The Energy of COPI for Budding Membranes

RESEARCH ARTICLE The Energy of COPI for Budding Membranes Abdou Rachid Thiam1,2*, Frédéric Pincet1,2* 1 Laboratoire de Physique Statistique, Ecole Normale Supérieure de Paris, Université Pierre et Marie Curie, Université Paris Diderot, Centre National de la Recherche Scientifique, 24 rue Lhomond, 75005, Paris, France, 2 Department of Cell Biology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT 06520, United States of America * (ART); (FP) Abstract As a major actor of cellular trafficking, COPI coat proteins assemble on membranes and locally bend them to bud 60 nm-size coated particles. Budding requires the energy of the coat assembly to overcome the one necessary to deform the membrane which primarily depends on the bending modulus and surface tension, γ. Using a COPI-induced oil nanodroplet formation approach, we modulated the budding of nanodroplets using various amounts and types of surfactant. We found a Heaviside-like dependence between the budding efficiency and γ: budding was only dependent on γ and occurred beneath 1.3 mN/m. With the sole contribution of γ to the membrane deformation energy, we assessed that COPI supplies ~1500 kBT for budding particles from membranes, which is consistent with common membrane deformation energies. Our results highlight how a simple remodeling of the composition of membranes could mechanically modulate budding in cells. OPEN ACCESS Citation: Thiam AR, Pincet F (2015) The Energy of COPI for Budding Membranes. PLoS ONE 10(7): e0133757. doi:10.1371/journal.pone.0133757 Editor: Ludger Johannes, Institut Curie, FRANCE Received: February 9, 2015 Accepted: July 1, 2015 Published: July 28, 2015 Copyright: © 2015 Thiam, Pincet. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by International Outgoing Fellowship n° PIOF-GA-2011-299292 to ART. Competing Interests: The authors have declared that no competing interests exist. Introduction Coat proteins, namely Clathrin coats, coat protein complex I (COPI) and II (COPII) perform a critical step of intracellular vesicle trafficking. They respectively form vesicles from the plasma, the Golgi and the endoplasmic reticulum membranes, exhibiting different morphology and mechanical properties. To induce vesicle formation, monomers of the coat protein machineries, called coatomers, assemble on the target membrane and polymerize to locally bud nanometer sized spherical caged-particles of given curvature [1, 2]. This budding process is biochemically and mechanically regulated [3, 4]. Biochemical regulation is inherent to local variation of one or several components of the coat protein machineries [4]. Mechanical regulation occurs by variations of the bending modulus κ, e.g. by remodeling of membrane composition, and the surface tension γ, e.g. by changing the membrane surfactant density [5, 6]. These mechanical parameters define the minimal energy for budding off a particle of radius r, E = 8πκ+4πγr2, the sum of the bending and stretching energies. This minimal energy which is presumably different for each organelle membrane has to be met by the polymerization energy of the coatomers, E
, to form spherical coats enclosing the particles. Knowing E
for each coat protein machinery will bring important and new knowledge on biochemical and biophysical regulation of cellular trafficking. Previous theoretical attempts based on the comparison PLOS ONE | DOI:10.1371/journal.pone.0133757 July 28, 2015 1 / 11 The Energy of COPI for Budding Membranes between the bending energy of bilayers and the elasticity of dilation of bilayer-bound coat proteins [7, 8] suggest E
to be of the order of 2000 kBT. Of the three coat proteins, only COPI was shown to act in vivo on both phospholipid bilayers and monolayers, namely on the Golgi apparatus and lipid droplets which are organelles at the core of cellular energy metabolism [4, 9–11]. Because the Golgi has a very low surface tension γ (<<1 mN/m) [12], deforming its membrane is almost solely dependent on the bending modulus[13, 14] κ, ~20 kBT, whose contribution to E is predominant. In contrast to the Golgi, lipid droplets are covered by a single phospholipid monolayer membrane. The surface tension of this type of membrane was determined for triolein emulsion droplets to be between 1 to 40 mN/m [5, 15], much higher than that of the Golgi bilayer. Hence, for lipid droplets, the contribution of γ becomes very important for the membrane deformation energy [5, 16]. The ability of COPI to bud nanoparticles from a monolayer or bilayer membrane can be predicted knowing E
COPI, the energy supplied by the polymerization of COPI coatomers. Measuring E
COPI in cells is experimentally challenging because the mechanical parameters are not controlled, membranes are dynamic systems and other proteins may interfere with them, and finally visualization of the coat formation is difficult. So far, various in vitro approaches, based on unilamellar vesicles [14, 17–19], or cell membrane extracts [20, 21], were used to exclusively study the ability of coat proteins to form vesicles. These approaches probed the biochemical triggering of budding and well described the molecular details of coatomer assembly mechanisms. The description of the energy landscape of the budding process is however still lacking because of the challenge to concomitantly visualize budded coat-vesicles with controlled membrane parameters. Using a recently developed COPI-induced oil nanodroplet formation approach [5], we worked with different amounts and types of surfactant in the oil, to vary membrane mechanical properties, and studied how they influence nanodroplets budding. We found that the efficiency of the budding reaction depends on the surfactant type. However, a direct Heaviside-like dependence between the budding efficiency and γ was found, independently of the surfactant. Budding was mainly opposed by γ and occurred only beneath 1.3 mN/m. This simple dependency upon γ was expected for the emulsion monolayer membrane in our experiment because it was presumed that other mechanical terms have a minor contribution to budding. Hence, we used the sole contribution of the stretching energy due to γ to determine that COPI supplies an energy of ~1500 kBT to bud membranes. Materials and Methods Preparation of the solutions Phospholipids (PLs) and the triolein solution: we chose a lipid composition close to that of cellular natural lipid droplets [5]: Dioleoylphosphatidylcholine, Dioleoylphosphatidylethanolamine, Cholesterol, Lyso-Phosphatidylinositol, Lyso-Phosphatidylethanolamine, LysoPhosphatidylcholine (50:20:12:10:5:3). All phospholipids were purchased from Ava (...truncated)