Spontaneous Protein Crowding in Liposomes: A New Vista for the Origin of Cellular Metabolism

Abstract

One question in the origin of life is the time at which membrane compartments came into the picture as hosts for the first forms of metabolism. If we assume the proteins and nucleic acids came first, then it is difficult to conceive how all the macromolecular components could have been entrapped at a later time in a single compartment. On the other hand, the hypothesis that metabolism originated from inside the compartment means that we would then have to conceive semipermeable, sophisticated membranes in prebiotic times, which does not appear plausible. With this study, we believe that we can offer a partial solution to this riddle, at the same time opening a new vista on the principles of the entrapment of solute in vesicles. We used cryo-TEM to study the entrapment of the protein ferritin in liposomes. The novel, surprising principle that appears is that when lipid surfaces close up in a proteincontaining solution to form vesicles, the entrapment frequency does not follow the expected Poisson distribution, but tends to assume a power-law behaviour, characterized by many "empty" vesicles (no or very little entrapped solute), and a long decreasing tail with extremely crowded vesicles. Cryo-TEM analysis shows indeed some extremely crowded liposomes adjacent to empty ones. The conclusion is that membrane closure can accumulate a remarkable number of solutes inside some compartments. The possible mechanism and relevance of this extreme local super-concentration effect for the origin of life are discussed.The spontaneous formation of lipid vesicles (liposomes) in an aqueous phase containing one or more solutes produces a heterogeneous population of liposomes in terms of solute content. Such entrapments have generally been studied by averaging techniques, such as batch absorbance or fluorescence, whereas little attention has been devoted to studying individual encapsulation.[1] This is partly due to the technical difficulty of directly counting molecules inside liposomes. The encapsulation of biomacromolecules inside liposomes, on the other hand, is an important issue in origins of life research (protocell models) as well as in recent studies on synthetic cells. [2] A series of recent experiments within our project on the construction of minimal living cells [3] revealed possible deviations from the number of macromolecules expected to be entrapped inside liposomes of diameter d < 200 nm. In particular, with the aim of producing green fluorescent protein (GFP) inside liposomes, we prepared liposomes in the presence of the transcription-translation macromolecular machinery, namely E. coli extracts as well as PURESYSTEM [4] (a cell-free protein synthesis kit containing 36 purified components, t-RNAs, ribosomes, for a total of about 80 different macromolecules). We showed that GFP was synthesised inside liposomes, despite of the fact that the Poisson probability of liposome co-entrapment of about 80 different macromolecules (each at a concentration of 0.1-1 mm) is vanishingly small (~10 À26). In order to explain the obs...

Quasi-cellular systems: stochastic simulation analysis at nanoscale range

“…In this case, where multiple co-encapsulation events occur, the "anomalous entrapment" phenomenon (seen before for single molecular species [13,19]) acts by creating nanoscale liposomes containing hundreds of different molecules in a very small volume.…”

“…It is more difficult to perform similar studies in submicron vesicles, where it is expected, however, a higher diversity due to the enhancement of stochastic events at a smaller scale. Quite interestingly, two recent studies demonstrated that when 200 nm (diameter) lipid vesicles are prepared in presence of single macromolecular species, like ferritin [13] or ribosomes [14], the solute encapsulation does not follow the expected behavior. It resulted, instead, in a dramatic dichotomy between many "empty" vesicles and few "super-crowded" ones (having an internal solute concentration up to 60 times higher than the expected value).…”

Development of an artificial cell, from self-organization to computation and self-reproduction

“…The internal structure of large multilamellar vesicles, obtained by the freeze-dry method, was studied by quantitative measurements of gene expression (84). The encapsulation of the PURE system inside small unilamellar vesicles of 100 nm in diameter revealed a strong heterogeneity of the entrapment below the micrometer size (85,86). Amphipathic peptides, which insert spontaneously into lipid bilayers, and autonomous pore-forming proteins have proven to work when they are expressed inside synthetic vesicles (87).…”

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

“…In fact, it has been recently discovered that a large heterogeneity exist in a population of vesicles prepared by these methods (Dominak & Keating, 2007;Lohse et al, 2008). A clear-cut evidence of superconcentration of proteins inside lipid vesicles has been reported in the case of ferritin (Luisi et al, 2010).…”

“…What is important is the demonstration that sets of free molecules and lipids spontaneously assemble into a cell-like particle, and that functions -initially not present in the bulk phase, become characteristic of compartmentalized systems. For example, the recently reported discovery (Luisi et al, 2010) of self-concentration of proteins (and ribosomes, Souza et al, submitted manuscript) inside liposomes points to the emergence of functional cells from diluted compounds. In other words, the formation of liposomes implies the formation of concentrated/segregated phase that might exhibit enhanced functionality when compared with the analogous system in bulk (see also Souza et al, 2009).…”