Membrane Remodeling Processes Research Articles

Curvature and Lipid Packing Modulate the Elastic Properties of Lipid Assemblies: Comparing HII and Lamellar Phases.

Accumulating evidence indicates that membrane reshaping and fusion processes, as well as regulation of membrane protein function, depend on lipid composition. Although it is widely accepted that cell membranes are under considerable stress and frustration and can be locally highly curved, experimental approaches to determine the material properties of lipids usually rely on their study in a relaxed environment or in flat bilayers. Here, we propose a computational method to determine the elastic properties of lipid assemblies of arbitrarily shaped interfaces and apply it to lipidic mixtures in the inverted hexagonal and lamellar phases. We find that the bending rigidity critically depends on the geometry of the system and correlates with the changes in lipid chain order imposed by the specific environment. Our results are relevant for resolving local lipid properties of deformed, stressed, or frustrated membranes that notably emerge around integral membrane proteins or during different membrane remodeling processes.

Endophilin-A1 BAR domain interaction with arachidonyl CoA.

Endophilin-A1 belongs to the family of BAR domain containing proteins that catalyze membrane remodeling processes via sensing, inducing and stabilizing membrane curvature. We show that the BAR domain of endophilin-A1 binds arachidonic acid and molds its coenzyme A (CoA) activated form, arachidonyl-CoA into a defined structure. We studied low resolution structures of endophilin-A1-BAR and its complex with arachidonyl-CoA in solution using synchrotron small-angle X-ray scattering (SAXS). The free endophilin-A1-BAR domain is shown to be dimeric at lower concentrations but builds tetramers and higher order complexes with increasing concentrations. Extensive titration SAXS studies revealed that the BAR domain produces a homogenous complex with the lipid micelles. The structural model of the complexes revealed two arachidonyl-CoA micelles bound to the distal arms of an endophilin-A1-BAR dimer. Intriguingly, the radius of the bound micelles significantly decreases compared to that of the free micelles, and this structural result may provide hints on the potential biological relevance of the endophilin-A1-BAR interaction with arachidonyl CoA.

Vps4 Stimulatory Element of the Cofactor Vta1 Contacts the ATPase Vps4 α7 and α9 to Stimulate ATP Hydrolysis

The endosomal sorting complexes required for transport (ESCRTs) function in a variety of membrane remodeling processes including multivesicular body sorting, abscission during cytokinesis, budding of enveloped viruses, and repair of the plasma membrane. Vps4 ATPase activity modulates ESCRT function and is itself modulated by its cofactor Vta1 and its substrate ESCRT-III. The carboxyl-terminal Vta1/SBP-1/Lip5 (VSL) domain of Vta1 binds to the Vps4 β-domain to promote Vps4 oligomerization-dependent ATP hydrolysis. Additionally, the Vps4 stimulatory element (VSE) of Vta1 contributes to enhancing Vps4 oligomer ATP hydrolysis. The VSE is also required for Vta1-dependent stimulation of Vps4 by ESCRT-III subunits. However, the manner by which the Vta1 VSE contributes to Vps4 activation is unknown. Existing structural data were used to generate a model of the Vta1 VSE in complex with Vps4. This model implicated residues within the small ATPase associated with various activities (AAA) domain, specifically α-helices 7 and 9, as relevant contact sites. Rational generation of Vps4 mutants defective for VSE-mediated stimulation, as well as intergenic compensatory mutations, support the validity of this model. These findings have uncovered the Vps4 surface responsible for coordinating ESCRT-III-stimulated Vta1 input during ESCRT function and identified a novel mechanism of Vps4 stimulation.

Direct interaction of actin filaments with F‐BAR protein pacsin2

Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.

Endophilin A1 induces different membrane shapes using a conformational switch that is regulated by phosphorylation.

Membrane remodeling is controlled by proteins that can promote the formation of highly curved spherical or cylindrical membranes. How a protein induces these different types of membrane curvature and how cells regulate this process is still unclear. Endophilin A1 is a protein involved in generating endocytotic necks and vesicles during synaptic endocytosis and can transform large vesicles into lipid tubes or small and highly curved vesicles in vitro. By using EM and electron paramagnetic resonance of endophilin A1, we find that tubes are formed by a close interaction with endophilin A1's BIN/amphiphysin/Rvs (BAR) domain and deep insertion of its amphipathic helices. In contrast, vesicles are predominantly stabilized by the shallow insertion of the amphipathic helical wedges with the BAR domain removed from the membrane. By showing that the mechanism of membrane curvature induction is different for vesiculation and tubulation, these data also explain why previous studies arrived at different conclusions with respect to the importance of scaffolding and wedging in the membrane curvature generation of BAR proteins. The Parkinson disease-associated kinase LRRK2 phosphorylates S75 of endophilin A1, a position located in the acyl chain region on tubes and the aqueous environment on vesicles. We find that the phosphomimetic mutation S75D favors vesicle formation by inhibiting this conformational switch, acting to regulate endophilin A1-mediated curvature. As endophilin A1 is part of a protein superfamily, we expect these mechanisms and their regulation by posttranslational modifications to be a general means for controlling different types of membrane curvature in a wide range of processes in vivo.

Membrane Remodeling Processes Induced by Phospholipase Action

Important cellular events such as division require drastic changes in the shape of the membrane. These remodeling processes can be triggered by the binding of specific proteins or by changes in membrane composition and are linked to phospholipid metabolism for which dedicated enzymes, named phospholipases, are responsible. Here wide-field fluorescence microscopy is used to visualize shape changes induced by the action of phospholipase A1 on dye-labeled supported membranes of POPC (1-palmitoyl-2-oleoly-sn-glycero-3-phosphocholine). Time-lapse imaging demonstrates that layers either shrink and disappear or fold and collapse into vesicles. These vesicles can undergo further transformations such as budding, tubulation, and pearling within 5 min of formation. Using dye-labeled phospholipases, we can monitor the presence of the enzyme at specific positions on the membrane as the shape transformations occur. Furthermore, incorporating the products of hydrolysis into POPC membranes is shown to induce transformations similar to those observed for enzyme action. The results suggest that phospholipase-mediated hydrolysis plays an important role in membrane transformations by altering the membrane composition, and a model is proposed for membrane curvature based on the presence and shape of hydrolysis products.

RNA-Seq-Based Analysis of Cold Shock Response in Thermoanaerobacter tengcongensis, a Bacterium Harboring a Single Cold Shock Protein Encoding Gene

BackgroundAlthough cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. Thermoanaerobacter tengcongensis MB4, a thermophile harboring only one known cold shock protein gene (TtescpC), can survive from 50° to 80°C, but has poor natural competence under cold shock at 50°C. We therefore examined cold shock responses and their effect on natural competence in this bacterium.ResultsThe transcriptomes of T. tengcongensis before and after cold shock were analyzed by RNA-seq and over 1200 differentially expressed genes were successfully identified. These genes were involved in a wide range of biological processes, including modulation of DNA replication, recombination, and repair; energy metabolism; production of cold shock protein; synthesis of branched amino acids and branched-chain fatty acids; and sporulation. RNA-seq analysis also suggested that T. tengcongensis initiates cell wall and membrane remodeling processes, flagellar assembly, and sporulation in response to low temperature. Expression profiles of TtecspC and failed attempts to produce a TtecspC knockout strain confirmed the essential role of TteCspC in the cold shock response, and also suggested a role of this protein in survival at optimum growth temperature. Repression of genes encoding ComEA and ComEC and low energy metabolism levels in cold-shocked cells are the likely basis of poor natural competence at low temperature.ConclusionOur study demonstrated changes in global gene expression under cold shock and identified several candidate genes related to cold shock in T. tengcongensis. At the same time, the relationship between cold shock response and poor natural competence at low temperature was preliminarily elucidated. These findings provide a foundation for future studies on genetic and molecular mechanisms associated with cold shock and acclimation at low temperature.

The free energy of nanopores in tense membranes.

Membrane nanopores are central players for a range of important cellular membrane remodeling processes as well as membrane rupture. Understanding pore formation in tense membranes requires comprehension of the molecular mechanism of pore formation and the associated free energy change as a function of the membrane tension. Here we propose a scheme to calculate the free energy change associated with the formation of a nanometer sized pore in molecular dynamics simulations as a function of membrane tension, which requires the calculation of only one computationally expensive potential of mean force. We show that membrane elastic theory can be used to estimate the pore formation free energy at different tension values from the free energy change in a relaxed membrane and the area expansion curves of the membranes. We have computed the pore formation free energy for a dipalmitoyl-phosphatidylcholine (DPPC) membrane at two different lateral pressure values, 1 bar and -40 bar, by calculating the potential of mean force acting on the head group of a single lipid molecule. Unrestrained simulations of the closing process confirm that the intermediate states along this reaction coordinate are reasonable and show that hydrophilic indentations spanning half the bilayer connected by a hydrophobic pore segment represent the corresponding high energy transition state. A comparison of the stability of simulated membranes to experiment at high loading rates show that, contrary to expectation, pores form too easily in small simulated membrane patches. This discrepancy originates from a combination of the absence of ions in the simulations and the small membrane size.

Abstract A186: Novel target to control lung tumor growth: Disruption of cell membrane remodeling by modulating myoferlin expression.

Abstract Introduction: Lung cancer is the leading cause of cancer death in human. One feature of tumor cells is their ability to rapidly proliferate. These fast dividing tumor cells undergo continuous membrane damage and remodeling cycles. Hence, disruption of membrane remodeling and repair could block tumor growth. Myoferlin has been shown to mediate membrane processes such as receptor trafficking and membrane repair via endocytosis and exocytosis. Loss of myoferlin expression in non-cancer cell lines leads to defects in processes essential for cell proliferation, such as trafficking of membrane receptors (1, 2) and repairing ruptured cell membrane (3). Involvement of myoferlin in multifaceted membrane events shared by cancer pathogenesis suggests that modulating myoferlin expression can block tumorigenic activities. Herein, we investigated whether interfering with normal myoferlin expression, membrane repair and remodeling provides therapeutically relevant antitumor effects. Purpose and Hypothesis: Myoferlin regulates lung tumor growth by mediating cell proliferation and membrane remodeling process. Methods and Results: To investigate the potential expression of myoferlin in tumor, we performed Western blot analysis on a range of mouse and human cancer cell lines and immunohistochemistry on mouse and human lung carcinoma tissues. We found that myoferlin was expressed in various human and mouse cancer cell lines as well as solid tumors. With the use of immunofluorescent assay, we visualized the localization of myoferlin expression around the peri-nuclear region, cytoplasm and Golgi apparatus in cultured mouse Lewis lung carcinoma (LLC) cells. To assess the role of myoferlin in tumor pathogenesis, loss of function studies were performed using a myoferlin silencing RNA (siRNA)-based approach. Briefly, mouse Lewis lung carcinoma (LLC) cells were transfected with myoferlin siRNA and the effects of myoferlin knockdown on tumor cell proliferation and membrane repair were evaluated. A mouse xenograft tumor model was also used to determine the effects of myoferlin knockdown on solid tumor growth. Knockdown of myoferlin caused a 90% decrease in proliferation of mouse LLC cells and disabled membrane resealing after membrane damage. In addition, myoferlin siRNA decreased solid lung tumor growth by 55%, which was attributed to substantial reduction of tumor cell proliferation. Conclusion: Our results identify the anti-proliferative effect associated with attenuated myoferlin expression in lung tumor growth both in vitro and in vivo. This opens up a new therapeutic approach for lung cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A186. Citation Format: Suk Kei Cleo Leung, Carol Yu, Michelle I. Lin, Cristina Tognon, Pascal Bernatchez. Novel target to control lung tumor growth: Disruption of cell membrane remodeling by modulating myoferlin expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A186.

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

Author SummaryRegulated secretion of peptide hormones, such as growth hormone (GH) and insulin, represents a fundamental process in controlling physiological homeostasis. In endocrine cells, hormone-containing vesicles bud from the Golgi apparatus to enable storage and regulated release into the blood stream. Here we show that two proteins with a lipid membrane-shaping BAR domain, PICK1 and ICA69, work together in the pituitary gland and the pancreas to facilitate the budding of early secretory vesicle from the Golgi apparatus. The physiological significance of our findings was borne out by showing that mice and Drosophila flies lacking the PICK1 encoding gene have marked growth retardation. PICK1-deficient mice showed increased fat accumulation, reduced body weight and length, as well as reduced glucose clearance from the blood stream. Consistent with these findings, we observed a severe reduction in GH storage in the pituitary and impaired secretion of both insulin and GH in response to physiological stimuli. Finally, we found that PICK1 expression levels were raised in a fly model of type 2 diabetes and in high-fat-diet-induced obese mice. These results indicate that alteration of PICK1 expression might play a role in pathophysiological processes of metabolic diseases and/or in a protective compensatory mechanism.

A Comparison of Coarse-Grained and Continuum Models for Membrane Bending in Lipid Bilayer Fusion Pores

To establish the validity of continuum mechanics models quantitatively for the analysis of membrane remodeling processes, we compare the shape and energies of the membrane fusion pore predicted by coarse-grained (MARTINI) and continuum mechanics models. The results at these distinct levels of resolution give surprisingly consistent descriptions for the shape of the fusion pore, and the deviation between the continuum and coarse-grained models becomes notable only when the radius of curvature approaches the thickness of a monolayer. Although slow relaxation beyond microseconds is observed in different perturbative simulations, the key structural features (e.g., dimension and shape of the fusion pore near the pore center) are consistent among independent simulations. These observations provide solid support for the use of coarse-grained and continuum models in the analysis of membrane remodeling. The combined coarse-grained and continuum analysis confirms the recent prediction of continuum models that the fusion pore is a metastable structure and that its optimal shape is neither toroidal nor catenoidal. Moreover, our results help reveal a new, to our knowledge, bowing feature in which the bilayers close to the pore axis separate more from one another than those at greater distances from the pore axis; bowing helps reduce the curvature and therefore stabilizes the fusion pore structure. The spread of the bilayer deformations over distances of hundreds of nanometers and the substantial reduction in energy of fusion pore formation provided by this spread indicate that membrane fusion can be enhanced by allowing a larger area of membrane to participate and be deformed.

Mesoscale Simulations of Curvature Inducing Proteins on Wavy Membranes

Curvature inducing cell membrane proteins migrate along mean curvature gradients during cell membrane remodeling processes such as endocytosis and cell motility. The curvature sensing abilities of these membrane proteins are vital for their localization and subsequent recruitment of other cell constituents to the membrane. Recent experiments have demonstrated the redistribution of curvature inducing proteins along a precurved membrane supported by a wavy substrate. These experiments can elucidate the behavior of curvature inducing proteins over a much smaller curvature range than was analyzed in previous experiments of segregation on pulled membrane tethers. Wavy substrates provide a model system for deconvolution of the slight effects of frame tension and pinning. The surface migration of these proteins along a wavy surface has been investigated using a curvilinear membrane model evolved with Monte Carlo techniques based on the Helfrich Hamiltonian. The migration and segregation of membrane proteins is quantified in simulations with free energy methods incorporating Widom ghost particle insertion and thermodynamic integration.

Temporal and Spatial Remodeling of Host Cell Plasma Membrane during HIV Assembly Revealed by Quantitative Superresolution Microscopy

The budding of enveloped retrovirus, such as HIV, involves oligomerization of the structural protein Gag at the plasma membrane and the incorporation of a patch of the host cell plasma membrane in the viral particle. Previous studies indicate that certain host proteins and viral envelope proteins are enriched in the membrane of virus particles. Since the viral membrane is derived from the host membrane, such specialized composition of viral membrane suggests viral assembly and budding involve differentiation of the host cell plasma-membrane at the assembly site. However, the mechanism of the membrane remodeling process during viral assembly is not understood. using a combination of quantitative superresolution microscopy (PALM and STORM) and diffraction-limited TIRF microscopy, we have investigated the spatial and temporal differentiation of the host cell plasma-membrane during budding of HIV. We find that in absence of Gag, the viral envelope protein is randomly distributed in the plasma membrane. However, oligomerization of Gag at the membrane leads to dramatic redistribution of the envelope proteins at the site of Gag assembly. We also see enrichment of GPI-anchored protein such as CD59 at the assembly site, while a model transmembrane protein EGFP-GT46 is actively excluded from the membrane patch. Gag is anchored to the plasma membrane via myristoylated lipid anchor and electrostatic interactions. Interestingly, we find that myristoylated inner leaflet anchored proteins are enriched while proteins with farnesyl and geranyl lipid anchors are depleted at the assembly site. Taken together, our results indicate that the oligomerization of HIV Gag at the plasma-membrane creates a specialized microenvironment, which leads to the differentiation of the local plasma membrane. This provides new insights about the temporal and spatial remodeling of plasma membrane during assembly and budding of retroviruses.

Bin2 Is a Membrane Sculpting N-BAR Protein That Influences Leucocyte Podosomes, Motility and Phagocytosis

Cell motility, adhesion and phagocytosis are controlled by actin and membrane remodelling processes. Bridging integrator-2 (Bin2) also called Breast cancer-associated protein 1 (BRAP1) is a predicted N-BAR domain containing protein with unknown function that is highly expressed in leucocytic cells. In the present study we solved the structure of Bin2 BAR domain and studied its membrane binding and bending properties in vitro and in vivo. Live-cell imaging experiments showed that Bin2 is associated with actin rich structures on the plasma membrane, where it was targeted through its N-BAR domain. Pull-down experiments and immunoprecipitations showed that Bin2 C-terminus bound SH3 domain containing proteins such as Endophilin A2 and α-PIX. siRNA of endogenous protein led to decreased cell migration, increased phagocytosis and reduced podosome density and dynamics. In contrast, overexpression of Bin2 led to decreased phagocytosis and increased podosome density and dynamics. We conclude that Bin2 is a membrane-sculpting protein that influences podosome formation, motility and phagocytosis in leucocytes. Further understanding of this protein may be key to understand the behaviour of leucocytes under physiological and pathological conditions.

Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth

BackgroundDuring nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.ResultsTo investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.ConclusionsThis study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.

Endosomal trafficking in animal cytokinesis

Cytokinesis is the terminal stage of eukaryotic cell division in which the cytoplasm of a dividing cell is partitioned between two daughter cells. In animal cells, this multifaceted cellular process is spatially and temporally regulated and requires dramatic remodeling of the cytoskeleton and plasma membrane. Animal cytokinesis proceeds when the acto-myosin contractile-ring, formed at the equatorial cortex of a dividing cell, advances inward like a 'purse string' and is a major driving-force for the separation of the two daughter cells. In this review, we highlight many of the recent advances in our understanding of the function and mechanisms of action of the endocytic protein machinery that control animal cytokinesis. This includes regulation of endosome delivery and targeting by Rab and ARF GTPases, their effectors FIP3, FIP4 and JIP4, the exocyst and centralsplindlin complexes and phosphoinositides. Roles for endosomal SNAREs, BRUCE and the ESCRT pathway in the membrane remodeling processes that lead to abscission are also discussed.

Endosomal trafficking in animal cytokinesis

Cytokinesis is the terminal stage of eukaryotic cell division in which the cytoplasm of a dividing cell is partitioned between two daughter cells. In animal cells, this multifaceted cellular process is spatially and temporally regulated and requires dramatic remodeling of the cytoskeleton and plasma membrane. Animal cytokinesis proceeds when the acto-myosin contractile-ring, formed at the equatorial cortex of a dividing cell, advances inward like a ‘purse string’ and is a major driving-force for the separation of the two daughter cells. In this review, we highlight many of the recent advances in our understanding of the function and mechanisms of action of the endocytic protein machinery that control animal cytokinesis. This includes regulation of endosome delivery and targeting by Rab and ARF GTPases, their effectors FIP3, FIP4 and JIP4, the exocyst and centralsplindlin complexes and phosphoinositides. Roles for endosomal SNAREs, BRUCE and the ESCRT pathway in the membrane remodeling processes that lead to abscission are also discussed.

Beclin 1-interacting autophagy protein Atg14L targets SNARE-associated protein Snapin to coordinate endocytic trafficking

Autophagy is a highly regulated membrane remodeling process that allows the lysosome-mediated degradation of cytoplasmic entities by sequestrating them in double-membrane autophagosomes. Autophagy is hence highly intertwined with the endocytic trafficking pathway, sharing similar molecular machinery. Atg14L, also known as Beclin 1-associated autophagy-related key regulator (Barkor), directly interacts with Beclin 1 through its coiled-coil domain and enhances phosphatidylinositol 3-phosphate kinase class III (PI3KC3) activity to induce autophagosome membrane nucleation, highlighting its essential role in the early stage of mammalian autophagy. Here, we report a novel function of Atg14L in the endocytic trafficking pathway wherein Atg14L binds to and colocalizes with the fusogenic SNARE effector protein Snapin to facilitate endosome maturation. Atg14L specifically binds to Snapin and this interaction effectively facilitates endosomal maturation without affecting autophagic cargo degradation. Consequently, atg14l knockdown significantly delayed the late stage of endocytic trafficking, as evidenced by the retarded kinetics of internalized surface receptor degradation. This phenotype was effectively complemented by wild-type Atg14L or Beclin 1-binding mutant, but not by its Snapin-binding mutant. Taken together, our study demonstrates that Atg14L functions as a multivalent trafficking effector that regulates endosome maturation as well as autophagosome formation, reflecting the complexity of the crosstalk between autophagic and endocytic vesicle trafficking in higher eukaryotes.

PEX11 proteins attract Mff and hFis1 to coordinate peroxisomal fission

Fission of membrane-bound organelles requires membrane remodeling processes to enable and facilitate the assembly of the scission machinery. Proteins of the PEX11 family were shown to act as membrane elongation factors during peroxisome proliferation. Furthermore, through interaction with fission factors these proteins coordinate progression of membrane scission. Using a biochemical approach, we determined the membrane topology of PEX11γ, one of the three human PEX11 proteins. Analysis of PEX11γ mutants, which localize to peroxisomes, revealed essential domains for membrane elongation including an amphipathic region and regulatory sequences thereof. Through pegylation assays and in vivo studies, we establish that the PEX11γ sequence includes two membrane-anchored domains, which dock an amphipathic region onto the peroxisomal membrane thereby regulating its elongation. The interaction profile of wild-type and mutant PEX11γ and reveals a rearrangement between homo- and heterodimerization and association with fission factors. We also demonstrate the presence of the mitochondrial fission factor Mff on peroxisomes and its interaction with PEX11 proteins. Our data reveal several features of the molecular mechanism of peroxisome proliferation in mammalian cells: (1) PEX11γ is required and acts in coordination with at least one of the other PEX11 proteins to protrude the peroxisomal membrane; (2) PEX11 proteins attract both Mff and human Fis1 (hFis1) to their site of action; and (3) the concerted interaction of PEX11 proteins provides spatiotemporal control for growth and division of peroxisomes.

A Subtle Interplay Between Three Pex11 Proteins Shapes De Novo Formation and Fission of Peroxisomes

The organization of eukaryotic cells into membrane-bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These organelles are formed either through a growth and division process from existing peroxisomes or de novo from the endoplasmic reticulum (ER). Among the proteins involved in the biogenesis of peroxisomes, peroxins, members of the Pex11 protein family participate in peroxisomal membrane alterations. In the yeast Saccharomyces cerevisiae, the Pex11 family consists of three proteins, Pex11p, Pex25p and Pex27p. Here we demonstrate that yeast mutants lacking peroxisomes require the presence of Pex25p to regenerate this organelle de novo. We also provide evidence showing that Pex27p inhibits peroxisomal function and illustrate that Pex25p initiates elongation of the peroxisomal membrane. Our data establish that although structurally conserved each of the three Pex11 protein family members plays a distinct role. While ScPex11p promotes the proliferation of peroxisomes already present in the cell, ScPex25p initiates remodeling at the peroxisomal membrane and ScPex27p acts to counter this activity. In addition, we reveal that ScPex25p acts in concert with Pex3p in the initiation of de novo peroxisome biogenesis from the ER.