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Plant Cell. May 2007; 19(5): 1580–1589.
PMCID: PMC1913742

Structure and Function of a Mitochondrial Late Embryogenesis Abundant Protein Are Revealed by Desiccation[W]

Abstract

Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into α-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.

INTRODUCTION

Desiccation tolerance is rather unusual in eukaryotes, with some exceptions found in lower plants, algae, lichens, and invertebrates. However, it is widespread among most higher plant seeds, which can survive for long periods without water and are thus authentic anhydrobiotes. Desiccation tolerance involves an array of mechanisms designed to protect and repair the structure and the components of cells and tissues. A class of hydrophilic proteins expressed in late seed development, the so-called late embryogenesis abundant (LEA) proteins, were discovered three decades ago and have been consistently associated with desiccation tolerance because of their expression profile and their hydrophilicity (Cuming, 1999). Hydrophilic proteins are indeed highly represented in prokaryotes and eukaryotes exposed to water deficit (Garay-Arroyo et al., 2000). LEA proteins are prominent in plants (there are >50 LEA genes in Arabidopsis thaliana) but have also been found and related to desiccation tolerance in anhydrobiotes belonging to other kingdoms, such as bacteria and nematodes (Battista et al., 2001; Browne et al., 2002). The major features of LEA proteins are low sequence complexity, occurrence of repeat motifs, high hydrophilicity, heat solubility, and an apparent lack of defined structure (Cuming, 1999; Wise and Tunnacliffe, 2004). A number of mechanisms have been proposed to justify their role in desiccation tolerance: water replacement, ion sequestering, macromolecules, and membrane stabilization (Close, 1996; Cuming, 1999). Experimentally, several LEA proteins were shown to behave in vitro as cryoprotectants (Kazuoka and Oeda, 1994; Houde et al., 1995; Bravo et al., 2003), stabilize glassy states (Wolkers et al., 2001), act as molecular sponges (Tompa et al., 2006), prevent protein aggregation (Goyal et al., 2005), and bind to lipid vesicles (Koag et al., 2003) or actin filaments (Abu-Abied et al., 2006). Structurally, several LEA proteins were found largely unfolded in solution (Eom et al., 1996; Lisse et al., 1996; Soulages et al., 2002; Goyal et al., 2003; Mouillon et al., 2006), and some were shown to undergo transition to a more folded state upon dehydration (Wolkers et al., 2001; Goyal et al., 2003; Shih et al., 2004). Thus, a great diversity of biochemical and structural features have been reported for LEA proteins. However, molecular mechanisms explaining the functions of individual LEA proteins are lacking.

In desiccation-tolerant eukaryotes, mitochondria are expected to be highly protected, since they are the primary energy-generating systems whose failure would compromise stress tolerance and recovery. Pea (Pisum sativum) seed mitochondria were indeed found to accumulate a LEA protein (LEAM) with repeat motifs predicted to form amphipathic helices (Grelet et al., 2005). Here, we show that LEAM is an intrinsically disordered protein that reversibly folds upon drying into amphipathic helices reminiscent of apolipoproteins in order to coat and protect the inner mitochondrial membrane upon desiccation.

RESULTS

Reversible Folding of LEAM

LEAM is a 37-kD protein monomer but behaves as a protein of higher relative molecular weight in gel filtration, suggesting a nonglobular structure. Its secondary structure was evaluated using circular dichroism (CD) spectroscopy. The CD spectrum of the protein in aqueous solution appeared typical of a random coil structure, with a 195-nm minimum of ellipticity (Figure 1A). LEAM is thus a monomer that is natively unfolded in solution. Natively unfolded polypeptides share common features, such as low complexity, high flexibility, a low mean hydrophobicity, and a relatively high net charge (Uversky et al., 2000; Romero et al., 2001). Although unfolded in solution, LEAM is expected with high confidence to fold into α-helical motifs, according to secondary structure program analysis (Grelet et al., 2005). Indeed, in the presence of a desolvating agent such as trifluoroethanol (TFE), the CD spectrum of LEAM was typical of α-helices, with high ellipticity at 191 nm and minima at 206 and 222 nm (Figure 1A). The TFE-induced folding of LEAM is fully reversible, since the protein shifted back to a random coil structure when TFE was removed by dialysis (Figure 1A).

Figure 1.
CD Analysis of LEAM.

We then searched for other conditions that promote the helical folding of LEAM in aqueous media. LEAM remained completely unfolded in a wide range of temperatures ranging from 20 to 100°C. Increasing osmolarity by the addition of 2.7 M glycerol, or lowering water potential to −1.7 MPa with polyethylene glycol, did not promote any structural change in LEAM, which remained unfolded. While several detergents (CHAPS, Triton X-100, and deoxycholate) and phospholipids (phosphatidylethanolamine, phosphatidylcholine, and cardiolipin) were ineffective, SDS proved to be a strong inducer of helical folding (Figure 1B; see Supplemental Figure 1 online). To compare the effect of SDS and TFE as helix inducers of LEAM, the molecular ellipticity at the maxima of 191 nm was plotted as a function of their concentration (Figure 1B). Clearly, the two compounds displayed different dose responses, with an almost linear correlation in the case of TFE contrasting with a sharp helix induction between 0.02 and 0.1% (v/v) for SDS, followed by a plateau at higher concentrations (Figure 1B). The effective SDS concentrations for helix formation were in the range of the critical micellar concentration of the detergent (0.06 to 0.07% = 2.0 to 2.5 mM). Using the DICHROWEB program (Lobley et al., 2002), the proportions of α-helix, β-sheet, turn, and random coil secondary structures were determined from the CD spectra. The results clearly show the predominance of random coil structures of the protein in solution and its propensity to fold into helices in the presence of SDS or TFE (Figure 1C). In agreement with the spectra (see Supplemental Figure 1 online), the number of helices with TFE was found to be higher than with SDS (Figure 1C). The β-sheet and turn proportions did not change significantly upon folding of the protein, suggesting that they may be artifacts and may possibly correspond to intermolecular structures in the case of β-sheets.

To evaluate whether LEAM is folded in the dry state, 500 μg of protein was allowed to air-dry directly in a CD measuring device. The CD spectrum of dried LEAM protein was almost identical to that obtained with TFE in solution, indicating that LEAM likely folds into helices in the dry state (Figure 1C; see Supplemental Figure 1 online). This was further confirmed using Fourier transform infrared (FTIR) spectroscopy. Analysis of deconvoluted spectra in the amide I band region (1700 to 1600 cm−1) allows deduction of the secondary structure from the peak positions (Byler and Susi, 1986). In solution, the LEAM spectra revealed a maximum at 1649 cm−1, with a main peak at 1647.5 cm−1 surrounded by two other peaks at 1663 and 1630 cm−1 (Figure 2). These values were assigned to random coil structures according to published data (Byler and Susi, 1986) and in light of the CD observations. The protein solution was then allowed to air-dry directly (15 min) on the diamond window, and a new spectrum was recorded. The disappearance of the deuterated water peak in the spectrum indicated that the dried protein was devoid of free water (data not shown). A significant change of the amide I band absorption was induced by dehydration, since the spectral maximum appeared at 1652.0 cm−1 (Figure 2). This well-resolved peak, which was not found in the spectrum of LEAM in the liquid state, can be attributed to the formation of an α-helix (Byler and Susi, 1986). Deconvolution of the spectrum revealed a main component at 1652.2 cm−1 and four other peaks of decreasing intensities at 1636, 1672, 1686, and 1614 cm−1 (Figure 2). While some of these peak positions suggest the presence of coiled-coil structures, the spectral maxima were not shifted to the characteristic lower values of coiled coils (Heimburg et al., 1999). Interestingly, the FTIR spectra of the lyophilized LEAM did not reveal the structural transition induced by air-drying, suggesting that rapid freezing prevents the helical folding of LEAM upon dehydration (see Supplemental Figure 2 online).

Figure 2.
FTIR Spectroscopy of LEAM in Solution and in the Dry State.

Together, both CD and FTIR spectroscopy demonstrate that LEAM is capable of folding into a mainly α-helical structure upon desiccation. The structure transition is fully reversible, since the dried and subsequently rehydrated protein was found to be unstructured using both techniques (data not shown).

Molecular Modeling of LEAM Structure

In the initial characterization of LEAM, the polypeptide was shown to contain five adjacent repeats that could be modeled as amphipathic helices (Grelet et al., 2005). Given the demonstrated ability of LEAM to fold into α-helices upon dehydration or in particular chemical environments (SDS or TFE), the whole structure was modeled on an α-helical template. As shown in Figure 3A, where LEAM is represented as a single helical rod, the distribution of residues along the polypeptide reflects a precise organization that maintains an amphipathic character all along the polypeptide. The distribution of charged residues displays a striking motif, since almost all of the negatively charged residues form a stripe along the axis of the helix, which is flanked on each side by two stripes of positively charged residues (Figure 3A). Such a peculiar arrangement is illustrated by an axial projection of a section of the protein (residues 50 to 150) displaying only the charged residues for the sake of clarity (Figure 3A, center). The stripes, and hence the amphipathic structure, are twisted to the left until around position 90, then proceed almost straight until positions 170 to 190, which exhibit a marked right twist (Figure 3A). Afterward, the stripes remain straight until around position 275, where a final left twist is observed.

Figure 3.
Helical Rod Model of LEAM.

As seen in Figure 3B, the amphipathic helix profile of LEAM closely resembles that of the class A amphipathic helices found in different plasma apolipoproteins, which are involved in their association to membranes (Segrest et al., 1992). The archetypal amphipathic peptide of apolipoproteins shown in Figure 3B was demonstrated to interact with membranes by lateral immersion into the phospholipid layer, with the hydrophobic face exposed toward the interior of the membrane and the positive charges interacting with the phosphate groups (Mishra et al., 1994; Hristova et al., 1999). The structural analogy of LEAM with the class A helix and the structural behavior of folding in membrane-mimic conditions (SDS or TFE) or upon drying led us to postulate that LEAM interacts with membranes during desiccation. A BLAST search for relatives of LEAM revealed a series of proteins whose primary sequences were examined for predictive structural features. A Medicago truncatula protein and an Arabidopsis protein (accession numbers Q1S7M8 and At5g44310, respectively) clearly appeared to be the mitochondrial orthologs of LEAM. Several other plant proteins were predicted to be targeted to plastid, endoplasmic reticulum, mitochondria, or cytosol (see Supplemental Table 1 online). A Caenorhabditis elegans protein, presumably cytosolic, was also included. All proteins were predicted to be unfolded proteins with a high propensity to form α-helices displaying a class A profile (see Supplemental Table 1 online). This indicates that the peculiar arrangement of LEAM is not restricted to the pea mitochondrial LEA protein but likely is shared by other proteins, even from distantly related species, suggesting functional conservation over evolutionary time.

Intrinsic Chemical Modifications of LEAM

Relatively little is known about posttranslational modifications of LEA proteins. In two-dimensional PAGE of pea seed mitochondria, LEAM appears as several major spots of 37 kD (pI between 4.9 and 5.2) and several minor spots of lower mass (Figure 4A). Strikingly, an almost identical pattern was observed whenever LEAM was ectopically expressed in several hosts, such as Escherichia coli, Saccharomyces cerevisiae, or Arabidopsis, and even when the protein was produced in vitro using a continuous coupled transcription–translation system (Figure 4A). LEAM is thus subjected to intrinsic chemical modifications that are independent of the organism. Major protein modifications were identified as deamidation of Asn-23, Asn-44, and Gln-250 and oxidation of Trp-20 and Trp-253. For instance, mass spectra of the peptide TLNGDVDSEDVK clearly show the different variants of the peptide, revealing the occurrence of the succinimide intermediate of Asn-44 and its conversion into Asp (see Supplemental Figure 2 online). Similar peptide variants were detected in the cases of Asn-23 and Gln-250 (data not shown). The modified Trp residues showed a characteristic oxidation sequence with the successive addition of oxygen (+16 D and +32 D) followed by decarboxylation to yield kynurenines, with an increment of 4 D (see Supplemental Figures 3 and 4 online). Although it would be difficult to quantify the exact ratio of the peptide variants and their relatedness, they are certainly responsible for the recurrent pattern of the protein on two-dimensional PAGE gels.

Figure 4.
Intrinsic Modifications of LEAM.

The structural model of LEAM was examined for the positions and possible functional implications of spontaneous posttranslational modifications. Oxidation of Trp-20 to kynurenine and deamidation of Asn-23 are expected to introduce a positive and a negative charge, respectively, which would reinforce the alignments of charge in the helix (Figure 4B). The residue Trp-253 protrudes in the middle of an almost perfect alignment of four positively charged lysines (Lys-239, Lys-246, Lys-257, and Lys-264), and again, its modification to kynurenine would restore the positive stripe (Figure 4B). In addition, the bulky Trp residue may interfere with Lys-257 as it appears in the model. As in the previous case, the deamidation of Gln-250 could reinforce the negative stripe as well. Interestingly, both Trp modifications appeared to be coupled to deamidation of residues located at position +3 or −3, which are spatially close in the helix motif. For Asn-44, the modification to a negative charge would fit well into an alignment of several Asp residues (Asp-33, Asp-37, Asp-48, Asp-51, and Asp-58). Since P. sativum and M. truncatula are both legumes with a high degree of synteny, the occurrence of corresponding sequences in the orthologous sequence was examined. The hot-spot Q250GAW253 is perfectly conserved, while W20AYN23 in LEAM becomes WAYD in the M. truncatula protein, confirming that deamidation of Asn-23 in pea is equivalent to replacement by a negatively charged Asp in M. truncatula. The situation is less clear for Asn-44, because several substitutions are found in the corresponding region. In Arabidopsis, which is evolutionarily less related, the sequence GRYD corresponding to the hot-spot pea sequence W20AYN23 still indicates the introduction of a negative charge in the Asn position. Collectively, in light of the three-dimensional model examination and the related protein sequences, the intrinsic modifications of LEAM likely have important structural relevance.

Protection of Liposomes and Membrane Interaction in the Dry State

Since structural features of LEAM suggest a role in membrane protection during desiccation, a liposome-drying assay was established. Liposomes were prepared from a mixture of phosphatidylcholine and phosphatidylethanolamine, and small unilamellar vesicles obtained by extrusion and sonication were analyzed by size distribution. When the liposomes were air-dried and subsequently rehydrated, the mean size of the population increased considerably, from 150 nm (before drying) to >8000 nm, revealing rearrangements such as membrane fusions (Figure 5A). Almost no liposomes of the original size survived the treatment. Similar results were obtained when the liposomes were supplemented with various amounts of lysozyme (25 to 100 μg protein/600 μg lipid) before drying (Figure 5A). Lysozyme was chosen as a control protein in the protection assays because it shows only a weak interaction with neutral phospholipids (Pap et al., 1996) and retains its structure even at very low hydration (Nagendra et al., 1998).

Figure 5.
Protection of Liposomes against Desiccation and Interaction with Phospholipids.

However, when similar amounts of LEAM were added to the liposome suspension before drying, the rehydrated liposomes remained much more in the range of untreated original populations, especially for the highest concentration of LEAM (Figure 5A). The two peaks observed for the highest concentration (100 μg) likely correspond to unaltered liposomes (mean size = 150 nm) and to moderate fusions for the larger population (mean size = 600). Added proteins had no effect on the liposome size distribution before drying (data not shown). It can be concluded that, under these experimental conditions, LEAM provides significant protection to liposomes against the damaging effects of desiccation.

The interaction of LEAM with phospholipids was further examined by differential scanning calorimetry (DSC) applied to dry liposomes. This technique allows the monitoring of phase transitions in phospholipid layers. Liposomes were prepared with 1-steroyl-2-oleoyl-phosphatidylcholine 18:0/18:1 (SOPC), which exhibits a gel–liquid crystalline phase transition at ~49°C in the dry state (Figure 5B). When SOPC liposomes were dried together with variable amounts of LEAM, thermograms showed clear modifications (Figure 5B). The presence of LEAM provoked both a decrease in the transition peak temperature (down to 46°C) and a major collapse of endothermic peaks (Figure 5B). Both events occurred in a dose-dependent manner, revealing a major interaction of LEAM with SOPC phospholipids in the dry state. Similar amounts of lysozyme did not induce such a strong shift in the transition temperature and had only moderate effects on the endothermic transition (Figure 5B).

DISCUSSION

The biochemical and spectroscopic analyses described herein demonstrate that LEAM is intrinsically disordered in solution and thus is a genuine natively unfolded protein. Most of the described functions for natively unfolded proteins concern regulatory mechanisms that take advantage of induced conformational changes (Wright and Dyson, 1999; Dunker et al., 2001). LEAM demonstrated an instant and reversible propensity to adopt an essentially α-helical structure in the presence of TFE or SDS or upon air-drying. Such behavior is similar to that reported for Gm PM16, a soybean (Glycine max) LEA protein, which was proposed to participate with sugars in the formation of glassy matrices in the dry state (Shih et al., 2004). Several other LEA proteins were found to assume secondary structures upon temperature increase (Soulages et al., 2002), desiccation (Wolkers et al., 2001; Goyal et al., 2003), or in the presence of TFE (Lisse et al., 1996; Soulages et al., 2002). Interestingly, when LEAM was lyophilized, it remained disordered, presumably because rapid freezing before dehydration prevents the transition to the α-helical structure. Whether slow freezing allows the folding of LEAM, and whether the protein would then be able to play a role in freezing tolerance, has not been established yet. Thus, natively unfolded LEAM adopts an α-helical structure upon drying, a condition experienced in seeds, and in the presence of SDS, which mimics a membrane environment. The structural modeling of LEAM as a helical rod suggested a membrane interaction, since it revealed a strong analogy with the class A amphipathic helices of apolipoproteins coating low-density lipoprotein particles (Segrest et al., 1992). However, no evidence of structural transition was found when LEAM was exposed to phospholipid membranes in conditions in which other natively unfolded proteins such as α-synuclein undergo structural transition (Nuscher et al., 2004). These data indicate that the membrane association cannot proceed directly from the native unfolded state of LEAM.

Thus, the more likely scenario is that LEAM needs to adopt a helical conformation upon drying in order to interact with membranes, providing a protective role similar to that afforded to low-density lipoprotein particles by apolipoproteins in aqueous solution. This hypothesis is supported by the capacity of LEAM to protect liposomes against the deleterious effects of drying and by the effect of LEAM on the thermodynamic properties of membranes in the dry state. A three-dimensional model was built on the assumption that the functional conformation of LEAM is essentially α-helical and presented as a single and continuous helical rod. It is very unlikely that the peculiar axial disposition of the residues would occur by chance; therefore, this architecture is a strong clue toward the function of the protein with respect to membrane protection. The possible role of the observed twists in the structure may be related to membrane curvature or could be modulated by more flexible regions not taken into account in the model. Interestingly, although Pro is generally overrepresented in natively unfolded proteins (Dunker et al., 2001), its absence in mature LEAM would prevent major breaking events in the α-helix structure. Similarly, the LEAM relatives in other organisms also have few Pro residues, which are mostly found in the beginning (transit peptide) or at the end of the polypeptides (see Supplemental Table 1 online).

It is also worthy of note that the spontaneous chemical modifications of LEAM maintain the amphipathic and charge properties of the helical structure, thus contributing to the functional conformation of the protein. Their presence in the protein produced in vitro confirms their chemical origin and rapid occurrence after protein synthesis. Chemical deamidation is normally a relatively slow process that requires days to years in physiological conditions, and it has been suggested that it could play a biological role as a protein molecular clock (Robinson, 2002; Reissner and Aswad, 2003). Therefore, the rapid deamidation of LEAM is unusual, although it is favored by the occurrence of small residues (Gly and Ser) immediately downstream of the targeted residues (Figure 4) and by the flexibility of the protein in the disordered state (Kosky et al., 1999; Robinson, 2002). The ubiquitous enzyme l-isoaspartyl O-methyltransferase, which is able to regenerate deamidated residues, has been identified as a key player in an essential damage-repair mechanism of protein in models as diverse as neurons (Kim et al., 1997; Yamamoto et al., 1998) and plant seeds (Mudgett et al., 1997). Our results suggest either that l-isoaspartyl O-methyltransferase is not found in plant mitochondria or that LEAM is not a substrate for the enzyme. The other major modifications of LEAM are oxidation of Trp residues, a phenomenon that is common in mitochondrial proteomes from animals and plants, presumably because they are a major production site of reactive oxygen species (Taylor et al., 2003; Møller and Kristensen, 2006). Both protein deamidation and oxidation are generally associated with protein damage during aging or in relation to oxidative stress (Reissner and Aswad, 2003). Since the modifications of LEAM are intrinsic to the protein (i.e., they appear regardless of how the protein was produced) and because they are expected to restore structural features in the helix model, we propose that they are not age- or stress-related but rather have a biological role. From an evolutionary perspective, such intrinsic modifications imposed by the chemical environment of the modified residue in the polypeptide do not exert evolutionary pressure, since the protein is perfectly functional with the encoded amino acid.

Together, the structural and biochemical features of LEAM strongly suggest that its major role is to protect the inner mitochondrial membrane during seed desiccation. LEAM is the only LEA protein found in intact pea seed mitochondria, and it is localized in the matrix space, where it accumulates during late seed filling, just before desiccation (Grelet et al., 2005).

It can be expected that sucrose, which forms a stabilizing glassy state during drying (Crowe et al., 1998), should gain access to the intermembrane space through porin and contribute to organelle protection on the outside of the inner membrane. During drying, LEAM would shift to a helical state and likely be excluded from the core of the soluble matrix proteins, whose concentration should increase tremendously because of dehydration. The structural features of folded LEAM, namely amphiphilicity and axial charge disposition, would then promote integration into the membrane, parallel to its plane. The dimensions of the membrane interfaces can easily accommodate α-helices in such an orientation (White et al., 2001). By analogy with the amphipathic class A helices of apolipoproteins, LEAM would be stabilized by ionic interactions with the phosphate groups and hydrophobic interactions with the membrane core, following the snorkel model (Segrest et al., 1990; Mishra et al., 1994). While the principle is similar, the situation with LEAM is dramatically different, since interaction with the membrane occurs exclusively at low hydration, with the purpose of reinforcing the inner membrane and/or protecting its protein-free domains. LEAM could indeed have a stabilizing effect on the lipid phase behavior, which is especially challenged at low hydration.

During seed imbibition, the replenishing of water would reverse the transition, forcing LEAM to exit the membrane and adopt its unfolded conformation. Such a model is attractive because it favors the stability of the inner mitochondrial membrane in the dry state without hampering its proper function in the hydrated state. Indeed, if LEAM could fold in the liquid state, it would be in the position to enter the membrane and compromise its primordial function in energy transduction and transport. For instance, the biocide activity of many antimicrobial peptides is based on their ability to form amphipathic helices capable of entering and disturbing membranes (Giangaspero et al., 2001). Such a protective role for LEAM requires that a sufficient amount of protein accumulates in mitochondria to allow coverage of the membrane surface. Simple calculations based on the size features of pea seed mitochondria and the projected surface occupied by a helical rod of LEAM show that 20,000 polypeptides are required to cover one-third of the inner membrane surface, a reasonable assumption of the protein-free area. In the hydrated state, and with a matrix protein concentration in the range of 400 g/L, the 20,000 LEAM polypeptides would represent 0.6% of the total mass of the matrix proteins. This figure agrees well with the observed abundance of LEAM in the two-dimensional PAGE map of pea seed mitochondria (Bardel et al., 2002), reinforcing the hypothesis.

In conclusion, we have provided strong evidence toward a membrane-protecting role for a mitochondrial LEA protein during desiccation. The reversible folding of LEAM upon drying allows alternation between a reservoir of the natively unfolded polypeptide, which is highly soluble and does not hamper membrane function, and a helical form able to enter and reinforce the membrane at low hydration. The identification of LEAM homologs in several organisms with diverse predicted subcellular localizations suggests wide conservation of this protective protein in diverse biological membranes.

METHODS

Overexpression of LEAM

Recombinant LEAM was overexpressed in Escherichia coli (Grelet et al., 2005) and purified to homogeneity by anion-exchange and gel-filtration column chromatography (HiTrapQ HP, Superdex200 10/300GL; Amersham Biosciences). The LEAM precursor was expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter using the pFP101 vector system (Bensmihen et al., 2004). LEAM was expressed in yeast (Saccharomyces cerevisiae strain W303-1A) transformed with an engineered pYes2/CT plasmid (Invitrogen) under the control of the Gal4 promoter. Mitochondrial localization of LEAM in Arabidopsis and yeast hosts was confirmed by SDS-PAGE analysis of purified mitochondria from yeast (Guérin et al., 1979) and Arabidopsis leaves (Keech et al., 2005). LEAM was produced in vitro using the Rapid Translation System 100 (Roche Diagnostics).

Two-Dimensional PAGE Analysis

Proteins were precipitated with 10% (v/v) trichloracetic acid in acetone and subjected to isoelectric focusing and SDS-PAGE using the Protean IEF Cell and Criterion Precast System (Bio-Rad). Gels were stained with Coomassie Brilliant Blue R 250, or LEAM was detected by immunoblotting (Grelet et al., 2005).

CD and FTIR Spectroscopy

CD spectra were acquired with a Jobin-Yvon CD6 dichrograph at 20°C in 5 mM phosphate buffer, pH 7.5, at a protein concentration of 1.2 mg/mL in a 0.2-mm cell. Estimation of secondary structure was done with the method of CDSTR (Sreerama and Woody, 2000) using DICHROWEB (Lobley et al., 2002). FTIR spectra (250 scans) of proteins in deuterated water were recorded at a resolution of 2 cm−1 on a Nicolet Magna IR 550 spectrometer equipped with a liquid nitrogen–cooled Mercury-Cadmium-Telluride detector. Spectra were obtained by attenuated total reflection using a single reflection accessory fitted with a diamond crystal. All data manipulations were performed with Grams/32 software (Galactic Corporation). Parameters of Fourier deconvolution were chosen to obtain enough band narrowing in the amide I region without introducing significant side lobes in the 1690 to 1720 cm−1 region devoid of the protein band.

Preparation and Size Analysis of Liposomes

Liposomes were prepared using either 7.5 mg of Lipoid 75 (soybean lecithin at 69% phosphatidylcholine and 10% phosphatidylethanolamine) or 7.5 mg of SOPC (Avanti Polar Lipids) dissolved in chloroform:methanol (9:1, v/v). Solvent was removed by evaporation (Rotavapor; Heidolph) at 53°C, and dry lipids were rehydrated at 53°C by vigorous agitation with water. Unilamellar liposomes were then prepared by sonication (5 × 5 s; Vibracell sonicator) and extrusion with a hand-held extruder (100-nm-pore polycarbonate membrane; Avanti Polar Lipids). Desiccation assays were performed by air-drying overnight 50 μL (600 μg) of Lipoid 75 liposomes in a hermetic box with Silicagel, followed by rehydration with 300 μL of distilled water prior to size measurement. Volume diameter and intensity of the liposome batches were determined by dynamic light scattering monitored with an Autosizer 4700 (Malvern Instruments) fitted with a 488-nm laser beam at a fixed angle (90°).

DSC

SOPC liposomes (30 μL = 380 μg) placed in a DSC metal cup were air-dried overnight in a hermetic box with Silicagel. The cup was sealed, and calorimetry analysis was performed using a DSC 822c (Mettler) calibrated with an indium standard. Samples were cooled to 10°C, then scanned while heating at 5°C/min up to 80°C.

Molecular Modeling of LEAM Structure

A helical rod model of pea (Pisum sativum) Ps LEAM was generated with the SWISS-PDBViewer (Guex and Peitsch, 1997) (http://www.expasy.org/spdbv/) by threading the primary sequences onto the three-dimensional template of filamin (Protein Data Bank accession number 1GK7). Figures were drawn using PyMOL version 0.98 (http://pymol.sourceforge.net/; DeLano Scientific). Helical wheel projections were built using the WinPep program (Hennig, 1999) (http://www.ipw.agrl.ethz.ch/~lhennig/winpep.html).

Mass Spectrometry

For nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS), chromatographic separation of digested proteins was accomplished by loading 0.15 μg of peptide mixture onto a 15-cm fused silica C18 column (75 μm i.d., 3 μm, 100 Å, and 360 μm o.d.; Dionex). Peptide elution was achieved using the following linear gradient: (a) from 10% to 40% solvent B (CH3CN:water, 90:10 [v/v] containing 0.1% s formic acid) for 40 min and (b) from 40% to 90% solvent B for 5 min. The remaining percentage of the elution solvent was made of solvent A (water:CH3CN, 5:5 [v/v] containing 0.1% formic acid). Flow rate through the nano-liquid chromatography column was 200 to 300 nL/min. For automatic liquid chromatography MS/MS analysis, the QTOF Ultima instrument was run in data-dependent mode with the following parameters: 1-s scan time and 0.1-s interscan delay for MS survey scans; 400 to 1400 and 50 to 2000 m/z mass ranges for the survey and the MS/MS scans, respectively; five components; MS/MS to MS switch after 5 s; switchback threshold, 30 counts/s; include charge states 2, 3, and 4 with the corresponding optimized collision energy profiles. The acquired data were postprocessed to generate peak lists (.pkl) with PeptideAuto, which is part of proteinLynx from Masslynx 4.0. To identify the spontaneous posttranslational modifications of LEAM, MS/MS spectra were manually examined using the PEAKS studio program (http://www.bioinformaticssolutions.com/). The algorithm can efficiently choose the best amino acid sequence, from all possible amino acid combinations, to interpret the MS/MS spectrum.

Accession Number

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number Q5NJL5.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Effects of SDS, TFE, and Drying on LEAM Structure Challenged by CD Spectroscopy.
  • Supplemental Figure 2. Lyophilization Does Not Induce Helical Folding of LEAM.
  • Supplemental Figure 3. Mass Spectrometry Analysis of Asn Deamidation and Trp Oxidation.
  • Supplemental Figure 4. Tandem Mass Spectra of NSSMDWAYNSTSK-Modified Peptides.
  • Supplemental Table 1. Structural Features of Proteins Showing Sequence Relatedness to LEAM.

Supplementary Material

[Supplemental Data]

Acknowledgments

We are thankful to P. Goloubinoff (Lausanne University) for stimulating discussions. This work was supported by the Contrat Etat-Région Pays-de-la-Loire region, Program Semences, and by Anjou Recherche Semences.

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: David Macherel (gro.oibavin@oibmd).

[W]Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.107.050104

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