Biochemical characterization of atypical biotinylation domainsin seed proteins

Claudette Job1, Stéphanie Laugel1, Manuel Duval2, Karine Gallardo1 and Dominique Job1*1Laboratoire Mixte CNRS/INRA/Aventis (UMR1932), Aventis CropScience, 14–20 rue Pierre Baizet, 69263, LyonCEDEX 9, France; 2Department of Biology, Texas A & M University, College Station, TX 77843, USA

Abstract

Homologues of the pea SBP65, a late embryogenesisabundant (LEA) biotinylated protein that behaves as aputative sink for the free vitamin biotin during embryodevelopment, were characterized biochemically invarious plant species, including soybean, lentil, peanut,rape, cabbage, carrot and sugarbeet. Based onsequence homologies, the genome of Arabidopsisthaliana contains a gene putatively encoding ahomologue of pea SBP65. These proteins exhibit tworemarkable features. First, they only accumulate inseeds, particularly during late stages of embryodevelopment. The results strongly suggest that theseseed-specific biotinylated proteins belong to the class ofplant proteins called seed maturation proteins, which arepresumed to play major roles in embryo development.Secondly, covalent attachment of biotin occurs at alysine residue within a conserved motif of (V/M)GKF,which shows no resemblance to the highly conservedAMKM tetrapeptide that houses the target lysine residuein the well-characterized biotin-dependent carboxylasesand decarboxylases. These findings highlight novelstructural features for protein biotinylation.

Keywords: seed maturation, germination, LEA proteins,biotinylated proteins, biotin, Arabidopsis thaliana,cabbage (Brassica oleracea), carrot (Daucus carota),lentil (Lens culinaris), pea (Pisum sativum), peanut(Arachis hypogaea), rape (Brassica napus), soybean(Glycine max (L.) Merrill), sugarbeet (Beta vulgaris)

Introduction

Biotin is a water-soluble vitamin, also called vitaminH or B8, that is required by all forms of life. Thisvitamin is synthesized by plants, most bacteria andsome fungi, and plays crucial metabolic roles, servingas a covalently bound cofactor for a small family ofenzymes involved in the transfer of CO2 duringcarboxylation, decarboxylation and transcarboxyla-tion reactions (Samols et al., 1988; Knowles, 1989;Wurtele and Nikolau, 1990; Chapman-Smith andCronan, 1999a). Although the occurrence of biotin-dependent enzymes is ubiquitous in nature, proteinbiotinylation is a rare post-translational modificationevent in cells. For example, the only biotin-dependentcarboxylase in Escherichia coli is acetyl-CoAcarboxylase (EC 6.4.1.2), a multisubunit enzyme, inwhich one of the subunits is biotinylated andcorresponds to the biotin carboxyl carrier protein(BCCP). This regulatory enzyme of lipogenesiscatalyses the ATP-dependent carboxylation of acetyl-CoA. Saccharomyces cerevisiae and mammals arereported to contain only up to five biotinylatedproteins (reviewed by Chapman-Smith and Cronan,1999a). Attachment of biotin to the apoprotein (apo)form of the biotin-dependent enzymes is catalysed bybiotin protein ligase (BPL; EC 6.3.4.15), also known asbiotin holoenzyme synthetase. This enzymecovalently links biotin to a specific lysine residue atthe active site of newly synthesized biotin enzymesvia an amide bond (Lane et al., 1964). Consistent withthe observed scarcity of naturally occurringbiotinylation targets, the BPL-catalysed biotinylationreaction is highly specific. Thus, for all biotin-dependent enzymes described so far, the target lysineresidue occurs in a highly conserved AMKMtetrapeptide (Chapman-Smith and Cronan, 1999a).However, mutation of either conserved methionineadjacent to the target lysine residue in thePropionibacterium shermanii 1.3S subunit oftranscarboxylase has little effect on biotinylation, butrather affects catalytic efficiency of this biotin-containing enzyme (Shenoy et al., 1988, 1992). Reche

Seed Science Research (2001) 11, 149–161 DOI: 10.1079/SSR200169

*CorrespondenceFax: (+33) 4 72 85 22 97Email: dominique.job@aventis.comAbbreviations: Apo = apoprotein; BCCP = biotin carboxylcarrier protein; BPL = biotin protein ligase; LEA = lateembryogenesis abundant; PAGE = polyacrylamide gelelectrophoresis; PBS = phosphate saline buffer; PBST = PBScontaining Tween 20; SBP65 = seed biotinylated protein of65 kDa; SMP = seed maturation protein; TBS = Tris–salinebuffer.

and Perham (1999) also demonstrated recently thatdistal sequences play an important role in governingthe specificity of BCCP biotinylation catalysed by theE. coli BPL, in addition to the AMKM motif.

As in other organisms, biotin also serves as acovalently bound cofactor for biotin-dependentenzymes in plants (Wurtele and Nikolau, 1990;Dehaye et al., 1994; Anderson et al., 1998; Alban et al.,2000). However, a unique feature of plants is theexistence of a seed-specific, biotinylated protein thatwas first documented in pea, SBP65 (forseed biotinylated protein of 65 kDa) (Duval et al.,1994b). SBP65, which is the major biotinylated proteinin the mature pea seeds, behaves as a sink for freebiotin during late stages of embryo development andis rapidly degraded during germination (Duval et al.,1994b). In support of a peculiar function for thisprotein is that it is devoid of any known biotin-dependent carboxylase activity, presumably becausecovalent binding of biotin to the apoprotein does notoccur within the consensus AMKM tetrapeptidesequence (Duval et al., 1994a). SBP65 may constitute astorage form of biotin necessary for germination.Alternatively, by depleting the free biotin pool duringlate embryo maturation, the protein might help theembryonic cells to enter into and/or to maintain aquiescent state characteristic of most mature dryseeds (Bewley and Black, 1994). Interestingly, SBP65shares many physiological and molecular featureswith LEA (late embryogenesis abundant) proteinsthat accumulate in maturing seeds when they start todesiccate and are proposed to protect seed tissuesagainst desiccation-induced damage (Galau andDure, 1981; Dure, 1993a, b). Thus, as for LEA proteins(Dure, 1993b), SBP65 is extremely hydrophilic and itsamino-acid sequence contains several repeats (Duvalet al., 1994a).

Some evidence suggests the existence of suchembryo-specific biotinylated proteins in plant speciesother than pea. Thus, putative homologues of peaSBP65 have been reported in soybean seeds (Neto etal., 1997; Shatters et al., 1997; Hsing et al., 1998), castorbean seeds (Roesler et al., 1996) and carrot somaticembryos (Wurtele and Nikolau, 1992). This finding issupported by the recent cloning of a soybean cDNAputatively encoding a protein called SMP (GenBankaccession no. U59626), which belongs to the family ofseed maturation proteins, a subclass of LEA proteins,and shows extensive sequence similarity with peaSBP65. However, biochemical evidence to suggest theexistence of an atypical biotinylation domain in theseplant proteins is lacking.

In the present study, seed-specific biotinylatedproteins were purified to homogeneity from a numberof species, including soybean, lentil, peanut, carrot,cabbage, rape and sugarbeet. The general finding isthat a conserved lysine residue within the (V/M)GKF

tetrapeptide motif constitutes the covalent attachmentsite for biotin in all these seed proteins, whichhighlights novel structural features for proteinbiotinylation.

Materials and methods

Plant material

Seeds [soybean (Glycine max (L.) Merrill) cv. Jack; pea(Pisum sativum) cv. Douce Valencia; lentil (Lensculinaris) cv. Verte du Puits; peanut (Arachis hypogaea)cv. Valencia; rape (Brassica napus) cv. Westar; cabbage(Brassica oleracea) cv. Bartolo; sugarbeet (Beta vulgaris)cv. Univers; carrot (Daucus carota) cv. Nandor;Arabidopsis thaliana (ecotype Landberg erecta)] wereobtained from the seed collection of AventisCropScience (Lyon, France).

Soybean plants were grown in soil in a greenhousefor 5 months at 25°C : 18°C day : night temperatureand a photoperiod of 12 h light (500 �E m–2 s–1).Different organs (cotyledons, leaves, roots, stems,flowers, pods and seeds) were harvested at varioustimes and stored at –75°C until use.

Purification of seed-specific biotinylated proteins

Seed-specific proteins were purified according toDuval et al. (1994b) and Capron et al. (2000) with somemodifications. Frozen (–75°C) mature soybean seeds(100 g) were finely ground in a Waring blender. To thepowder, 500 ml of chilled buffer A (50 mM Tris–HCl,pH 7.8; 1 mM EDTA, pH 8.0) containing 500 mMNaCl was added, and the mixture was homogenizedwith a Polytron® (Kinematica GmbH, Kreins,Switzerland) homogenizer. After centrifugation(Sigma type 3K30) at 9000 g for 15 min at 4°C, theresulting supernatant (30 g of protein) was heated to90°C for 20 min, cooled to 20°C and then centrifuged(35,000 g, 15 min, 4°C). The supernatant (7.9 g ofprotein) was brought to 500 g l�1 (NH4)2SO4,incubated for 1 h at 4°C and then centrifuged(35,000 g, 15 min, 4°C). The pellet was resuspended in100 ml of buffer A containing 250 mM NaCl; then thesuspension was clarified by centrifugation (35,000 g,15 min, 4°C). The supernatant (2.2 g of protein)corresponded to the soluble heat-stable proteinextract. This extract was subjected to monomericavidin affinity chromatography on ImmunoPure®

Immobilized Monomeric Avidin (Pierce, Rockford,USA) according to Alban et al. (1993). The sample wasloaded (flow rate 0.1 ml min�1) onto the column (6 cm� 1 cm) equilibrated in buffer A containing 250 mMNaCl. After the column was washed (0.1 ml min�1)with 150 ml of buffer A containing 250 mM NaCl andthen with 60 ml of 50 mM citrate-phosphate (pH 6.1)

150 C. Job et al.

buffer, bound proteins (200 �g) were eluted with25 ml of 50 mM citrate-phosphate (pH 6.1) buffercontaining 2 mM D-biotin. SDS–PAGE analysisrevealed the presence of two major proteins, of about72 and 71 kDa, in the biotin eluate. Antibodiesdirected against the 72-kDa protein were raised in aguinea-pig as described previously (Job et al., 1997).

Heat-soluble protein extracts were also preparedfrom different organs of soybean plants (cotyledons,leaves, roots, stems, flowers, pods and seeds)harvested at different stages of development, asdescribed above for the mature dry seeds.

Seed-specific biotinylated proteins from otherplant species (pea, lentil, peanut, carrot, rape,cabbage, Arabidopsis and sugarbeet) were purifiedaccording to the protocol described above for thepurification of soybean biotinylated proteins.

Protein analyses

Protein concentrations in the various extracts weremeasured according to Bradford (1976). Bovine serumalbumin was used as a standard. SDS–PAGE of theprotein extracts was carried out according to Laemmli(1970), using a Mighty Small II SE250 electrophoresiscell (Hoefer Scientific Instruments, San Francisco,USA). Samples were mixed with load buffer [10 mMTris–HCl, pH 7.8, 1 mM EDTA, 2.5% (v/v) SDS,50 mM dithiothreitol (DTT) and 0.01% (w/v)bromophenol blue], heated to 100°C for 5 min andloaded onto gels (12% homogeneous polyacrylamidegels). Electrophoresis was conducted at a constantcurrent intensity of 30 mA, for 90 min at 25°C.

Biotinylated proteins eluted from the monomericavidin affinity column were concentrated and furtherpurified by SDS–PAGE onto 10% homogeneouspolyacrylamide gels. They were visualized bystaining with amido black. Following digestion of thegel-purified proteins by sequencing-gradeendoproteinase Lys-C (Boehringer Mannheim,Meylan, France) (100 pmol biotinylated protein,0.4 �g enzyme in 350 �l of 50 mM Tris–HCl buffer,pH 8.6, containing 0.03% SDS; 18 h incubation at37°C), the reaction mixture was injected onto a DEAEC18 high performance liquid chromatography(HPLC) column (2.1 mm diameter). Peptides wereeluted at 0.2 ml min�1 with a gradient of 2–45%acetonitrile (35 min) in 0.1% trifluoroacetic acid (TFA),and elution was monitored by absorbencymeasurements at 214 nm and 280 nm. Peptidescontained in fractions with a symmetrical peak formwere directly sequenced. Amino-acid sequencing wasperformed by Dr Jacques d’Alayer (Institut Pasteur,Paris, France) by automated Edman degradation ofthe peptides, using a PE Applied Biosystemsequencer.

Characterization of biotinylated proteins by ELISA

Biotinylated proteins were analysed by ELISA usingstreptavidin conjugated to horseradish peroxidase(Sigma, Saint Quentin Fallavier, France) as a specificreagent for biotin, and a peroxidase substrate solutioncontaining 1.8 mM 2,2�-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Aldrich, SaintQuentin Fallavier, France), and 0.003% (w/w) H2O2(Sigma) in 0.1 M citrate-phosphate buffer, pH 4.0 (Duvalet al., 1994b). Colour development was monitored at405 nm using a microplate reader (EL340 from Bio-TekInstruments, Winooski, USA) driven by a MacintoshIICx microordinator equipped with the DeltaSoftsoftware (version 4.1, BioMetallics, Inc., Princeton,USA).

Characterization of biotinylated proteins by Westernblotting

Following SDS–PAGE, proteins were transferred fromthe polyacrylamide gel on to nitrocellulose (BioTraceTM

from Gelman Sciences, Ann Arbor, USA) using asemi-dry electroblotter (Towbin et al., 1979). Blotswere rinsed twice for 5 min in 50 mM Tris–HCl,150 mM NaCl, pH 7.5 (TBS), then incubated for 1 h at 25°C in TBS containing 1% (v/v) Blocking Solution(Boehringer Mannheim). After incubation for 1 h with streptavidin conjugated to horseradishperoxidase (25 �g l�1 in TBS containing 0.5% BlockingSolution), blots were washed twice for 10 min in TBScontaining 0.1% Tween 20 and twice for 10 min in TBScontaining 0.5% Blocking Solution. Biotinylatedproteins were detected using the BM chemilumin-escence kit from Boehringer Mannheim, according tothe instruction booklet BM Chemiluminescence BlottingSubstrate (POD) from Boehringer Mannheim.

Identification of biotinylated peptides by indirectsolid-phase biotin assay

Following digestion of the purified seed-specificbiotinylated proteins by endoproteinase Lys-C, theresulting peptides were purified by HPLC asdescribed above and analysed for biotin content by anindirect solid-phase biotin assay (Duval et al., 1994b).ELISA plates were incubated for 3 h at 25°C with afixed amount of biotin-labelled �-galactosidase(usually 100 ng per well) in 100 µl of PBS (150 mMNaCl/10 mM Na2HPO4–NaH2PO4, pH 7.0), and thenwashed four times with PBS containing 0.1% (v/v)Tween 20 (PBST). Each peptide fraction (5 �l) wasmixed with 115 �l PBST containing a fixed amount(usually 10 ng) of streptavidin conjugated tohorseradish peroxidase, followed by incubation for1 h at 25°C. Portions (100 �l) of these mixtures werethen transferred to each �-galactosidase-coated well

Seed-specific biotinylated proteins 151

of the plates. After incubation for 1 h at 20°C, plateswere processed as for the direct ELISA, i.e. they werewashed four times with PBST and, after addition ofthe peroxidase/substrate solution, A405 was measuredas above. In this indirect biotin assay only assayscontaining biotinylated peptides will remaincolourless, while those containing an unbiotinylatedpeptide develop a green colour. Control experimentswere carried out using serial dilutions (0–0.2 ng) offree D-biotin in 100 �l PBST. The biotinylated peptidesthus identified were sequenced as described above.The amino acid modified by biotin in these peptideseluted near the phenylthiohydantoin (PTH) deriva-tive of arginine and was identified as biotinyl lysineby using biocytin (biotinyl lysine) as a control in theHPLC determinations (Newman et al., 1990).

Identification of biotinylated peptides by matrix-assisted laser desorption time of flight massspectrometry (MALDI-TOF)

Spectra were acquired by Dr Jean-Marc Strub(Laboratoire de Spectrométrie de Masse Bio-organique, Strasbourg, France) on a Bruker (Bremen,Germany) BIFLEX MALDI-TOF spectrometeroperated in reflector mode. Ionization wasaccomplished with the 337-nm beam from a nitrogenlaser with a repetition rate of 3 Hz. The peptides inwater/acetonitrile (0.5 �l) were deposited on a thinlayer of �-cyano-4-hydroxycinnamic acid made byrapid evaporation of a saturated solution in acetone.The droplets were dried under gentle vacuum beforeintroduction into the mass spectrometer (Goumon etal., 2000; Wilm, 2000).

Results

Purification of the seed-specific biotinylatedproteins from soybean and characterization of theirbiotinylation sequence

One of the distinguishing features of many LEAproteins is their solubility in water after boiling (Dure,1993a, b; Russouw et al., 1997; Walters et al., 1997). Aheat-soluble protein fraction was prepared frommature soybean seeds and analysed by SDS–PAGE.Biotinylated proteins were revealed on Western blotsusing streptavidin conjugated to horseradishperoxidase as a specific reagent for biotin. From thisextract two major biotinylated proteins of about 72and 71 kDa were detected (Fig. 1, lane 2). Followingpurification by affinity chromatography on amonomeric avidin Sepharose column, they wereseparated by SDS–PAGE, gel purified and submittedto digestion in the presence of endoproteinase Lys-C.The HPLC profiles for the two resulting peptide

mixtures were nearly identical (data not shown),suggesting a precursor–product relationship betweenthe two heat-soluble biotinylated proteins. In supportof this finding, antibodies raised against the 72-kDaprotein cross-reacted with the 71-kDa protein (datanot shown). In perfect agreement with the results ofShatters et al. (1997) and Neto et al. (1997) for soybeanseed biotinylated proteins, these proteins onlyaccumulated in seeds, primarily during desiccation(Fig. 2), and exhibited the same spatial and temporalaccumulation pattern reported for pea SBP65 (Duvalet al., 1994b). These features suggested that theseproteins corresponded to the soybean seedmaturation protein called SMP recently described byHsing et al. (1998) and for which a cDNA sequence isavailable (GenBank accession no. U59626). To addressthis question, the biotinyl domain of the 72-kDasoybean protein was characterized biochemically.Digestion of the pure protein by endoproteinase Lys-C yielded 33 peptides, out of which one (peptideno. 26) proved to be biotinylated according to theindirect solid-phase biotin assay. The sequence of this25 amino-acid long peptide is shown in Fig. 3A. Up tothe amino acid removed at step 15, the peptidesequence was identical to that predicted from thecDNA for soybean SMP (Fig. 3A). Amino acidsremoved at steps 16 to 25 were also identical to thosepredicted by this cDNA. However, according to thecDNA sequence, the amino acid at position 15 of thebiotinylated peptide should be a lysine residue,

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Figure 1. Characterization of heat-soluble biotinylatedproteins from mature seed extracts. Heat-soluble proteinswere extracted, separated by SDS–PAGE on a 12%polyacrylamide gel, subjected to Western blotting andstained for biotinylated proteins, using aperoxidase–streptavidin conjugate as a specific reagent forbiotin. Molecular weights of protein standards are given inkDa. Lanes 1–9, heat-soluble proteins from pea (12 �g),soybean (12 �g), lentil (12 �g), peanut (85 �g), rape (12 �g),cabbage (1 �g), Arabidopsis (85 �g), carrot (85 �g) andsugarbeet (85 �g), respectively.

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namely Lys125 in SMP (GenBank accessionno. U59626). Instead, this residue eluted near the PTHderivative of arginine and was identified as biotinyllysine by using biocytin as a control. To furtherascertain this result, amino-acid sequencing of a 17amino-acid long biotinylated peptide released bydigestion of pea SBP65 with endoproteinase Lys-Cwas performed. In agreement with previous results(Duval et al., 1994a), the sequence of this peptide(EDFGGVRDMGXFQMESK) was identical to thatpredicted by the cDNA sequence (Fig. 3A), except forthe X residue (corresponding to Lys103 in the peaprotein), which eluted near the PTH derivative ofarginine, as for Lys125 of soybean SMP. The amino-acid sequence of the biotinyl peptide isolated fromthe soybean biotinylated protein of about 71 kDa wasidentical to that determined for the soybeanbiotinylated protein of about 72 kDa (data notshown).

The isolated biotinyl peptides from pea SBP65 and

soybean biotinylated protein of about 72 kDa (Fig.3A) were also subjected to matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF)analysis. The spectra are shown in Fig. 4. Measuredmasses were 2186.9 Da and 3034.3 Da for the biotinylpeptides from the pea and soybean proteins,respectively. Assuming attachment of a single biotinylgroup to the lysine residue within the GKF motifpredicted by the cDNA sequences for the twopeptides, the theoretical mass of the modified peapeptide is 2186.9 Da and that for the modifiedsoybean peptide is 3034.4 Da. Thus, in both cases theexperimentally determined molecular masses are inexcellent agreement with the theoretical masses. Allthese results provide conclusive evidence that Lys125is the covalent attachment site for biotin in soybeanSMP.

Purification of the seed-specific biotinylatedproteins from various plant species andcharacterization of their biotinylation sequences

The protocol for the purification of the soybean SMPwas also used to investigate the existence of seed-specific biotinylated proteins in various plant species.Heat-stable biotinylated proteins were detected inmature seeds of lentil, peanut, carrot, rape, cabbage,Arabidopsis and sugarbeet (Fig. 1). Followingpurification by monomeric avidin affinity chroma-tography, some of these proteins were submitted todigestion by endoproteinase Lys-C and theirrespective biotinylated peptides characterized by theindirect solid-phase biotin assay. The amino-acidsequences of these peptides are shown in Fig. 3A. Asfor pea SBP65 (Duval et al., 1994b), soybean SMP(Hsing et al., 1998) and sugarbeet seed biotinylatedprotein (Capron et al., 2000; Job et al., 2000), all of theseproteins were found to disappear rapidly duringgermination and early seedling growth (data notshown).

For the heat-soluble biotinylated protein of about65 kDa from lentil seeds, the sequence of the biotin-containing peptide showed extensive homology withthat of the pea and soybean biotinylated peptides.However, the sequence of the biotinyl domain of the70-kDa biotinylated protein from peanut seeds wasmarkedly different, although pea, soybean, lentil andpeanut all belong to the same family of leguminousplants. The amino-acid sequences of the biotinyldomains of the 70-kDa biotinylated protein isolatedfrom mature sugarbeet seeds and of two biotinylatedproteins of about 60 kDa and 32 kDa from maturecarrot seeds were different from each other (Fig. 3A).They were also different from those for pea SBP65 andsoybean SMP (Fig. 3A). Although the two carrotsequences are very similar, they are not identical(Fig. 3A), which excluded the possibility of a

Seed-specific biotinylated proteins 153

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Figure 2. Spatial and temporal accumulation of soybeanheat-soluble biotinylated proteins. Heat-soluble proteinextracts were prepared from different organs of soybeanplants. From these extracts, levels of soybean heat-solublebiotinylated proteins were quantitated by ELISA usingspecific antibodies. They were expressed on a per mgprotein basis in arbitrary units. Results were normalized tothe level measured in mature dry seeds (MS). E 15 mg, E60 mg, E 120 mg, E 210 mg, E 250 mg, E 310 mg, E 410 mg,E 400 mg, E 370 mg, E 340 mg, E 315 mg and E 200 mg =developing embryos of the indicated fresh weight. The seeddesiccation phase of embryo development on the motherplant started at the 400-mg stage and ended at the 200-mgstage. The latter is equivalent to the mature dry-seed stage.Pod colour during development was green from E 15 mg toE 410 mg, green–yellow for E 370 mg, yellow for E 340 mg,yellow–brown for E 315 mg and brown for E 200 mg (forfurther details on developmental stages in soybean embryos,see Neto et al., 1997). R 3d, roots after 3 d of germination;R 7d, roots after 7 d of germination; Aerial cot, aerialcotyledons after 6 d of germination; F, flowers; L, leaves; P,pods; S, stems.

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Figure 3. Biotinylation domains of heat-soluble seed-specific biotinylated proteins, biotin enzymes and synthetic peptidesactive in the E. coli BPL biotinylation reaction. (A) Biotinyl domains of the seed proteins: pea SBP65, soybean biotinylatedprotein of about 72 kDa, lentil biotinylated protein of about 65 kDa, peanut biotinylated protein of about 70 kDa, carrotbiotinylated proteins of about 60 kDa and 32 kDa, rape biotinylated protein of about 60 kDa, cabbage biotinylated protein ofabout 80 kDa and sugarbeet biotinylated protein of about 70 kDa. The sequences for the pea, soybean, lentil, peanut, carrot,rape, cabbage and sugarbeet biotinyl peptides were experimentally determined. In all cases, the X residue eluted near the PTHderivative of arginine and was identified as biotinyl lysine by using biocytin as a control in the HPLC experiments. The proteinsequences predicted from the cDNA sequences of pea SBP65 (accession no. X75880) and soybean SMP (accession no. U59626),and the protein sequence from genomic database for the putative Arabidopsis thaliana seed maturation protein (accessionno. AC007087.5) are also shown. The biotinyl peptides from the pea and soybean proteins were used for molecular massdeterminations by MALDI-TOF (see Fig. 4). (B) Biotinyl domains of biotin-dependent enzymes. The target lysine residue isshown in bold. ACC, acetyl-CoA carboxylase (EC 6.4.1.2); BCCP, biotin carboxyl carrier protein of ACC; MCC,methylcrotonoyl-CoA carboxylase (EC 6.4.1.4); ODC, oxalacetate decarboxylase (EC 4.1.1.3); PC, pyruvate carboxylase (EC6.4.1.1); PCC, propionyl-CoA carboxylase (EC 6.4.1.3); TC, transcarboxylase (EC 2.1.3.1); UA, urea amidolyase (EC 6.3.4.6). (C)Peptides active in the E. coli BPL-catalysed biotinylation reaction. The ‘consensus’ sequence for biotinylation obtained fromscreens of combinatorial peptide libraries is from Schatz (1993). Peptide 85 is the peptide used in Beckett et al. (1999), which wasbiotinylated by the E. coli BPL as efficiently as the natural BBCP substrate.

precursor–product relationship between the twobiotinylated proteins of about 60 kDa and 32 kDa. Theonly common feature shared by all these sequenceswas the existence of a conserved (V/M)GXF motif(Fig. 3A), where in all cases the X residue eluted nearthe PTH derivative of arginine and was identified asbiotinyl lysine by using biocytin as a control in theHPLC experiments.

The 80-kDa biotinylated cabbage protein and the60-kDa biotinylated rape protein contained identicalbiotinylation domains (Fig. 3A). This presumablyreflected the fact that cabbage and rape both belong tothe same family (Brassicaceae). Of interest in thiscontext is the fact that during the course of theArabidopsis genome-sequencing programme, arecently released genomic BAC clone (F14N22,mapped on chromosome II) was found to contain asequence of which conceptual translation (GenBankaccession no. AC007087.5; gene F14N22.17; protein ID22997.1) indicates significant similarity with soybeanSMP and pea SBP65 (Fig. 5). This putative Arabidopsisseed maturation protein is highly hydrophilic andexhibits a hydrophilicity pattern remarkably similarto that of pea SBP65 and soybean SMP (Fig. 6). Inaddition, the predicted amino-acid sequence of theArabidopsis protein contains a MPHSVGKFV motif,which is highly homologous to the biotinylationsequence of the seed-specific biotinylated proteinsfrom rape and cabbage (Fig. 3A). Thus, not only doesthis genomic sequence provide the first indication forthe existence of an Arabidopsis homologue of the peaSBP65, but it also reinforces the finding that the Xresidue present in all seed biotinylated peptideswithin the (V/M)GXF motif (Fig. 3A) corresponds toa biotinyl lysine residue. By using the extractionprotocol described under Materials and methods, theexistence of a heat-soluble biotinylated protein frommature Arabidopsis seeds was revealed; its molecularmass was approximately 72 kDa (Fig. 1), thusmatching closely the molecular mass of the putativeArabidopsis seed maturation protein (theoreticalmolecular mass of 67,195 Da). This protein was heat-soluble, disappeared rapidly during germination andwas absent from immature embryos and leaves (datanot shown). Unfortunately, it was present at too low alevel in mature Arabidopsis seeds to performbiochemical characterization of its biotin attachmentsite.

Similarities between the seed-specific biotinylatedproteins within domains different from thebiotinylation domain

Pea SBP65 (GenBank accession no. X75880), soybeanSMP (GenBank accession no. U59626) and putativeArabidopsis seed maturation protein (GenBankaccession no. AC007087.5) exhibit extensive sequence

similarity (Fig. 5). To further substantiate thesequence similarity between the purified seed-specificbiotinylated proteins, additional amino-acidsequencing experiments were carried out. Inparticular, an examination of the HPLC profilesrecorded at 280 nm for the endoproteinase-Lys-C-digested biotinylated proteins revealed that all thepurified seed biotinylated proteins only exhibited afew (one or two) tryptophan-containing peptides.

Seed-specific biotinylated proteins 155

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Figure 4. MALDI-TOF spectra obtained for the biotinylpeptides of soybean biotinylated protein of about 72 kDaand pea SBP65 from mature seeds. Following digestion ofthese proteins in the presence of endoproteinase Lys-C, theirrespective biotinyl peptides were purified by HPLC. Onepart of the preparations was used for amino-acidsequencing, with results shown in Fig. 3A. The other partwas used for MALDI-TOF analyses. a.i., absolute intensity.(A) Biotinylated peptide from the soybean seed protein. (B)Biotinylated peptide from the pea seed protein.

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Figure 5. Amino-acid sequence comparison of seed-specific biotinylated proteins. The sequences shown are those of pea SBP65(GenBank accession no. X75880), soybean SMP (GenBank accession no. U59626) and putative seed maturation protein fromArabidopsis (GenBank accession no. AC007087.5). Alignment was produced by the Dialign 2.0 program (Morgenstern et al.,1996).

These peptides were sequenced and found to containseveral conserved residues (Fig. 7). The predictedsequence of putative Arabidopsis seed maturationprotein also exhibited this conserved motif (Fig. 7).

Other peptides were also sequenced, for whichFASTA and BLAST analyses yielded the followingresults. For example, the sequences TQRASDYARE,GRETGITAAE and EGTGKKEEEEQERATLE-DIQGFRANAQQK found from the biotinylated

cabbage protein of about 80 kDa showed extensivehomology (in the range of 73–78%) with the putativeArabidopsis seed maturation protein; furthermore, thethird of these three peptides exhibited significanthomology (52%) with pea SBP65. In addition, thesequence RETGITAAEQAARAK found from the rapebiotinylated protein of about 60 kDa showed highhomology (87%) with the putative Arabidopsis seedmaturation protein. Finally, the sequenceEESWREYEAK from the 65-kDa biotinylated proteinof mature lentil seeds showed 100% identity with peaSBP65.

Search for pea SBP65 homologues in yeast andanimals

The above results clearly indicated that seed-specificbiotinylated proteins containing an atypicalbiotinylation domain are widely represented in theplant kingdom. The BLAST program has been used tosearch for homologues of these plant biotinylatedproteins in other kingdoms. Running the BLASTPprogram with each of the biotinyl domains of theplant species referred to this report against the non-redundant protein databases at NCBI yielded nomatches. Furthermore, from using the searchBLASTN program with the pea SBP65 and soybeanSMP cDNA sequences encoding the respectivebiotinylated domains against the non-redundantnucleotide databases at NCBI, the output indicated anabsence of any homologue of these biotinyl-domain-containing genes in animal or yeast systems.

Discussion

Considering the high degree of similarity in theprimary structure of biotin attachment domains of themany carboxylases, decarboxylases andtranscarboxylases for which sequence data are nowavailable, the post-translational modification of specific

Seed-specific biotinylated proteins 157

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Figure 6. Hydropathy plots for the deduced proteinsequence of soybean SMP, pea SBP65, and putative seedmaturation protein from Arabidopsis. Hydropathy values(Kyte and Doolittle, 1982) are plotted against amino-acidposition using a window of nine residues. Positive valuesindicate hydrophobic regions and negative valuescorrespond to hydrophilic regions. Sequence data are from(A) soybean SMP (GenBank accession no. U59626); (B) peaSBP65 (GenBank accession no. X75880); and (C) putativeseed maturation protein from Arabidopsis (GenBankaccession no. AC007087.5).

Figure 7. Sequences in the vicinity of tryptophan residues insome heat-soluble seed-specific biotinylated proteins and inthe putative seed maturation protein from Arabidopsis(GenBank accession no. AC007087.5).

lysine residues in the biotinyl domains of biotin-dependent enzymes is highly selective. Indeed, thespecific biotinylated lysine residue occurs in a highlyconserved AMKM tetrapeptide [Fig. 3B; reviewed byChapman-Smith and Cronan, 1999a). However, twointriguing results raise the question of whether thisAMKM motif is the unique feature required for proteinbiotinylation in vivo. First, screening of peptidelibraries for activity in BPL-catalysed biotinylation in E.coli has led to the identification of a consensussequence of 13 amino acids, which is sufficient tospecify biotinylation (Schatz, 1993). Yet, the primarysequence of these peptides has little resemblance to thesequence around the biotinylated lysine residue in thebiotin-dependent enzymes, with the only strictlyconserved residue being the lysine itself (see Fig. 3C).Despite such divergence of amino-acid sequence,transient kinetic analysis of one of these peptides (seeFig. 3C) demonstrated that the biotinylation kinetics inthe presence of the E. coli BPL are very similar to thosemeasured for the natural BCCP substrate: in both casesthe values of kcat/Km are of the order of 10,000 M�1 s�1

(Beckett et al., 1999). Secondly, biotinidase (EC 3.5.1.12;an enzyme that hydrolyses endogenous and dietarybiocytin or short biotinyl peptides, thereby recyclingbiotin in animals; Craft et al., 1985) has recently beenshown to display two functions, acting either as abiotinyl-hydrolase or as a biotinyl-transferase,depending on the pH of the cell compartment and theavailability of specific protein acceptors (Hymes et al.,1995; Hymes and Wolf, 1998). In particular, histonesH2A, H2B, H1, H2 and H3 proved to be efficientlybiotinylated when incubated with human serumbiotinidase and biocytin at pH > 7 (Hymes and Wolf,1998). The amino-acid residues modified by biotin inhistones have not yet been characterized biochemically.Although it is unlikely that histones contain theAMKM motif found in biotin-dependent enzymes, ithas been proposed that, as for BPL, biotinidasetransfers biotin to the >-amino group of lysyl residues(Hymes and Wolf, 1998).

The present work demonstrates that naturallyoccurring protein domains different from the AMKMmotif may undergo specific post-translationalbiotinylation. Thus, in marked contrast to the biotin-dependent enzymes, the biotinylated lysine residue ofall seed-specific biotinylated proteins presentlyanalysed occurs within a highly conservedtetrapeptide sequence of (V/M)GKF (Fig. 3A). This(V/M)GKF motif also shows no resemblance to thepeptide sequences revealed through a combinatorialapproach and that served as substrates for the E. coliBPL (see Fig. 3C) (Schatz, 1993; Beckett et al., 1999).From the data obtained with nine plant species (Fig.3A), we conclude that this novel biotinyl domain iswidely distributed in the plant kingdom.

Previous studies indicated that neither the E. coli

BPL (Duval, 1995; Dehaye et al., 1997) nor theArabidopsis plastid-targeted BPL (Tissot et al., 1998) canuse the substrate apo-SBP65, the unbiotinylated formof SBP65. Similarly, Hsing et al. (1998) reported that theE. coli BPL was unable to biotinylate the apoproteinform of soybean SMP. These findings raise thequestion of the mechanism of biotinylation of theseseed-specific apoproteins. In plants, different forms ofBPL are found in chloroplasts, mitochondria and thecytosol (Tissot et al., 1996, 1997). While the precise roleof these different enzyme forms is unclear, such acompartmentalization of BPL activity might reflect thesubcellular location of the various proteinbiotinylation targets in plant cells. In pea, for example,different forms of the biotin-dependent carboxylaseshave been purified from chloroplasts, mitochondriaand the cytosol of leaf cells (reviewed by Alban et al.,2000), while SBP65 was localized to the cytosol ofembryonic cells (Duval et al., 1995). Therefore, thepreviously characterized plastid-targeted ArabidopsisBPL would be responsible for the biotinylation ofplastid acetyl-CoA carboxylase (Tissot et al., 1998). Themechanisms for targeting BPL to the cytosol or mitochondria have not yet beenelucidated in plants. We carried out a BLASTN search against the Arabidopsis sequence database atThe Arabidopsis Information Resource (TAIR;http://www.arabidopsis.org/blast/) using the cDNAsequence coding for the plastid-targeted ArabidopsisBPL (Tissot et al., 1997). Interestingly, this searchmatched two genomic BAC clones. The BAC cloneF3N11 (accession no. AC006053) contains the genecoding for the previously characterized plastid-targeted isoform. It is located on chromosome II,between the mi139 and m283 markers. This gene hadbeen identified by TIGR using gene predictionprograms and was referred to as F3N11.16. The othermatch is a locus contained in the BAC clone F28L22(GenBank accession no. AC007505), located onchromosome I and mapping close to the NIA2 marker.This second BPL gene putatively encodes a proteinshowing 82% sequence identity with the plastid-targeted Arabidopsis BPL. It is apparently devoid ofany transit peptide for targeting the protein to eitherthe plastids or the mitochondria and, therefore,putatively corresponds to a cytosolic BPL. Thismultiplicity of BPL-encoding genes in plants is inmarked contrast to the situation found inmicroorganisms and in other eukaryotes, where thereis strong experimental evidence for the existence of asingle gene encoding BPL (Chapman-Smith andCronan, 1999b). It will be interesting to determine theapoprotein substrate specificity of the plant BPLisoforms, particularly concerning their role in thebiotinylation of the seed-specific biotinylated proteins.

This work shows that while the seed-specificbiotinylated proteins appear to be widely distributed

158 C. Job et al.

in plants, they do not seem to have any counterpartsin animal and yeast systems. As the complete genomesequences of Caenorhabditis elegans and yeast areavailable at the NCBI databases for such similaritysearches, we can conclude that this novel biotinylateddomain is specific to the plant kingdom. Based onsequence homology and on the specific patterns ofspatial and temporal accumulation exhibited by theseed-specific biotinylated proteins, it is likely thatthese heat-stable biotinylated proteins belong to thesame family of seed maturation proteins (Rosenbergand Rinne, 1986; Blackman et al., 1991). Our worksuggests that the function of these biotinylatedproteins is specifically related to seed development.The existence of a homologue of pea SBP65 inArabidopsis will allow this question to be addressed bya reverse genetics approach. The crucial role of biotinin seed development and germination has alreadybeen exemplified by the characterization of biotinauxotroph mutants (bio1, bio2) of Arabidopsis, in whichbiosynthetic genes for biotin are inactivated(Schneider et al., 1989; Patton et al., 1998). Theseembryos could be rescued when grown in thepresence of biotin (Shellhammer and Meinke, 1990;Patton et al., 1998).

Acknowledgements

This work has been supported in part by grants fromthe Région Rhône-Alpes (Programme ‘Biotech-nologies’) and from the European Community (FAIRproject CT97–3711 ‘Genetic and molecular markers forseed quality’).

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Received 24 July 2000,accepted after revision 8 January 2001

© CAB International, 2001

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