effect on peroxisome biogenesis or proliferation in cells grown at peroxisome‐inducing growth conditions (methanol). However, the absence of Pex37 results in a reduction in peroxisome numbers and a defect in peroxisome segregation in cells grown at peroxisome‐repressing conditions (glucose). Conversely, overproduction of Pex37 in glucose‐grown cells results in an increase in peroxisome numbers in conjunction with a decrease in their size. The increase in numbers in PEX37‐overexpressing cells depends on the dynamin‐related protein Dnm1. Together our data suggest that Pex37 is involved in peroxisome fission in glucose‐grown cells. Introduction of human PXMP2 in H. polymorpha pex37 cells partially restored the peroxisomal phenotype, indicating that PXMP2 represents a functional homologue of Pex37. H.polymorpha pex37 cells did not show aberrant growth on any of the tested carbon and nitrogen sources that are metabolized by peroxisomal enzymes, suggesting that Pex37 may not fulfill an essential function in transport of these substrates or compounds required for their metabolism across the peroxisomal membrane.

Label-Free Proteomics of the Fetal Pancreas Identifies Deficits in the Peroxisome in Rats with Intrauterine Growth Restriction.

Aim The objective of the present study was to identify differentially expressed proteins (DEPs) in the pancreas of a fetus with intrauterine growth restriction (IUGR) and to investigate the molecular mechanisms leading to adulthood diabetes in IUGR. Methods The IUGR rat model was induced by maternal protein malnutrition. The fetal pancreas was collected at embryonic day 20 (E20). Protein was extracted, pooled, and subjected to label-free quantitative proteomic analysis. Bioinformatics analysis (GO and IPA) was performed to define the pathways and networks associated with DEPs. LC-MS results were confirmed by western blotting and/or quantitative PCR (q-PCR). The principal parameters of oxidative stress-superoxide dismutase (Sod) were determined in blood samples of fetal rats. Results A total of 57 DEPs (27 upregulated, 30 downregulated) were identified with a 1.5-fold change threshold and a p value ≤ 0.05 between the IUGR and the control pancreas. Bioinformatics analysis revealed that these proteins play important roles in peroxisome biogenesis and fission, fatty acid beta-oxidation (FAO), mitotic cell cycle, and histone modification. The peroxin Pex14 was downregulated in the IUGR pancreas as confirmed by western blotting and q-PCR. Pmp70, a peroxisomal membrane protein involved in the transport of fatty acids, was upregulated. Hsd17b4 and Acox1/2, which catalyze different steps of peroxisomal FAO, were dysregulated. Sod plasma concentrations in the IUGR fetus were higher than those in the control, suggesting partial compensation for oxidative stress. Multiple DEPs were related to the regulation of the cell cycle, including reduced Cdk1, Mcm2, and Brd4. The histone acetylation regulators Hdac1/2 were downregulated, whereas Sirt1/3 and acetylated H3K56 were increased in the IUGR fetal pancreas. Conclusion The present study identified DEPs in the fetal pancreas of IUGR rats by proteomic analysis. Downregulation of pancreas peroxins and dysregulation of enzymes involved in peroxisomal FAO may impair the biogenesis and function of the peroxisome and may underlie the development of T2 diabetes mellitus in adult IUGR rats. Disorders of cell cycle regulators may induce cell division arrest and lead to smaller islets. The present data provide new insight into the role of the peroxisome in the development of the pancreas and may be valuable in furthering our understanding of the pathogenesis of IUGR-induced diabetes.

Peroxisome Maintenance Depends on De Novo Peroxisome Formation in Yeast Mutants Defective in Peroxisome Fission and Inheritance.

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes.

Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis.

The yeast dynamin-like protein Vps1 has roles at multiple stages of membrane trafficking including Golgi to vacuole transport, endosomal recycling, endocytosis and in peroxisomal fission. While the majority of the Vps1 amino acid sequence shows a high level of identity with the classical mammalian dynamins, it does not contain a pleckstrin homology domain (PH domain). The Dyn1 PH domain has been shown to bind to lipids with a preference for PI(4,5)P2 and it is considered central to the function of Dyn1 in endocytosis. The lack of a PH domain in Vps1 has raised questions as to whether the protein can function directly in membrane fusion or fission events. Here we demonstrate that the region Insert B, located in a position equivalent to the dynamin PH domain, is able to bind directly to lipids and that mutation of three lysine residues reduces its capacity to interact with lipids, and in particular with PI(4,5)P2. The Vps1 KKK-AAA mutant shows more diffuse staining but does still show some localization to compartments adjacent to vacuoles and to endocytic sites suggesting that other factors are also involved in its recruitment. This mutant selectively blocks endocytosis, but is functional in other processes tested. While mutant Vps1 can localise to endocytic sites, the mutation results in a significant increase in the lifetime of the endocytic reporter Sla2 and a high proportion of defective scission events. Together our data indicate that the lipid binding capacity of the Insert B region of Vps1 contributes to the ability of the protein to associate with membranes and that its capacity to interact with PI(4,5)P2 is important in facilitating endocytic scission.

ER-mitochondria contacts are required for pexophagy in Saccharomyces cerevisiae.

Peroxisomes play important roles in lipid metabolism. Surplus or damaged peroxisomes can be selectively targeted for autophagic degradation, a process termed pexophagy. Maintaining a proper level of pexophagy is critical for cellular homeostasis. Here we found that endoplasmic reticulum (ER)-mitochondria contact sites are necessary for efficient pexophagy. During pexophagy, the peroxisomes destined for degradation are adjacent to the ER-mitochondria encounter structure (ERMES) that mediates formation of ER- mitochondria contacts; disruption of the ERMES results in a severe defect in pexophagy. We show that a mutant form of Mdm34, a component of the ERMES, which impairs ERMES formation and diminishes its association with the peroxisomal membrane protein Pex11, also leads to defects in pexophagy. The dynamin-related GTPase Vps1, which is specific for peroxisomal fission, is recruited to the peroxisomes at ER-mitochondria contacts by the selective autophagy scaffold Atg11 and the pexophagy receptor Atg36, facilitating peroxisome degradation.

Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission

Dynamin-related protein 1 (Drp1) is essential for mitochondrial and peroxisomal fission. Recent studies propose that Drp1 does not sever but rather constricts mitochondrial membranes allowing dynamin 2 (Dnm2) to execute final scission. Here, we report that unlike Drp1, Dnm2 is dispensable for peroxisomal and mitochondrial fission, as these events occurred in Dnm2 knockout cells. Fission events were also observed in mouse embryonic fibroblasts lacking Dnm1, 2 and 3. Using reconstitution experiments on preformed membrane tubes, we show that Drp1 alone both constricts and severs membrane tubes. Scission required the membrane binding, self-assembling and GTPase activities of Drp1 and occurred on tubes up to 250 nm in radius. In contrast, Dnm2 exhibited severely restricted fission capacity with occasional severing of tubes below 50 nm in radius. We conclude that Drp1 has both membrane constricting and severing abilities and is the dominant dynamin performing mitochondrial and peroxisomal fission.

Infection-Induced Peroxisome Biogenesis Is a Metabolic Strategy for Herpesvirus Replication

Viral proteins have evolved to target cellular organelles and usurp their functions for virus replication. Despite the knowledge of these critical functions for several organelles, little is known about peroxisomes during infection. Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism. Here, we discovered that the enveloped viruses human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) induce the biogenesis of and unique morphological changes to peroxisomes to support their replication. Targeted proteomic quantification revealed a global virus-induced upregulation of peroxisomal proteins. Mathematical modeling and microscopy structural analysis show that infection triggers peroxisome growth and fission, leading toincreased peroxisome numbers and irregular disc-like structures. HCMV-induced peroxisome biogenesis increased the phospholipid plasmalogen, thereby enhancing virus production. Peroxisome regulation and dependence were not observed for the non-enveloped adenovirus. Our findings uncover a role of peroxisomes in viral pathogenesis, with likely implications for multiple enveloped viruses.

Clinical and Neuroimaging Spectrum of Peroxisomal Disorders.

Peroxisomes play vital roles in a broad spectrum of cellular metabolic pathways. Defects in genes encoding peroxisomal proteins can result in a wide array of disorders, depending upon the metabolic pathways affected. These disorders can be broadly classified into 2 main groups; peroxisome biogenesis disorders (PBDs) and single peroxisomal enzyme deficiencies. Peroxisomal enzyme deficiencies are result of dysfunction of a specific metabolic pathway, while PBDs are due to generalized peroxisomal dysfunction. Mutations in PEX1 gene are the most common cause of PBDs, accounting for two-thirds of cases. Peroxisomal fission defects is a recently recognized entity, included under the subgroup of PBDs. The aim of this article is to provide a comprehensive review on the clinical and neuroimaging spectrum of peroxisomal disorders.

Steric interference from intrinsically disordered regions controls dynamin-related protein 1 self-assembly during mitochondrial fission

The self-assembling, mechanoenzymatic dynamin superfamily GTPase, dynamin-related protein 1 (Drp1), catalyzes mitochondrial and peroxisomal fission. Distinct intrinsically disordered regions (IDRs) in Drp1 substitute for the canonical pleckstrin homology (PH) domain and proline-rich domain (PRD) of prototypical dynamin, which cooperatively regulate endocytic vesicle scission. Whether the Drp1 IDRs function analogously to the corresponding dynamin domains however remains unknown. We show that an IDR unique to the Drp1 GTPase (G) domain, the ‘extended 80-loop’, albeit dissimilar in location, structure, and mechanism, functions akin to the dynamin PRD by enabling stable Drp1 mitochondrial recruitment and by suppressing Drp1 cooperative GTPase activity in the absence of specific partner-protein interactions. Correspondingly, we find that another IDR, the Drp1 variable domain (VD), in conjunction with the conserved stalk L1N loop, functions akin to the dynamin PH domain; first, in an ‘auto-inhibitory’ capacity that restricts Drp1 activity through a long-range steric inhibition of helical inter-rung G-domain dimerization, and second, as a ‘fulcrum’ for Drp1 self-assembly in the proper helical register. We show that the Drp1 VD is necessary and sufficient for specific Drp1-phospholipid interactions. We further demonstrate that the membrane-dependent VD conformational rearrangement essential for the alleviation of Drp1 auto-inhibition is contingent upon the basal GTP hydrolysis-dependent generation of Drp1 dimers from oligomers in solution. IDRs thus conformationally couple the enzymatic and membrane activities of Drp1 toward membrane fission.

The N-terminal amphipathic helix of Pex11p self-interacts to induce membrane remodelling during peroxisome fission

Pex11p plays a crucial role in peroxisome fission. Previously, it was shown that a conserved N-terminal amphipathic helix in Pex11p, termed Pex11-Amph, was necessary for peroxisomal fission in vivo while in vitro studies revealed that this region alone was sufficient to bring about tubulation of liposomes with a lipid consistency resembling the peroxisomal membrane. However, molecular details of how Pex11-Amph remodels the peroxisomal membrane remain unknown. Here we have combined in silico, in vitro and in vivo approaches to gain insights into the molecular mechanisms underlying Pex11-Amph activity. Using molecular dynamics simulations, we observe that Pex11-Amph peptides form linear aggregates on a model membrane. Furthermore, we identify mutations that disrupted this aggregation in silico, which also abolished the peptide's ability to remodel liposomes in vitro, establishing that Pex11p oligomerisation plays a direct role in membrane remodelling. In vivo studies revealed that these mutations resulted in a strong reduction in Pex11 protein levels, indicating that these residues are important for Pex11p function. Taken together, our data demonstrate the power of combining in silico techniques with experimental approaches to investigate the molecular mechanisms underlying Pex11p-dependent membrane remodelling.