Plant Molecular Biology 19: 1079-1083, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

1079

Update section Short communication

Sequence of an oleosin cDNA from Brassica napus James S. Keddie l, Eira-Wyn Edwards 2, Terry Gibbons 2, Charles H. Shaw 2 and Denis J. Murphy 1 1Cambridge Laboratory, John Innes Centre, Colney Lane, Norwich NR 4 7UJ, UK; 2Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK Received 23 January 1992; accepted in revised form 27 April 1992

Key words: oleosin, embryogenesis, cDNA, Brassica napus, oil-body protein

Abstract Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryoderived 2gtl 1 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 bp and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3' untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.

The protein profile of mature oilseed rape (Brassica napus) seeds is dominated by three sets of proteins, namely cruciferin, napin and oleosin [10]. Immunogold labelling of B. napus embryo sections has demonstrated that oleosins are located on the surface of the oil-body [11] and molecular characterisation of oleosins from a number of oilseed species has shown that the protein consists of a highly hydrophobic central domain flanked by amphipathic or polar C- and N-terminal domains [2, 3, 4, 5, 12, 13, 15]. Oleosins show striking homology in the central domains [ 12], with homology also being found in a C-terminal amphipathic a-helix and an N-terminal polar a-helix. The central domain, because of

its hydrophobicity, is proposed to be embedded in the lipid phase of the oil-body whereas the flanking regions are believed to lie on the lipid/ lumen interface or in the lumen [ 12]. Recent resuits from circular dichroism and Fourier transform infrared spectroscopic analysis of purified oleosin protein [8] have shown that this domain consists mostly of r-sheet structure. The function of the oleosin protein is unclear (see [12] for a discussion). Oleosin protein was prepared from mature B. napus cv. Bienvenu seeds by repeated centrifugation/flotation steps and antibodies were prepared against SDS-PAGE gel-purified oleosin protein as previously described [ 11]. The anti-

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63779.

1080 1

CCAGCTAGAACCCATCACGATATCACC~ACCCGAGATCAGTATCCATTGA%~TAGCCGAGAC

60

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CGAGACC~GTATGGGATGA~GGTC~GACC~KGTA~TATGTCCGGCC2%A2uACTACTCC p R D Q Y G M I G R D Q Y N M S G Q N Y S

40

~2%GTCTAGGCAGA~fGCTAAAGCTACCAC~fGC-~AGTCACCGCEGGGGA~fCT~CCTTGI~I

180

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61' 21 121 41 181 61 241 81

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CTCTCCAGTCTCACCCTTGTGGGAACGGTC2%~"EGC2%~GATTGTTGCCACTCCACTGCTT

240

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80

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GTTATCTTTAGCCC2tATCCTAGTCCCCGCTCTCATCACCGTCGCACTGCTGATCACCGGC

300

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301

TTTCTCTCCTCTGGTGCATTTGGCATTGCAGCTATAACGGTCTTCTCTTGGATCTAC~G

101

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361

TATG~CGGGAGAGCACCCACAGGGGTCAGAT~AAGTTGGACAGTGC~GGATGAAGCTG

121

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421

GG~GC2h~GGCTCAGGATATG~GGACAGAGCTTACTACTATGGACA~.C~CATACAGGT

141

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481

GAGG~CACGATCGGGACCGTGACCACCGC~CCGACCGTGACCGCACCCGGGGAACCCAA

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Fig. lA. D N A sequence and d ~ i v e d a m i n o acid s e q u e n c e o f a n o l e o s m cDNA, BnV, ffom B~ss~anapus. An N-terminalamino acid tandem r e p e ~ isindicated by arrows. BnV

549 TTAattacgccaagactat ...............................

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I illill I I II BnIV BnV BnIV BnV

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tctatatggtaaa

Fig. lB. The 3' u n ~ a n s l ~ e d r e , o n of BnV has been aligned with the 3' u n ~ a n s l ~ e d r e , o n of a previously reposed oleosin cDNA, BnlV [ 12]. The TAA termination codons of both cDNAs ~ e m ~ c a t e d as upper-case letters. Full stops in ~ e nucleotide sequence indic~e where gaps have been introduced to ~cilit~e the ~ignment.

1081 body was prepared as described by Sambrook et al. [14] and its specificity was determined by western blotting as previously reported [ 11]. The antiserum was used to screen a 2gtl 1 expression library (Amersham) made from mRNA

Bnl Bnll Bnlll BnIV BnV ZmI ZmIl Dcl GmI Psi Hal

purified from late-stage B. napus embryos [ 12]. Plaques were identified due to their immunoreactivity to the antiserum and inserts were cloned into Bluescript K S - (Stratagene) for further analysis. The sequence presented in this paper is

- -X IHLQ P Q Y E G D V G Y G Y G Y G G R A D Y K S R G P S K N Q

--XRQ M T D T A R T H H D I T SRDQY P R D R U Q Y S M I G R D R D Q Y S I M G R D R D Q Y N M Y G R D Y S K S R Q --RDQYPRDRDQYSMIGRDRDKYSM IGRDRDQYNMYGRDYSKSRQ - - P A R T H H D I T T R D Q Y P ....... L I S R D R D Q Y G M I G R . . EQYIqMSGQNYSKSRQ - -R ~ Y G D L Q R ~ G E A Q Q Q Q K Q G A M M T MADRDRSG IYGGAHATY~RPMGEQVKKGMLHD M A E R G T Y A H Q V Q V H PQQTANQPC43VKS L L P K N S P S T SQ M T T V P P H SVQV/TFI'rtlRYEAGVVPPARFEAPRYEAG I K A P S S I Y H S E R --SRGPSXNQ - -TTTTYDRI~, "I"IQPHYRQDDRSRYDQQTHSQ

conserved hydrophobio domain BnI Bnll Bnlll BnIV BnV Zml ZmII DCl GraI GmIl Psi Hal

.... ~ IVGVPVGGSLLALAGLTLAGSVIGLMLSVPLFLLF S PVIVPAAIXX ..... I~TAVTAGGSLLVLS SLTAVGTVI LLTVAT - . . . . . I A ~ A V T A V T A G G SLLVLS S L T L V G T V I A L T V A T P L L V I F S P I L V P A L I T V ..... IA~AVTAVTAGG SLLVLSSLTLVGTVIALTVAT PLLVIF S P ILVPAL ITV . . . . . I A ~ A T T A V T A G D S L L V L S S LTLVG'TVIAL IVAT PLLVI F S P I L V P A L I T V ..... ~TAATFC-GSMLVLSGLI L A G T V I A L T V A T PVLVI F S P V L V P A A I A L G PTASQAL~TLF P L G G L L L V L S G L A L T A S V V G L A V A T P V F L IF S P V L V P A A I A L ...... ~ _ A V V T L L ~ L A G I T L V G T L IGLAVAT P L F L L F S P V L V P A A L T I G PTT S ~ / L A V V A G L P V G G Ir .r.r.rA G L T L A G T L T G L V V A T P L F I I F S P V L I P A T V A I I- - N S L A G T L T G L A V A T P L F V L F S P V L V P A T V A I ...... ~/VAL I V G V P V G G S I / A I ~ G I T L A G S V I G S M L S X P L F L L F S PVIV- • .S T S ~ T L A I I A L L P V G G I L L G L A A L T F I G T L I G L A L A T P L F V I F S P I I V P A V L T I

Bnl Bnlll BnIV BnV ZmI Zmll Dcl Gml Grail Hal

G L A V T A I~ % S G L F G ~ X L S S V V W X I AMLITGFLSSGGFGIAAITVFSWI3 ALLITGFLLSGGFGIAAITVFSWI5 A L L I T G F L S S G A F G IAAITVFSWI'% A L M A A G F V T SGGI.GVAALSVFSWM3 A L A V M G F L T S G A L G L G G L S S L T C LZ G L A V T G F L G S G A F G L T G L S S L SWVI G L A V A G F LT S G V F G L T ~ L S W I I G L A V A G F LT S G A F G L T A L S SF SWI I G L A V T G F LASGTFGLTGLSSLSYLI

BnIII BnIV BnV ZmI ZmII DcI GrnII HaI

........ D R T R G T Q H T T e n d G Q Q Q T G ........ G E H D R ........ D R T R G T Q H T T e n d G Q Q H T G ........ E D H D R D R D H R T D R D R T R G e n d DQAQGSE end TAQAGQAIQGRAQEAGTC-GC~GAGAGGGGRAS S e n d T K E V G D T IQ S K A A Q A Q D T T A T T A G R D T R S T A R D T SRT e n d TKEVC-QKTKEVGQDIQSKAQDTREAAARDARD.. A ~ T TKEVC-QKTKEVGQDI Q S K A Q D T TKDFGQKI Q S T ~ G ~ ~ K E G R K S G D R T end

GraI Gr~II

TVTATTATA end TVTATTATA end

amphlpathic

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Fig. 2. Amino acid sequence alignment of all oleosins sequenced to data. BnI, BnlI, BnlV [12], BnlII [5] and BnV [this paper] are oleosin sequences from Brassica napus. Other oleosin sequences are as follows: ZmI [15] and Z m l l [ 13] are from Zea maya, DcI [3] is from Daucus carom, GmI and G m l l [4] are from Glycine max, RsI [12] is from Raphanus sativus (radish) and Hal [2] is from Helianthus annuus. The conserved hydrophobic central domain and the conserved C-terminal amphipathic a-helix are indicated as boxes. A potential leucine zipper motif is indicated by asterisks. To facilitate the alignment gaps in the amino acid sequence were introduced and are shown as full stops.

1082 of one selected and purified clone, termed BnV. Analysis of the 781 bp c D N A (Fig. 1A and 1B) identified an open reading frame of 549 bp followed by an untranslated region of 222 bp and a poly(A) region of 10 bp. The open reading frame encodes a polypeptide of 183 residues, of 20.3 kDa with a pI of 10.0. As there are no methionine codons in the immediate vicinity of the 5' end of the open reading frame and the predicted molecular weight of the protein is similar to that determined by SDS-PAGE analysis [ 11 ] the c D N A appears to be near full-length. Using G C G programs on a VAX system the sequence was compared to the sequence of a second oleosin cDNA, BnlV, previously cloned from the same rapeseed library [ 12]. At the D N A level the entire cDNAs are 77% identical. At the amino acid level the entire proteins are 86% identical and when conservative amino acids are included this increases to 91%. The 70 residue conserved central hydrophobic domain displays 94~o identity and 97% similarity. A tandem repeat of 8 amino acids in the N-terminal region of unknown function was also identified (see Fig. 1A). This motif is also found as a trimer in BnlV (see Fig. 2). The untranslated 3'-end regions of the two cDNAs displayed some striking homology (see Fig. 1B). Apart from the sequence immediately downstream of the termination codon, which is absolutely conserved, there are also many other conserved motifs. This sequence conservation may be due to BnlV and BnV having evolved from a common ancestor and the conserved elements may be important for m R N A stability or processing. The conservation of these elements suggests that they are important for the translational control of oleosin expression. Figure 2 shows an amino acid sequence alignment of all oleosin sequences reported to date [2, 3, 4, 5, 12, 13, 15 and this paper]. The central hydr0phobic region is strongly conserved between all oleosins (indicated by a box). The high level of sequence conservation is evidence that this domain is vital to oleosin function, for example possibly to anchor it to the oil-body. Within the central domain is a run of leucine residues or conservatively substituted leucine residues, occur-

ring on average every seventh residue. This structure is very similar to the leucine zipper motif that is commonly associated with the dimerisation domain of many DNA-binding proteins [7] although this motif has also been identified in transmembrane proteins [1, 6, 9]. The SDS-PAGE gelpurified oleosin protein has been shown to dimerise on storage [4, M. Li and D.J. Murphy, unpublished results]. It is possible that it is through this motif that oleosin/oleosin protein interactions occur, although since the motif is found in a t-strand domain, the dimerisation mechanism is likely to be different from that found in classical leucine zipper proteins. Also indicated as a box in Fig. 2 is a region of each protein containing a conserved amphipathic a-helical domain. Each 18 residue amino acid sequence, when plotted onto an Edmunson wheel, produces a perfect amphipathic a-helix. This helix, because of its amphipathicity, is predicted to lie half in and half out of the oil-body. The divergence of the amino acid sequence of this motif between oleosins of different species, and the conservation of its position relative to the central domain suggests that the C-terminal amphipathic a-helix is very important for oleosin function.

Acknowledgements This work was funded by the Agricultural and Food Research Council. The authors extend their thanks to Mr Joseph Satriani for his continuing help.

References 1. Buckland R, Wild F: Leucine zipper motif extended. Nature 338:547 (1989). 2. Cummins I, Murphy DJ: c D N A sequence of a sunflower oleosin and transcript tissue specificity. Plant Mol Biol, in press. 3. Hatzopoulos P, Franz G, Choy L, Sung RZ: Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot. Plant Cell 2: 457467 (1990).

1083 4. Kalinski A, Loer DS, Weisemann JM, Matthews BF, Herman E: Soybean oil body membrane protein 24 kDa is expressed as isoforms of closely related genes. Plant Mol Biol 17:1095-1098 (1991). 5. Keddie JS, Hubner G, Slocombe SP, Jarvis RP, Cummins I, Edwards E-W, Shaw CH, Murphy DJ: Cloning and characterisation of an oleosin gene from Brassica napus. Plant Mol Biol, in press. 6. Kossel H, Dory I, Igloi G, Maier R: A leucine-zipper motif in photosystem I. Plant Mol Biol 15:497-499 (1990). 7. Landshultz WH, Johnson PF, McKnight SC: The leucine-zipper: A hypothetical structure common to a new class of DNA-binding proteins. Science 243:1681-1688 (1988). 8. Li M, Smith LJ, Clark DC, Wilson R, Murphy DJ: Secondary structure of a new class of lipid body proteins from oilseeds. J Biol Chem 267: 8245-8253. 9. McCormack K, Campanelli JT, Ramaswami M, Mathew MK, Tanouye MA: Leucine-zipper motif update. Nature 340:103 (1989).

10. Murphy DJ, Cummins I: Biosynthesis of seed storage products during embryogenesis in rapeseed, Brassica napus. J Plant Physiol 135:63-69 (1989). 11. Murphy DJ, Cummins I: Purification and immunogold localisation of the major oil-body membrane protein of oilseed rape. Plant Sci 60:47-54 (1989). 12. Murphy DJ, Keen JN, O'Sullivan J, Au DMY, Edwards E, Jackson PJ, Cummins I, Gibbons T, Shaw CH, Ryan AR: A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins. Biochim Biophys Acta 1088: 8694 (1991). 13. Qu R, Huang AHC: Oleosin kD 18 on the surface ofoil bodies in maize. Genomic and cDNA sequences. J Mol Biol 265:2238-2243 (1990). 14. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). 15. Vance VB, Huang AC: The major protein from lipid bodies of maize: characterisation and structure based on cDNA cloning. J Biol Chem 262:11275-11279 (1987).