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NUTRIENT UPTAKE BY PROTOCELLS: A LIPOSOME MODEL SYSTEM PIERRE-ALAIN MONNARD∗ and DAVID W. DEAMER Department of Chemistry and Biochemistry, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 96064, U.S.A. (∗ Author for correspondence) (Received 2 November, 1999) Abstract. Over the past decade, several liposome-based models for protocells have been developed. For example, liposome systems composed of polymerase enzymes encapsulated with their substrates have demonstrated that complex compartmentalized reactions can be carried out under conditions in which polymeric products are protected from degradation by hydrolytic enzymes present in the external medium. However, such systems do not have nutrient uptake mechanisms, which would be essential for primitive cells lacking the highly evolved nutrient transport processes present in all contemporary cells. In this report, we explore passive diffusion of solutes across lipid bilayers as one possible uptake mechanism. We have established conditions under which ionic substrates as large as ATP can permeate bilayers at rates capable of supplying an encapsulated template-dependent RNA polymerase. Furthermore, while allowing the permeation of monomer substrates such as ATP, bilayer vesicles selectively retained polymerization products as small as dimers and as large as a transfer RNA. These observations demonstrate that passive diffusion could be used by the earliest forms of cellular life for transport of important nutrients such as amino acids, phosphate, and phosphorylated organic solutes. Keywords: liposome, membrane, nucleotide triphosphates, passive diffusion, protocell 1. Introduction The pathway by which prebiotic molecular systems undergoing chemical evolution assembled into the earliest forms of cellular life remains an unsolved problem. The general assumption is that, some 3.8 billion years ago, simple self-replicating systems of polymeric molecules appeared spontaneously on the early Earth. It is also assumed that the polymerization processes by which growth and replication occurred required a source of chemical energy, were catalyzed in some way, and were directed by a primitive genetic system. At some point the polymerization reactions began to take place in membrane-bounded compartments (Deamer and Oro, 1980; Deamer, 1997). Compartmentalization is a necessary prerequisite for maintaining the integrity of such interdependent molecular systems and for permitting the variations required for speciation (Tawfik and Griffiths, 1998). The components of the boundary structures defining the earliest cellular compartments are unknown. However, the availability of self-assembling amphiphilic molecules on the early Earth has been Origins of Life and Evolution of the Biosphere 31: 147–155, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 148 P.-A. MONNARD AND D. W. DEAMER established, produced either by abiotic synthesis, (Hargreaves et al., 1977) or by delivery of extraterrestrial organic material during late accretion (Deamer, 1985; Deamer and Pashley, 1989). Furthermore, the fact that all contemporary living cells are defined by lipid-bilayer membranes suggests that protocellular structures could be modeled in the laboratory by dispersing lipid molecules as bilayer vesicles in aqueous phases (Deamer and Oro, 1980; Lazcano, 1994a, b; Luisi et al., 1999). However, the use of lipid bilayers for this purpose has a significant drawback, which is their relative impermeability to polar or ionic molecules (Deamer et al., 1994; Mouritsen et al., 1995). To overcome this permeability barrier, complex protein transport systems have evolved in all contemporary cells (Aidley and Stanfield, 1996) which were presumably absent in early cells. Therefore, the latter necessarily relied on simple transport mechanisms such as passive diffusion across bilayer membranes to take up nutrients and sources of chemical energy from the environment. At the same time, early membranes must have provided a selective permeability barrier that permitted permeation of monomers while retaining polymeric products of metabolism. Otherwise newly synthesized substances would diffuse into the surrounding bulk phase and the potential for interactive systems and speciation would be lost (Deamer et al., 1994). The permeability properties of bilayers have been extensively investigated for cations, anions (Kanehisa and Tsong, 1978; Rosenquist et al., 1981; Deamer and Bramhall, 1986; Paula et al., 1996) and small molecules (Langner and Hui, 1993). Permeability has been shown to depend on the gel-fluid phase transition (Mouritsen et al., 1995), the length of the lipid hydrocarbon chains (Paula et al., 1996), and on the nature of the solutes, especially size and charge density. Solute molecules such as water, glycerol, urea and glucose can cross the membrane barrier either by partitioning and diffusing in the bilayers (Brunner et al., 1980)) or by using transient defects resulting from disturbances in the lipid packing order (Paula et al., 1996) whose frequency increases at the lipid phase transition temperature Tm (Kanehisa and Tsong, 1978; Mouritsen et al., 1995). What solutes might have been important to early cellular life? Several obvious examples include phosphate, simple carbohydrates like glyceraldehyde, and perhaps amino acids. Of these, phosphate and amino acids are ionic, and bilayer membranes have been shown to represent significant permeability barriers to their free diffusion (Chakrabarti and Deamer, 1992). In the work reported here, we will show that under certain conditions molecules as large as nucleoside triphosphates can permeate across bilayers composed of an intermediate-chain length lipid (DMPC). Furthermore, the selectivity of these membranes is very high: oligonucleotides as small as dimers are unable to permeate under the same conditions. NUTRIENT UPTAKE BY PROTOCELLS: A LIPOSOME MODEL SYSTEM 149 2. Materials and Methods 2.1. R EAGENTS 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids. ATP, adenyl(30 → 50 )uridine 30 -monophosphate (AU), adenyl(30 → 50 )-cytidyl (30 → 50 )-cytidine (ACC), myristic acid (MA), and sodium cholate were purchased from Sigma Chemical Co (St. Louis, MO). tRNA (from wheat germ) was obtained from Fluka (Buchs, Switzerland). BioGel 15 A (mesh 200–400) was purchased by Bio-Rad. Other chemicals used were of the highest grade commercially available. 2.2. L IPOSOME PREPARATIONS . Pure lipids or lipid mixtures were prepared by dissolving the lipids in CHCl3 or MeOH/CHCl3 (9:1) followed by evaporation to form thin films on the interior surface of the glass vessel. Multilamellar liposomes were formed by swelling the film with 40 mM Tris, pH 8.0 (or pH 8.75 when MA was present), and 1.75 mM MgCl2 to obtain a total concentration of 160 mM lipid. In permeation experiments monitoring efflux of encapsulated solutes, the permeant solute was also present at this step. The suspensions were dried under N2 -flow, and carefully rehydrated, first using a dampened fabric above the film at 37 ◦ C for 20 min, followed by deionized water addition to obtain a final lipid concentration of 160 mM. These suspensions were then extruded ten times through 200-nm pore size polycarbonate filters (LiposoFastTM , Avestin, Inc.). 2.3. P ERMEABILITY MEASUREMENTS : U PTAKE OF EXTERNALLY ADDED SOLUTES Permeability measurements were usually performed at 23.3 ◦ C with 33 mM pure DMPC, or DMPC/MA mixed liposomes, or at 32 ◦ C for those composed of DMPC/DPPC. The solutes were added from concentrated stock solutions to produce the following final concentrations: 7 or 40 mM ATP (from a 200-mM stock solution, pH 8), 1 mg mL−1 AU or ACC, or 11.55 mg mL−1 tRNA. Samples were taken after incubation times up to 16 hr at specified temperatures. External solutes that were not encapsulated were removed on a spin column (Bio-gel A 15 m, mesh 200– 400). Over 99.5% of non-entrapped nucleotides or tRNA can be removed by this procedure (Chonn et al., 1991; Monnard et al., 1997) at 12–16 ◦ C. Encapsulation efficiency was determined by UV absorption at 260 nm after solubilization of the liposomes with cholate (molar ratio of cholate to surfactant was always higher than 1.8). 150 P.-A. MONNARD AND D. W. DEAMER Figure 1. Permeability of DMPC liposomes to ATP and tRNA at phase transition temperature (23.3 ◦ C): Uptake and release (insert). #: 7 mM ATP; : 11.55 mg mL−1 tRNA. 2.4. P ERMEABILITY MEASUREMENTS : E FFLUX OF ENCAPSULATED SOLUTES Liposome preparations containing trapped solutes such as ATP were incubated at 23.3 or 32 ◦ C (DMPC/DPPC (1:1) mixed liposomes) for up to 16 hr. Released solute molecules were separated on a spin column at 12–16 ◦ C, and solute that remained entrapped was spectroscopically determined at 260 nm after vesicle solubilization with cholate as described above. 3. Results The permeability measurements of nucleoside triphosphates were conducted using only ATP at the transition temperature of each liposome suspension (as established in earlier studies by anisotropy measurements with diphenylhexatriene – data not shown). Because ionic groups on solutes are by far the major factor governing permeability, we assume that the permeation rates of all nucleotides are similar. In Figure 1, the release (insert) and uptake of ATP in 200-nm liposome preparations are shown. After 16 hr incubation at 23.3 ◦ C, the concentration of ATP had clearly equilibrated across the membrane with a half-time of 33 min, and 3.5% (about 245 nmol) of the initial externally added ATP eluted with the liposomes. In control experiments, where the free ATP was separated directly after addition of the solute (that is, without incubation), less than 0.5% of ATP eluted with the vesicles. In other experiments, the partition coefficient (K) of ATP in octanol was measured. The value of 8.4 × 10−5 suggests that solute molecules rarely partition into the DMPC bilayers. These results demonstrated that measurable amounts of ATP NUTRIENT UPTAKE BY PROTOCELLS: A LIPOSOME MODEL SYSTEM 151 TABLE I Permeability coefficients calculated from uptake data with 7 mM ATP as solute Liposome composition DMPC DMPC DMPC:DPPC (50:50) DMPC:MA (95:5) DMPC:MA (89:11)c DMPC:MA (80:20)c Temperature Half-time k × 10−4 (◦ C) (min) (1 s−1 )a Permeability coefficient (P) × 10−9 (cm s−1 )b 23.3 37.0 32.0 23.3 23.3 23.3 33.3 53.7 36.6 10.8 4.1 18.6 3.90 0.03 0.55 4.63 35.90 5.69 1.3 0.01 0.2 1.6 12.0 1.9 a k-values were obtained by linear regression on a plot ln(([ATP] at equilibrium – [ATP]t )/(([ATP]at equilibrium) versus t (s). b P values were calculated assuming a monodisperse size distribution of unilamellar 200-nm extruded liposome according to P = (k r)/3. c At pH 8.75. diffuse across bilayers at the phase transition temperature (Tm) by the transient defect model (Mouritsen et al., 1995; Paula et al., 1996). The permeability coefficients (see Table I) calculated from these data were independent of the solute gradient (7 and 40 mM ATP) across the bilayers. As expected, the flux and the total amount of encapsulated ATP increased as the solute gradient increased (data not shown). Furthermore, magnesium present in the buffer markedly inhibited ATP permeation when the ratio of Mg2+ to ATP was 1:3.5 or greater, which indicates that ATP molecules do not permeate if they have bound a magnesium ion. This observation is not consistent with earlier studies carried out with mixed lipid (lecithin based) planar membranes in the presence of large magnesium concentrations (500 mM), or small SUVs of mixed lecithin/cholesterol (Petkau and Chelack, 1972; Stillwell and Winter, 1974). In these studies, the increase of ATP permeability was found to be related to the presence of divalent cations in the solutions. However, these bilayers contained lipids with charged headgroups that could have directly interacted with metal ions, thereby causing disruption of membrane, so that the results are not directly comparable. The permeability of DMPC bilayers at the phase transition temperature could be only useful if it is sufficiently selective to permit monomer permeation yet prevent the release of larger oligomers produced in the liposomes. To check this critical aspect, the permeation rates of AU (1 mg mL−1 ), ACC (1 mg mL−1 ), and tRNA (11.55 mg mL−1 ) were investigated. Under the same conditions used to demonstrate ATP permeation, tRNA data showed no significant uptake (see Figure 1) or release (see insert Figure 1). Furthermore, no uptake or release could be detected 152 P.-A. MONNARD AND D. W. DEAMER using either the dinucleotide (AU), or the trinucleotide (AAC). The absence of released tRNA after 16 hr incubation also demonstrated that DMPC bilayers were stable even in the presence of large amounts of encapsulated polynucleotides. It should be noted that no hydrolysis of RNA molecules could be detected during the incubation period. The temperature dependence of bilayer permeability to ATP was determined at two critical temperatures: The bilayer phase transition temperature (Tm = 23.3 ◦ C) and the optimal temperature for typical enzyme catalyzed reactions (37 ◦ C). The liposomes exhibited a decrease of permeability to ATP permeability exceeding 2 orders of magnitude when the temperature was increased from Tm to 37 ◦ C (see Table I) This result is consistent with the suggestion that the passive diffusion of ATP occurs through transient defects in the bilayer that are most numerous at Tm. 3.1. P ERMEABILITY OF MIXED LIPID SYSTEMS It is probable that lipid-like molecules were available only in the form of mixtures in the prebiotic environment. Furthermore, mixed lipid systems would have markedly different permeability properties than the pure DMPC used in the experiments described above. Therefore we also tested lipid compositions in which a single chain lipid – myristic acid (MA) – or a second phosphatidylcholine derivative (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) was added. As shown in Table I, the permeability coefficient of mixed DMPC/DPPC (1:1) systems was significantly decreased, presumably because of the higher Tm of the DPPC (41 ◦ C). In the presence of MA – at molar ratios where no changes in the morphology of the liposomes were observed – the permeability coefficient first increased as the molar ratio of MA to DMPC increased to approximately 11 mol %, then decreased to a value similar to that of pure DMPC liposomes. 4. Discussion The passive diffusion of NDPs across the bilayers of membranes composed of intermediate-chain length lipids (DMPC) or fatty acids (oleic acid/oleate) has been already established (Chakrabarti et al., 1994; Walde et al., 1994). For instance, in earlier work from our laboratory, the half-time for ADP entry into DMPC liposomes was approximately 2 hr. (The temperature was not maintained at the Tm of 23.3 ◦ C, so the half-time was longer than reported here for ATP.) In oleic acid/oleate systems, available data show that about 4% of externally added ADP permeated after 30 hr incubation at 25 ◦ C. Again, the longer half-life reflects the fact that the lipid was 18 carbon chain length, rather than the 14 carbon chains of DMPC used here. The present study extends these results to NTPs bearing an additional negative charge. We found that ATP can diffuse across bilayers of pure DMPC or DMPC/MA mixtures, and that the rate was greatest at Tm, suggesting NUTRIENT UPTAKE BY PROTOCELLS: A LIPOSOME MODEL SYSTEM 153 that the mechanism was transient defects in the bilayer rather solubility-diffusion (Paula et al., 1996). It follows that transcription of DNA templates by a templatedirected enzyme, such as T7 RNA polymerase, should be possible with the substrates being supplied in the external bulk medium. These are currently underway in our laboratory and will be reported elsewhere. In contrast to enzyme-catalyzed reactions with limited amounts of entrapped substrates (Oberholzer et al., 1995a, b), the substrate permeation rates described here would permit catalyzed reactions to continue as long as the enzymes retain catalytic activity. The possibility that continuous polymerization, replication and transcription reactions might be achieved in liposome model systems lends plausibility to the idea that passive diffusion across lipid bilayers would allow early cells to have access to ionic nutrients in the environment. It remains uncertain whether the observed permeation rates could sustain the activity of an enzyme with a relatively high turnover rate, and we are now testing this possibility. The ATP permeability coefficients (calculated here under the assumption of monodisperse and unilamellar 200-nm liposomes) mean that 5 ATP molecules per s entered each pure DMPC liposome at Tm. With mixed DMPC/MA (89:11) liposomes, approximately 60 molecules should cross the bilayer each second, a value that approaches the turnover of NTPs by a single entrapped T7 RNA polymerase molecule. Permeation of ionized substrates through transient defects at the Tm of pure lipids provides a useful laboratory model, but early cells must have relied on a more general mechanism to ensure nutrient uptake. The simplest such permeation process also incorporates the idea of transient defects, but uses shorter chains and lipid mixtures to increase their frequency, rather than Tm. For instance, DMPC was used here for experimental simplicity, and it is obviously implausible the early cells would have membranes composed of a pure lipid with a phase transition temperature. More likely is that their membranes were composed of mixtures of relatively short chain lipid-like amphiphiles that happened to have a sufficiently high passive permeability to be able to support internal metabolic activity with external nutrients. For instance, in the experiments reported here, mixed DMPC/MA liposomes containing a significant amount of single-chain fatty acids (11 mol %) displayed markedly enhanced permeability toward NTPs. Therefore, membrane-forming compounds such as fatty acids with simpler chemical structures than DMPC, could be used to increase bilayer permeability. Single chain lipids also answer two major concerns about a protocell membrane consisting of long-chain phosphatidylcholines. First, the longest chains reported for meteoritic monocarboxylic acids so far are octanoic and nonanoic acid (Lawless and Yuen, 1979), and these would need to condense with a linker species (glycerol, phosphate) to form a phosphatidyl molecule. Second, a source of phosphate groups for phospholipid synthesis is not at all clear (Deamer et al., 1994). We are therefore undertaking investigations of mixed fatty acid/fatty alcohol vesicles with shorter chains (8–12 carbon single 154 P.-A. MONNARD AND D. W. DEAMER chain amphiphiles), and initial results indicate that such vesicles readily encapsulate macromolecules like nucleic acids and enzymes (C. Apel, unpublished). An important result of the present study is related to the selectivity of the passive diffusion. Although ATP is permeable under the conditions described here, DMPC bilayers exhibited a surprisingly high barrier to larger solutes. For example, even the shortest oligomer, a dimer, remained encapsulated with no measurable loss over periods of many hours. It follows that if longer nucleotide oligomers are produced within the aqueous compartment of a liposome, they would remain entrapped. This is an essential prerequisite for a protocell which would then be able to gradually increase molecular complexity in the interior volume, while releasing metabolic end products. The indefinite encapsulation of tRNA even at high solute concentrations (466 µM assuming a tRNA length of 75 nt) demonstrates that a lipid bilayer could have withstood the osmotic pressure related to captured macromolecules and associated metabolism, thereby preventing the osmotic disruption of protocells suggested by some authors (Hartman, 1995). Furthermore, experiments, such as the entrapped PCR reaction by Oberholzer et al. (1995a), show that lipid bilayers alone can preserve the integrity of a complex enzymatic system, even though they are exposed to wide temperature variations. Therefore, liposomes seem to be a suitable model for a protocell during early evolution leading to cellular life. Acknowledgement This work was supported by NASA grant NAG5-4665. References Aidley, D. J. and Stanfield, P. R.: 1996, Book Ion Channels. Molecules in Action, Cambridge University Press, New York. Brunner, J., Graham, D. E., Hauser, H. and Semenza, G.: 1980, Ion and Sugar Permeabilities of Lecithin Bilayers: Comparion of Curved and Planar Bilayers, J. Membrane Biol. 57, 133–141. Chakrabarti, A. C., Breaker, R. R., Joyce, G. F. and Deamer, D. W.: 1994, Production of RNA by Polymerase Protein Encapsulated Within Phospholipid Versicles, J. Mol. Evol. 39, 555–559. Chakrabarti, A. C. and Deamer, D. W.: 1992, Permeability of Lipid Bilayer to Amino Acids and Phosphate, Biochim. Biophys. Acta 1111, 171–177. Chonn, A., Semple, S. C. and Cullis, P. R.: 1991, Separation of Large Unilamellar Liposomes from Blood Components by a Spin Column Procedure: Toward Identifying Plasma Proteins which Mediate Liposomes Clearance in vivo, Biochim. Biophys. Acta 1070, 215–222. Deamer, D. W.: 1985, Boundary Structures are Formed by Organic Components of the Murchison Carbonaceous Chondrites, Nature 317, 792–794. Deamer, D. W.: 1997, The First Living Systems: A Bioenergetic Perspective, Microbiol. Mol. Biol. Rev. 61, 230–261. Deamer, D. W. and Bramhall, J.: 1986, Permeability of Lipid Bilayers to Water and Ionic Solutes, Chem. Phys. Lipids 40, 167–188. NUTRIENT UPTAKE BY PROTOCELLS: A LIPOSOME MODEL SYSTEM 155 Deamer, D. W., Mahon, E. H. and Bosco, G.: 1994, Self-Assembly and Function of Primitive Membrane Structures, in S. Bengtson (ed.), Early Life on Earth, Columbia University Press, New York, Chichester, West Sussex, Nobel Symposium No. 84, pp. 107–123. Deamer, D. W. and Oro, J.: 1980, Role of Lipids in Prebiotic Stuctures, Biosystems 12, 167–175. Deamer, D. W. and Pasley, R. M.: 1989, Amphiphilic Components of the Murchison Carbonaceous Chondrite: Surface Properties and Membrane Formation, Origins of Life and Evolution of the Biosphere 19, 21–38. Hargreaves, W. R., Mulvihill, S. J. and Deamer, D. W.: 1977, Synthesis of Phophosplipids and Membranes in Prebiotic Condition, Nature 266, 78–80. Hartman, H.: 1995, Speculations on the Origin of the Genetic Code, J. Mol. Evol. 40, 541–544. Kanehisa, M. I. and Tsong, T. Y.: 1978, Cluster Model of Lipid Phase Transition with Application to Passive Permeation of Molecules and Structure Relaxations in Lipid Bilayers, J. Am. Chem. Soc. 100, 424–432. Langner, M. and Hui, S. W.: 1993, Dithionite Penetration through Phospholipid Bilayers as a Measure of Defects in Lipid Molecular Packing, Chem. Phys. Lipids 65, 23–30. Lawless, J. G. and Yuen, G. U.: 1979, Quantitation of Monocarboxylic Acids in the Murchison Carbonaceous Meteorite, Nature 282, 431–454. Lazcano, A.: 1994a, The Transition from Nonliving to Living, in S. Bengtson (ed.), Early Life on Earth, Columbia University Press, New York, Chichester, West Sussex, Nobel Symposium No. 84, pp. 60–69. Lazcano, A.: 1994b, The RNA World, its Precessors, and its Descendants, in S. Bengtson (ed.), Early Life on Earth, Columbia University Press, New York, Chichester, West Sussex, Nobel Symposium No. 84, pp. 70–80. Luisi, P. L., Walde, P. and Oberholzer, T.: 1999, Lipid Vesicles as Possible Intermediates in the Origin of Life, Current Opinion in Colloid and Interface Science 4, 33. Monnard, P.-A., Oberholzer, T. and Luisi, P. L.: 1997, Encapsulation of Polynucleotides in Liposomes, Biochim. Biophys. Acta 1329, 39–50. Mouritsen, O. G., Jorgenseb, K. and Honger, T.: 1995, Permeability of Lipid Bilayers Near the Phase Transition, in E. A. Disalvo and S. A. Simon (eds.), Permeability and Stability of Lipid Bilayers, CRC Press, Boca Raton, 1, pp. 137–160. Oberholzer, T., Albrizio, M. and Luisi, P. L.: 1995a, Polymerase Chain Reaction in Liposomes, Chemistry and Biology 2, 677–682. Oberholzer, T., Wick, R., Luisi, P. L. and Biebricher, C. K.: 1995b, Enzymatic RNA Replication in Self-Reproducing Vesicles: An Approach to a Minimal Cell, Biochim. Biophys. Res. Commun. 207, 250–257. Paula, S., Volkov, A. G., Van Hoek, A. N., Haines, T. H. and Deamer, D. W.: 1996, Permeation of Proton, Potassium Ions, and Small Polar Molecules through Phospholipid Bilayers as a Function of Membrane Thickness, Biophys. J. 70, 339–348. Petkau, A. and Chelack, W. S.: 1972, Can. J. Biochem. 50, 615. Rosenquist, K., Gabran, T. and Rydhag, L.: 1981, Studies of Permeability Across Bilayers of Lecithin, Lipidforum, Scandinavian forum for lipid research and technology, Bergen, Norway, 1, pp. 85– 89. Stillwell, W. and Winter, H. C.: 1974, Biochem. Biophys. Res. Commun. 56, 617. Tawfik, D. S. and Griffiths, A. D.: 1998, Man-Made Cell-like Compartments for Molecular Evolution, Nature Biochtechnology 16, 652–656. Torchilin, V. P., Klibanov, A. L., Huang, L., O’Donell, S., Nossif, N. D. and Khaw, B. A.: 1992, FASEB J. 6, 2716. Walde, P., Goto, A., Monnard, P.-A., Wessicken, M. and Luisi, P. L.: 1994, Oparin’s Reaction Revisited: Enzymatic Synthesis of Poly(adenyl acid) in Micelles and Self-Reproducing Vesicles, J. Am. Chem. Soc. 116, 7541–7547.