Search for the most 'primitive' membranes and their reinforcers: a review of the polyprenyl phosphates theory.

Terpenoids have an essential function in present-day cellular membranes, either as membrane reinforcers in Eucarya and Bacteria or as principal membrane constituents in Archaea. We have shown that some terpenoids, such as cholesterol and α, ω-dipolar carotenoids reinforce lipid membranes by measuring the water permeability of unilamellar vesicles. It was possible to arrange the known membrane terpenoids in a 'phylogenetic' sequence, and a retrograde analysis led us to conceive that single-chain polyprenyl phosphates might have been 'primitive' membrane constituents. By using an optical microscopy, we have observed that polyprenyl phosphates containing 15 to 30 C-atoms form giant vesicles in water in a wide pH range. The addition of 10 % molar of some polyprenols to polyprenyl phosphate vesicles have been shown to reduce the water permeability of membranes even more efficiently than the equimolecular addition of cholesterol. A 'prebiotic' synthesis of C10 and C15 prenols from C5 monoprenols was achieved in the presence of a montmorillonite clay. Hypothetical pathway from C1 or C2 units to 'primitive' membranes and that from 'primitive' membranes to archaeal lipids are presented.

Introduction

Life is cellular, and a boundary separates the living organism from the outside world. Yet most discussions on the molecular origins of life center around questions such as how amino acids, sugars and nucleotides were formed in interstellar space or on Earth, how proteins originated and how nucleic acids arose and became able to self-replicate. Here we consider a fourth question: how did membranes originate? (Deamer 1986; Morowitz et al. 1988).

Understanding the origin of membranes is central to an understanding of the origin of life. Life may have begun as a two-dimensional system limited by a mineral surface, but in a three-dimensional system in water, segregation of a separate compartment is achieved by a closed lipidic membrane or vesicle. Lipid bilayers have very low permeability to ions, polar solutes and biopolymers, and thus define what is inside as the living organism, what is around as the membrane, and what is outside as the rest of the world. This article will focus on the early formation and evolution of membranes.

On the way of deducing the origins of membrane constituents, it is obvious to consider first how contemporary cell membranes are built. A high schematic representation - ignoring all the complexity of plasma membranes - is shown in Fig. 1 (Left). Cell membranes are composed of basically two main classes of molecules, lipids and proteins, independent on whether the cells belong to one of the three domains of living organisms: Eucarya, Bacteria and Archaea (Woese et al. 1990; Yamagishi et al. 1998). Eucaryotic membranes are formed by the self-assembly of amphiphilic di-acyl phospholipids, containing sterols like cholesterol. Bacterial membranes rarely contain cholesterol, but often contain hopanoids (Ourisson and Albrecht 1992; Ourisson and Rohmer 1992) or carotenoids (Liaane-Jensen 1979) (Fig. 2 Right). Archaeal membranes lack n-acyl lipids and instead consist of polyprenic diphytanylglyceryl or bis(diphytanylglyceryl) phospholipids (Fig. 3). Cholesterol, hopanoids, carotenoids, phytane and bisphytane are belonging to terpenoids (isoprenoids), which represent the most diverse family of natural products. These observations suggest that terpenoids are important for membrane function, either as membrane reinforcers in Eucarya and Bacteria or as principal membrane constituents in Archaea.

Fig. 1

(Left, Ourisson and Nakatani 1994) Very simple model of eucaryotic membranes. Membranes separate ‘inside’ from ‘outside’. Eucaryotic membranes usually consist of a bilayer of di-acyl phospholipids and sterols like cholesterol. Amphipathic proteins are also present in the bilayer. (Right) Topography of cholesterol in lipid bilayers

Fig. 2

(Left, Ourisson and Nakatani 1994) Hypothetical mechanism of the reinforcement of lipidic membranes by cholesterol, hopanoids, α, ω-dipolar carotenoids, and bis(diphytanylglyceryl) phospholipids. (Right) Structure and molecular dimension of three membrane reinforcers: cholesterol, bacteriohopane tetrol, bacterioruberine

Fig. 3

Structure of archaeal lipids: diphytanylglyceryl phospholipids in Halobacterium and bis(diphytanylglyceryl) phospholipids in Sulfolobus, Thermoplasma, Methanogene. Both the diether phophatidylcholine (left hand) and the 72-membered macrocyclic tetraether di-phosphates (right hand) formed vesicles in water (Dannenmuller et al. 2000; Eguchi et al. 2000)

The Role of Cholesterol in Membranes: Membrane Reinforcer

Let us first consider the effect of cholesterol on eucaryotic phospholipid membranes; it is incorporated in the bilayer, its OH group forms a hydrogen bond with the head-group of a phospholipid (Rohmer et al. 1979; Nakatani et al. 1996). Its dimensions allow cooperative van der Waals attractive forces to elongate and order the neighboring lipidic chains by decreasing their ability to undergo trans-gauche isomeric rotations (Scott HL 1991) (Fig. 1 Right).

There are different types of membrane reinforcement (Fig. 2 Left). Cholesterol and hopanoids act as ‘nails’, inserting into one half of the lipid bilayer. α, ω-Dipolar carotenoids cross both halves of the bilayer like ‘rivets’ (Milon et al. 1986a). Some archaeal lipids (bola-amphiphilic type molecules: Fuhrhop JH and Wang T 2004) form both halves of the bilayer themselves and these membranes are automatically reinforced like ‘struts’.

This reinforcement of membrane architecture has been shown by a wide variety of physical methods (Kwok and Evans 1981; Demel and De Kruyff 1976). We developed a new direct method to evaluate the mechanical properties of vesicles in the presence of cholesterol or its surrogates (Sun et al. 1986; Milon et al. 1986b). More precisely, we employed a stopped-flow/light scattering method to follow the swelling of unilamellar vesicles in response to osmotic pressure (Fig. 4).

Fig. 4

Recording of scattered light versus time during vesicle swelling upon osmotic pressure. Analysis : I(t) – Io = ΔI [1 – e(−t/τ)], I: light scattering intensity; Apparatus : stopped-flow: t ½ : reaction half-time. After rapid mixing (3 ms) of a vesicle solution with the same volume of a hypotonic buffer, the scattered light decreases upon vesicle swelling as a result of the decreasing bilayer thickness (Milon et al. 1986b)

We first established that the kinetics of vesicle swelling is controlled by the water permeability. We then observed that cholesterol and α, ω-dipolar carotenoids lower the water permeability, showing that they reinforce the membrane (Milon et al. 1986a; Lazrak et al. 1987). The reinforcement of membranes by hopanoids was shown on monolayer studies (Kannenberg et al. 1983).

The amphiphilic molecules in primitive membranes must have been very different from the molecules in membranes of the present-day living organisms. As was mentioned above, there are striking differences in the membrane lipid structures found in Archaea (polyprenic phospholipids) and those in Eucarya (di-acyl phospholipids). The “fatty acid scenario” has been well discussed for the origin of di-acyl phospholipids (Gebicki and Hicks 1973; Hargreaves and Deamer 1978; Walde et al. 1994; Walde 2006; McCollom et al. 1999). Recently, it was shown by a stopped-flow method that single-chain fatty acid membranes are more permeable to ribose than di-acyl phospholipid membranes (Budin and Szostak 2011). We present here our hypothesis that polyprenyl phosphates could be the precursors of archaeal membrane lipids. This scenario would be an alternative of the former one.

Search for the Most ‘primitive’ Membranes

It was possible to arrange the known membrane terpenoids in a ‘phylogenetic’ sequence, and a retrograde analysis led us to conceive that polyprenyl phosphates might have been ‘primitive’ membrane constituents (Fig. 5).

Fig. 5

(adapted from Ourisson and Nakatani 1994) Hypothetical evolution of membrane terpenoids (membrane components and reinforcers). Di-acylglyceryl phospholipids are shown for comparison

Starting from these polyprenyl phosphates, the step-wise recruitment of novel reactions, non-enzymatic at first, could have led to changes in the polar head-group, to cyclizations, dimerizations, and oxidations, and finally to archaeal lipids, carotenoids, hopanoids, and cholesterol.

‘Prebiotic’ Synthesis of Polyprenols

Polyprenyl chains of Archaea are biosynthesized by successive C5 increments of C5 isoprene units. The chemistry involved in these elongation steps is electrophilic alkylations of double bonds. The duplication of C5 units has been realized in 57 % yield (a mixture of isomeric C10 prenols) by simple treatment of monoprenols in the presence of montmorillonite clay (Fig. 6). These steps have been repeated, and led from geraniol (C10) to C15 prenols in 15 % yield (Désaubry et al. 2003). These steps should be tested with C5 and C10 prenylphosphates or pyrophosphates (Fig. 7).

Fig. 6

Synthesis of polyprenols under ‘prebiotic’ conditions (adapted from Désaubry et al. 2003)

Fig. 7

(adapted from Ourisson and Nakatani 1994) Hypothetical pathway from C1 or C2 units through C5 units to ‘primitive’ membranes. Farnesyl phosphate (C15) is synthesized and accumulated on the mineral surface

Search for the Origin of C5 Isoprene Units

What compounds might be the starting materials of C5 isoprene units in the prebiotic synthesis ? In present-day living organisms, the C5 unit is made either by the mevalonate pathway (Bloch 1992) or by the methylerythritol phosphate pathway (Rohmer 2003). Both are too complex to have close prebiotic analogues. However, these C5 units could be derived from simpler precursors (Fig. 7). Indeed, they (isopentenol and dimethylallyl alcohol) were synthesized by acid catalyzed Prins reaction from formaldehyde and isobutene (Blomquist and Verdol 1955). Isobutene itself was obtained by olefin homologation on zeolite, either from methanol (C1) or from ethylene (C2) in high temperature (Cui et al. 2008; Ilias and Bhan 2013; Oikawa et al. 2006). These abiotic conditions are potentially prebiotic, and isobutene, ethylene and formaldehyde do occur in volcanic gases (Capaccioni et al. 2004, 2011; Minissale et al. 2007; Tassi et al. 2009; Kiyosu and Asada 1995; Oro 1994). These steps should be tested experimentally in similar conditions on the prebiotic Earth (Martin et al. 2008; Mulkidjanian et al. 2012).

Hypothetical Pathway for the Formation of ‘primitive’ Membranes

We postulated that the simplest possible polar head could be a phosphate and synthesized a series of polyprenyl phosphates. We have shown by optical and confocal microscopies that single-chain polyprenyl phosphates containing 15 C-atoms (three C5 units) to 30 C-atoms (six C5 units) form giant vesicles in a wide pH range of 2 to 9 (Pozzi et al. 1996; Streiff et al. 2007) (Fig. 8). Based on the above observations, we postulated a plausible scheme for the chain elongation of polyprenols leading to the formation of ‘primitive’ membranes (Fig. 7).

Fig. 8

Phase contrast image of giant vesicles of farnesyl phosphates (C15) with spherical and discoid shapes at pH = 3.6 and T = 25 °C. Bar: 10 μm

The synthesis of polyprenyl phosphates (and pyrophosphates) with progressively longer chains (C5 increments) could be achieved by anchoring one end of the molecule to a solid mineral surface through a phosphate group. Once critical length and concentration have been achieved, polyprenyl phosphates would peel off the surface to form vesicles (Fig. 7). Note that the intermediates involved closely simulate those operating, with enzymes, in the biosynthesis of polyprenols (Porter and Spurgeon 1981). In contemporary organisms, the stepwise condensation of C5 phosphate units is achieved by specific prenyl-transferases (Liang et al. 2002). It is probably not a coincidence, but a reminiscence that the modern biosynthesis of polyprenols involves pyrophosphates at all stages.

Polyprenols as Reinforcers of Primitive Membranes

We have above observed that single-chain polyprenyl phosphates occupy a central position in the ‘phylogenetic’ sequence of membrane terpenoids (Fig. 5) and they form vesicles by themselves. Which components could have been reinforcers of polyprenyl phosphates ? We speculated that ‘primitive’ membranes might have been made up of a mixture of polyprenyl phosphates and polyprenyl alcohols (polyprenols), in much the same way, n-acyl carboxylates gave vesicles when mixed with equimolecular amounts of n-alcohols of the same chain length (Hargreaves and Deamer 1978; Apel et al. 2002). We have shown that the addition of the corresponding polyprenols increases the stability of vesicles of polyprenyl phosphates at higher pHs up to pH 12 (Streiff et al. 2007; Ribeiro et al. 2007), which is in good agreement with the ratio of hydrophilic and hydrophobic volume to predict the vesicle formation (Israelachivili et al. 1977). Using the stopped-flow/light scattering method, we demonstrated that the addition of 10 mol % of some polyprenols decreases the water permeability of polyprenyl phosphate vesicles (Table 1). This study thus provided a favorable argument that polyprenols might have been reinforcers of polyprenyl phosphate membranes. It may be noted that phytol and tricyclohexaprenol are as much as or better reinforcers than cholesterol in geranylgeranyl phosphate membrane (Fig. 9).

Table 1

(adapted from Ribeiro et al. 2007) Water permeability of vesicles made of geranylgeranyl phosphate alone or with 10 mol% of polyprenols, measured at 15.0 ± 0.1 °C in a stopped-flow/light scattering apparatus

Lipid composition of vesicles	Vesicle diameter a (nm)	t1/2 b (ms)
Geranylgeranyl phosphate (GGP)	180 ± 16	20 ± 1
GGP + 10 mol% phytol	165 ± 24	234 ± 7
GGP + 10 mol% tricyclohexaprenol	170 ± 39	250 ± 20
GGP + 10 mol% cholesterol	162 ± 35	193 ± 8

aAverage diameter ± standard deviation (evaluated by photon correlation spectroscopy)

bAverage t1/2 ± standard deviation. t ½: reaction half-time

Fig. 9

Structure of geranylgeranyl phosphate, phytol and tricyclohexaprenol

What is the actual advantage for a membrane to be“reinforced” by a sterol or a polyprenol ? One could propose several arguments to this issue. At a physical level, the membrane is subject to a number of physical stressing events during a cell’s life. Cells have to maintain the interior integrity, as well as transmembrane potential at all time. Thus it is essential for the membrane to resist to any harsh situation and the optimization of membrane reinforcers obviously has been nature’s strategy. It is routinely observed that hyperosmotic shocks on non-reinforced plain phospholipid bilayers lead to their disruption and leakage of their content. On an evolutionary point of view, it is amazing to observe how eucaryotic cells have been optimizing cholesterol structure to make it a perfect membrane reinforcer. The history of this optimization over millions of years can be traced in cholesterol metabolic pathway, and especially in the successive removal of methyl groups on its alpha face (Fig. 5) which lowered its membrane interactions (Bloch 1983). Our hypothesis here is that even before eucaryotic cells and cholesterol appeared on earth, at the level of procaryotic cells, this strategy of incorporating in membranes and optimizing membrane reinforcers was already in place.

Prevalence of Phosphates and Phosphorylation of Alcohols

Phosphates are present, in the form of an ester, in adenosine triphosphate (ATP), nucleic acids, and membrane lipids of all living organisms. The indispensable role of phosphates in many aspects of biochemistry is assigned to the specific properties of this group (Westheimer 1987). But, this ubiquitous role of phosphates is in sharp contrast with the low abundance of phosphorus in the Earth crust (0.12 %). There are, however, some scenarios in which phosphates could have been made available for prebiotic chemistry (Schwartz 2006). Pyrophosphate, triphosphate and tetraphosphate have been found in volcanic gases (Yamagata et al. 1991). The concentration of oligophosphate ions in layered minerals could be much increased resulting from the weathering of magmatic rocks (Arrhenius et al. 1997). Polyphosphates are also produced when orthophosphate salts are heated (Rabinowitz et al. 1968). The phosphorylation of polyprenols may have initially involved polyphosphoric acid (PPA), and phosphorylation of adenosine with polyphosphate salts in aqueous solution afforded a mixture of 5′adenosine monophosphate (5′AMP), 2′AMP and 3′AMP (Schwartz and Ponnamperuma 1968). Recently, PPA was employed for the synthesis of phosphoglycolamide from glycolamide (Weber et al. 2003; Krishnamurthy et al. 1999). Phosphorylation of adenosine into adenosine-5′-triphosphate was performed using trimetaphosphate (cyclotriphosphate) (Saffhill 1970; Etaix and Orgel 1978). Phosphorylation of some nucleosides with urea-inorganic phosphate mixtures gave nucleoside phosphates (Lohrmann and Orgel 1970). C5-C20 isoprenoid phosphates along with their pyrophosphates were simply obtained from the condensation of the corresponding alcohols with bis-triethylammonium phosphate (Keller and Thompson 1993) or tetrabutylammonium dihydrogen phosphate (Danilov et al. 1989). The conversion of alcohols into the corresponding phosphate esters was achieved using acetic anhydride activation (Dueymes et al. 2008) or imidoyl phosphate activation (Powner and Sutherland 2011) of inorganic phosphate. Phosphitylation of alcohols followed by oxidation could be an alternative pathway for the formation of phosphates (Yoza et al. 1992; De Graaf and Schwartz 2000; Bryant et al. 2010). Recently, it was proposed that meteorites would have provided the reduced iron-nickel phosphide schreibersite (Pasek et al. 2013). They have shown that schreibersite reacts with glycerol in an aqueous solution to give glyceryl phosphate. In spite of these observations, the origin of the phosphate in prebiotic system and the non-enzymatic synthesis of organophosphates continue to remain unresolved issues.

From ‘primitive’ Membrane Lipids to Archaeal Lipids

The next stage of elaboration of polyprenyl phosphates (‘primitive’ membrane lipids) could have been to change the head-group. There could be several plausible ways in which the polyprenyl phosphates might have been converted to di-polyprenyl-glyceryl phosphates, contemporary membrane constituents of Halobacterium. One hypothetical pathway could involve polyprenyl-glyceryl phosphates (Fig. 10). Starting compounds would be glycerol and a polyprenol. Glyceryl phosphate could be obtained by the phosphorylation of glycerol, for example, with amido-triphosphate derived from metatriphosphate in the presence of Mg 2+ (Mullen and Sutherland 2007). Polyprenyl phosphate and polyprenyl pyrophosphate could be obtained by phosphorylation by polyphosphate (Schwartz and Ponnamperuma 1968) or simply by phosphate (Keller and Thompson 1993; Danilov et al. 1989). The OH group of glyceryl phoshate is known to be a good nucleophile for the acylation (Ichihara et al. 2005). The nucleophilic attack on polyprenyl pyrophosphate (complexed with Mg 2+) by glyceryl phoshate could afford polyprenyl-glyceryl phosphates in a similarity with the biosynthesis of geranylgeranyl-glyceryl phosphate from geranylgeraniol and glyceryl phosphate (Zhang et al. 1990). The hydrophobic/hydrophilic balance in the structure of polyprenyl-glyceryl phosphates might not be favorable for the formation of vesicles (Tanaka et al. 2004). It is likely, however, that polyprenyl-glyceryl phosphates could also form vesicles, perhaps when combined with suitable amounts of polyprenols. These scenarios will have to be experimentally examined.

Fig. 10

Hypothetical pathway from polyprenyl pyphosphates to archaeal lipids and mechanism for the formation of a glyceryl head-group. In the transition state for the reaction of polyprenyl pyrophosphate and glyceryl phosphate, the carbocation intermediate at C1-C3 carbons of polyprenyl pyrophosphate is formed and the negative charge developed on pyrophosphate group is stabilized by coordination with Mg 2+. The pyrophosphate complexed with Mg 2+ could serve as a good leaving group. PPA polyphosphoric acid

Conclusion

In contrast to the fatty acid ester motif found in Eucarya and Bacteria, archaeal lipids are composed of diphytanylglyceryl units bearing phosphates as polar head group. We present here our hypothesis that polyprenyl phosphates could be the precursors of archaeal membrane lipids. We first observed that single-chain polyprenyl phosphates occupy a central position in the ‘phylogenetic’ sequence of membrane terpenoids (Fig. 5). We then confirmed that polyprenyl phosphates form vesicles in water alone or with polyprenols. Polyprenols were shown to be good membrane reinforcers. These observations led us to hypothesize that primitive membranes might have been formed by polyprenyl phosphates and polyprenols. A hypothetical pathway from C1 or C2 units through C5 isoprene units to ‘primitive’ membrane was discussed. Finally, we proposed a hypothetical pathway from polyprenyl phosphates (‘primitive’ membrane lipids) to archaeal lipids. These questions are important and they should be examined in a significant manner when the issue of prebiotic reactions is clarified. Nobody knows which the first membrane-forming molecules were. But ‘polyprenyl phosphates scenario’ could be a good and reasonable alternative to the ‘fatty acid scenario’.

Furthermore, once vesicles are formed, a remarkable series of new properties are automatically brought into operation and make it possible to progressively complexify the system towards proto-cells (Walde et al. 1994; Ourisson and Nakatani 1999, 2006; Szostak et al. 2001; Luisi 2002; Takakura et al. 2003; Walde 2006; Luisi and Stano 2011).