THE JOURNAL OP B~LOGICAL CHEM~~Y 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 4, Issue of February 5, pp. 2238-2243, 1990 Printed in U.S. A.

Oleosin KD 18 on the Surface of Oil Bodies in Maize GENOMIC AND cDNA SEQUENCES AND THE DEDUCED PROTEIN STRUCTURE*

(Received for publication, May 30, 1989)

Rongda Qu$ and Anthony H. C. Huang From the Department of Botany and Plant Sciences, University of California, Riverside, California 92521

Oleosins are newly discovered, abundant, and small M, hydrophobic proteins localized on the surface of oil bodies in diverse seeds. So far, most of the studies have been on the general characteristics of the proteins, and only one protein (maize KD 16) has been studied using a cDNA clone containing an incomplete coding se- quence. Here, we report the sequences of a genomic clone and a cDNA clone of a new maize oleosin (KD 18). There is no intron in the gene. The 5’-flanking region contains potential regulatory elements includ- ing RY repeats, CACA consensus, and CATC boxes, which are presumably involved in the specific expres- sion of the proteins in maturing seeds. The deduced amino acid sequence was analyzed for secondary struc- tures. We suggest that KD 18 of 187-amino acid resi- dues contains three major structural domains: a largely hydrophilic domain at the N terminus, a hydrophobic hairpin a-helical domain at the center, and an amphi- pathic a-helix domain at the C terminus. These struc- tural domains are very similar to those of oleosin KD 16. However, the KD 18 and KD 16 amino acid se- quences as well as nucleotide sequences are highly similar only at the central domain (72 and 71%, re- spectively). The similarities are highest at the loop region of the a-helical hairpin. These results suggest that KD 18 and KD 16 are isoforms, encoded by genes derived from a common ancestor gene. We propose that the hairpin domain acts as an indispensible internal signal for intracellular trafficking of oleosins during protein synthesis as well as an anchor for oleosins on the oil bodies. The other two domains can undergo relatively massive amino acid substitutions without impairing the structure/function of the oleosins or have evolved to generate oleosins having different func- tions.

Most plant seeds store oils (triacylglycerols) which are synthesized during seed maturation and mobilized during postgerminative seedling growth. The storage oils are con- fined to subcellular organelles called oil bodies (lipid bodies, oleosomes, and spherosomes). The spherical oil body is about 1 pm in diameter, and consists of a matrix of triacylglycerols surrounded by a “half-unit” membrane (l-4). This unique

* This work was supported by the United States Department of Agriculture Grant 88-37262-3530. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505212.

$ Ph.D. Candidate of the University of South Carolina. Present address: Biology Dept., Washington University, St. Louis, MO 63130.

membrane has only one layer of phospholipids, and their acyl moieties are supposed to face inside to interact with the hydrophobic triacylglycerol core. Embedded tightly in the membrane is a small group of proteins which are unique to the oil bodies (5).

We have initiated an intensive study on the oil body pro- teins using the scutellum (the oil storage tissue) of maize kernel as a model system (5,6). The proteins of isolated maize oil bodies can be resolved into four major proteins by SDS’ polyacrylamide gel electrophoresis. The protein with a higher M, (40,000) is designated as H protein, while the other three proteins having lower M, values (19,000, 18,000 and 16,000) are called Ll, L2, and L3, respectively. Together, these four proteins constitute about 5-10% of the total scutellum pro- tein. L3, L2, Ll, and H represent about 50, 20, 10, and 20%, respectively, of the total oil body proteins. The L proteins are only found in oil bodies, whereas the H protein is also detected in the microsomes. In addition to their low M, values, the three L proteins share other common features, including their alkaline pl values, resistance to solubilization from the oil bodies after repeated washing, and partitioning into the hy- drophobic phase in Triton X-114 extraction. Subsequent stud- ies on the oil bodies from soybean (7) and rapeseed (8) found proteins with characteristics very similar to those of the maize L proteins. The maize L proteins, as well as similar proteins of other seed oil bodies are termed oleosins. In this report, we refer to the L proteins by their M, values (i.e. L2 as KD 18 and L3 as KD 16).

So far, most of the studies on oleosins have been on the general characteristics of the proteins. The structure of the oleosin genes has not been studied. Only one oleosin (maize KD 16) has been studied using a cDNA which contains an incomplete coding sequence (6). The deduced amino acid sequence of KD 16 suggests that the protein contains three structural domains: a hydrophilic domain at the N terminus, a hydrophobic hairpin a-helical domain at the center, and an amphipathic a-helix domain at the C terminus. Whether these secondary structures are unique to KD 16 or also present in other oleosins of maize and other seed species is unknown. This information is important in the elucidation of the struc- ture/function relationship of oleosins.

In this report, we present findings on a new maize oleosin (KD 18), including the sequence of a cDNA clone and that of a 3.1-kb segment of a genomic clone containing the complete coding sequence of 0.56 kb as well as 1.5 kb of the 5’-flanking region and 1.0 kb of the 3’-flanking region. We compare the deduced structures of KD 18 and KD 16 and propose some structural/functional features of the various structural do- mains of the oleosins.

1 The abbreviations used are: SDS, sodium dodecyl sulfate; kb, kilobase; bp, base pair.

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Maize Oil Body Proteins 2239

-1403% -333-CACA

-256 -RY repeats -167-CATC -122-CATC -105-FIY repeats

-40 -TAtA

i

1 -transcript starts 96 -ATG

1043 -AATAAT : 1059 -transcript stops

D 1709

FIG. 4. Essential features of the KD 18 gene. A 3.1-kb frag- ment of a KD 18 genomic clone was sequenced (see Fig. 3), and its essential features including the potential regulatory elements are shown. The positions of the corresponding mRNA and its coding sequence and of the KD 18 cDNA clone are indicated. Neither intron nor appreciable sequence for N-terminal signal peptide is present. The numbers along the genomic DNA represent bp upstream or downstream of the initiation point of transcription. The figure is not drawn to scale.

EXPERIMENTAL PROCEDURES’

RESULTS

Structure of KD 18 Gene-The KD 18 genomic clone con- tains a maize DNA insert of about 14 kb long. Restriction mapping and Southern blotting reveal that KD 18 gene is located in a 4-kb Hind111 fragment. A 3.1-kb section of this Hind111 fragment, which was estimated to contain the coding sequence in the middle, was sequenced. The sequencing strat- egy (Fig. 1) and the sequence (Fig. 3) are shown. The essential features of KD 18 gene are summarized in Fig. 4. Transcrip- tion starts at the site indicated in Fig. 3, as revealed by Sl mapping (Fig. 2). Translation is suggested to start at 98 bp downstream of the transcription site for the following reasons: (a) +98 represents the first ATG (14) on the transcript; (b) the sequence surrounding the suggested initiation codon (AGCAATGGC) matches the consensus sequence surround- ing the translation initiation codon of plant genes (15, 16); (c) the M, calculated from the deduced amino acid sequence concurs with the M, of KD 18 determined by SDS-polyacryl- amide gel. The open reading frame (Fig. 3) is genuine since the deduced amino acid sequence matches with the sequence of a peptide obtained after endoproteinase Lys-C digestion. Transcription appears to terminate at any one of the three nucleotides of 1059-1061 bp (the sequence is TAA), as judged from the nucleotides preceding immediately the poly(A) tail of the cDNA. A polyadenylation signal AATAAT is found at 1043 bp, which is 17-19 bp preceding the transcription ter- mination site. AATAAT is not the most commonly used polyadenylation signal, but it has been found in plant genes (15). The GC content of the whole transcript is 58.4% overall and 74.1% in the coding region.

* Portions of this paper (including “Experimental Procedures,” Figs. 1-3, 5, 7, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass.Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

The KD 18 cDNA clone contains an 862-bp insert excluding 30 bp of a poly(dA) tail and the synthetic EcoRI linkers. The insert covers most of the KD 18-coding region and the com- plete 3’-noncoding region (Fig. 4). A stretch of 197 bp of 5’- terminus of the transcript (including 100 bp coding sequence) is missing from the cDNA clone. The complete identical sequences between the genomic DNA and cDNA, the similar- ities between the M, values of authentic KD 18 and that calculated from the deduced amino acid sequence, and the results from Sl mapping, all indicate the absence of introns in the KD 18 gene.

Potential regulatory elements of short sequences are pres- ent at the 5’-flanking region of KD 18 gene (Figs. 3 and 4). A sequence CTATATAT is found at -40 bp, which is similar to the plant consensus TATA box sequence (17). A perfect RY repeat (18) sequence (CATGCATG) is present at -265 bp, and a similar one (CATGCAAG) is at -105 bp. These repeats are present in almost all of the legume seed-protein genes (19) and some cereal seed-protein genes (20) and are thought to play a role in the regulation of transcription of seed-protein genes. A “CATC” box has been found as a highly conserved tetranucleotide sequence in the 5’-flanking regions of many cereal seed-protein genes (20). Two such CATC boxes (at -122 bp and -167 bp) are found in the 5’-flanking region of the KD 18 gene. A sequence TAACACAACA is found at -333 bp, which perfectly matches the so-called CACA consensus sequence. This consensus sequence has been found in many legume seed protein genes (21).

Characteristics of the Deduced KD 18 Amino Acid Se- quence--The deduced amino acid sequence (Fig. 3) shows that KD 18 has 187 amino acids and a M, of 18,615. This M, is very close to the experimentally determined M, values of in vivo and in vitro synthesized KD 18 (5). The finding reinforces the idea that there is no characteristic N-terminal signal sequence of KD 18 (17). The protein is rich in Gly, Ala, and Leu (Table I). The proportion of hydrophobic amino acids (22) is 45.7%, which matches the prediction that KD 18 is a moderately hydrophobic protein based on its partitioning in a Triton X-114 system (5). Computer analysis of the local hydropathy of the amino acid sequence by the method of Kyte and Doolittle (23) using a moving window of 7 suggests that the protein consists mainly of three structural domains: a hydrophobic domain in the middle flanked on either side by a relatively hydrophilic domain (Fig. 5). The secondary struc- tures in the three major domains in KD 18 were empirically predicted based on the Chou and Fasman rules (24). A model of the conformation of KD 18 on the surface of an oil body is proposed (Fig. 6) based on the following analytical results. Our proposal is speculative. Nevertheless, it does seem very

3nm I 1

OIL BODY

TAG

PL

CYTOSOL

FIG. 6. A proposed model of the conformation of KD 18 protein on the surface of an oil body. Cylinders are used to depict ol-helices. The phospholipid (PI,) and triacylglyercol (TAG) moieties of the oil body are shown. The amino acids as termini of a-helices are indicated by their numbers.

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2240 Maize Oil Body Proteins

logical for a protein residing on the surface of an oil body, and it provides a model for future experimentation.

The N-terminal peptide of 48 amino acids is largely hydro- philic except a short hydrophobic segment (No. 7 to 18), as judged from the local hydropathic indices. This segment is predicted to be inserted into the phospholipid layer. The two amino acids flanking this segment (No. 6, Arg; No. 19, Gln) are either basic or polar and could interact with the phosphate or the functional groups of the phospholipids. The remaining N-terminal peptide is hydrophilic, and a 2.2-turn a-helix is predicted to occur at amino acids No. 32-39. No cleavage of an N-terminal signal sequence is appreciable. The in vitro and in vivo synthesized KD 18 have indistinguishable M, by SDS-polyacrylamide gel electrophoresis (5). Our initial at- tempts at sequencing KD 18 suggest that the N-terminal amino acid is blocked. It is possible that Met (No. 1) in the newly synthesized KD 18 is removed and Ala (No. 2) is acetylated (14).

The central domain of KD 18 is a strongly hydrophobic segment of 78 amino acids (No. 49-126). Most of the amino acids in this region are individually hydrophobic or neutral, but all possess local hydrophobic values. In the absence of an appropriate data base of known structures of membrane- associated proteins, the empirical prediction of secondary structures is unreliable (25, 26). Based on the studies of Engleman and Steize (27), and the suggestion on the KD 16 protein of maize oil body (6), we present the hydrophobic domain of KD 18 protein as a helical hairpin. In this hairpin structure, each of the two ol-helices is composed of 33 amino acids which constitute a length of about 5 nm. The three Pro (No. 82,89, and 93) localized at the center of this hydrophobic domain would enable the breaking of the a-helix and the forming of a loop of 12 amino acids.

The C-terminal peptide of 61 amino acids (No. 127-187) consists of a 9.2-turn amphipathic a-helix (No. 137-169) and a hydrophobic segment of 14 amino acids (No. 170-183). The latter segment is predicted to be buried inside the phospho- lipid layer; the entry (No. 167, Gln, starting from the amphi- pathic a-helix) and exit (No. 184, Arg) amino acids, being either positively charged or polar, would be able to interact with the phosphate or functional groups of the monolayer phospholipids on the surface of an oil body. The amphipathic nature of the 9.2-turn a-helix is proposed based on the follow- ing observations. First, there is an alternation of hydrophobic and hydrophilic amino acids along the polypeptide (Fig. 7A). Second, in a helical wheel analysis (28), the 9.2-turn a-helix can be viewed in three continuous helical wheels (Fig. 7B). The central 18 amino acids represent a complete helical wheel, and the amino acids flanking this wheel partially fill the positions of two other wheels. From these wheels, we can see the following characteristics. (a) The hydrophobic amino acids are located on one side while the hydrophilic amino acids are present on the other side. (b) The hydrophilic amino acids flanking the hydrophobic face are either positively charged or polar. (c) The hydrophobic face is larger in the central wheel and smaller in the two flanking wheels. Third, a cylindrical plot (29) shows the presence of a hydrophobic face which is larger in the center than at the opposite ends (Fig. 7C). Together, these observations suggest that the 9.2-turn a-helix is amphipathic. Its hydrophobic portion should face the inte- rior of the oil body and the hydrophilic portion be exposed to the cytosol. The positively charged or polar functional groups of the amino acids at the junction of the amphipathic phase would interact with the negatively charged phosphate groups or functional groups of the surface phospholipids. For maxi- mal hydrophobic interactions, the center of the a-helix would

bend toward the interior of the oil bodies. The abundant presence of Gly and Ala, the two amino acid residues contrib- uting to maximal flexibility in protein conformation (30), in the a-helix would facilitate such bending. The hydrophobic face consists of numerous Ala (11 out of 14 residues), and the amphipathic helix is thus less hydrophobic than any of the amphipathic helix found in mammalian apolipoproteins (31, 32).

Comparison between Maize KD 18 and KD 16 Amino Acid Sequences-Both KD 18 and KD 16 possess alkaline p1 values as observed by two-dimensional SDS-polyacrylamide gel elec- trophoresis (5). They are hydrophobic proteins as judged by their partitioning in Triton X-114 (5) and the polarity indices (12) of their deduced amino acid compositions (37.0 and 34.5, respectively). They are both rich in Gly, Ala, and Leu (Table I).

The amino acid sequences of KD 18 and KD 16 were aligned (Fig. 8), and the similarity in amino acids (identical plus similar amino acids) (33) was computed using the computer program of “Bestfit” based on the algorithm of Smith and Waterman (34). There is 57.1% similarity between the aligned amino acid sequences of KD 18 and KD 16 (full length of KD 18 and almost full length of KD 16).

The degree of similarity between KD 18 and KD 16 in each of the three predicted structural domains is distinct. There is only 36% similarity between the N-terminal hydrophilic do- mains and 39% similarity between the C-terminal amphi- pathic domains. These are low values of sequence similarity and are considered of little significance in sequence compari- sons (35). In contrast, the KD 18 and KD 16 central hydro- phobic hairpin ol-helices are 72% similar. Fifty % of the amino acids in this region are identical. The two proteins are most similar in the “loop” and its flanking region, where 15 of the 17 amino acids (88%) are identical and the remaining two amino acids are also similar (giving a total of 100% similarity). The high degree of similarity and identity of the amino acid sequences in the hairpin domain is considered to be of distinct phylogenetic homology (36).

Although the predicted secondary structures of KD 18 and KD 16 are strikingly similar in having three major domains, the C-terminal 18 amino acids in KD 18 is missing from KD 16 (Fig. 8). This feature alone can account for most of the difference between the M, values of the two proteins.

The N-terminal hydrophilic domain and the C-terminal amphipathic domain are relatively more hydrophilic than the central domain and are predicted to be antigenic (14). Since KD 18 and KD 16 are highly similar in amino acid sequences only in the central hairpin domains, antibodies raised against one protein would be less likely to recognize the dominant antigenic determinants of the other protein. This is consistent

KD18 1

KD16 1

FIG. 8. An alignment of amino acid sequences of KD 18 and KD 16. The similar amino acids are shown by bar connections, while the identical ones are also boxed.

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N-terminal csntral C-terminal domain domain domain

i

-____-___^____-___LCLL________LL__I__L__~~-~--~~~, 1

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FIG. 9. A dot matrix plot showing similarity of coding nu- cleotide sequences between KD 18 and KD 16. Each dot repre- sents a 75% similarity (37) with a moving window of 20 nucleotides. The two long diagonals in the center of the plot shows a high degree of similarity between the KD 18 and KD 16 nucleotide sequences encoding the central domain. The dense dots at the lower left corner reflect localized similarity between the two sequences encoding the N-terminal domain, where each sequence has a tandem array of CAG codons (5 in KD 18 and 4 in KD 16) encoding Gln.

with our findings that KD 18 and KD 16 antibodies and antigens are not cross-reactive (data not shown).

Comparison between KD 18 and KD 16 Coding Nucleotide Sequences-When the coding nucleotide sequences for KD 18 and KD 16 were aligned for best fit, the divergence for the three structural domains observed in KD 18 and KD 16 amino acid sequences was also detected at the nucleotide level, although less strikingly. The overall nucleotide identity is 56.2%. The identities in the regions encoding the N-terminal, central hairpin, and C-terminal domains are 42.2, 71.0, and 48.8%, respectively. When the two nucleotide sequences were compared by a dot matrix analysis (37), only the sequences encoding the central hairpin domain show a high degree of similarity (Fig. 9).

DISCUSSION

Oleosins belong to a new class of seed proteins related to the deposition of storage oils. They are apparently encoded by a small gene family. In this report, we present, for the first time, the complete genomic sequence of a member of the gene family. The gene contains no intron. Several potential regu- latory elements of short sequences at the noncoding region are identified. Some of these elements, such as the TATA box and the AATAAT sequence, are likely to be universal signals for the general transcriptional and translational controls. Other elements, including the RY repeats, the CACA consen- sus sequence, and the CATC boxes, have been found at the 5’-flanking regions of genes encoding storage proteins in seeds. Their presence in the KD 18 gene indicates that they are not unique to the expression of storage protein genes but rather may participate in the general controls of gene expres- sion in maturing seeds. These controls include the quantity, tissue specificity, temporal control, and hormonal regulation, and they are shared, to a great extent, between the genes for storage proteins and the genes for storage oil-body proteins in seeds.

Earlier we showed that maize KD 18 and KD 16 share many similar chemical and biological properties (2, 5, 38). The similar chemical properties include alkaline p1 values, low M, values, and hydrophobicity. The similar biological

properties are localization in oil bodies, expression in a tem- poral and tissue-specific manner, and hormonal regulation. In this paper, we make a direct comparison between their encoded nucleotide sequences and amino acid sequences. The two proteins both possess three very similar structural do- mains. However, the degree of similarities among the three domains are quite different. Specifically, the central hairpin domains are very similar in nucleotide and amino acid se- quences, whereas the other two domains are more diverged. We propose that KD 18 and KD 16 genes are derived from a common ancestoral gene and that KD 18 and KD 16 are isoform proteins.

A common origin of KD 18 and KD 16 has its implications. First, the extensive amino acid substitutions in the N-termi- nal hydrophilic domain and the amphipathic a-helix domain suggests that these regions have fewer structural constraints relative to the central hairpin region for carrying out meta- bolic functions. The metabolic functions of oleosins may include maintaining the integrity of the oil bodies and provid- ing recognition signals for specific lipase anchorage in lipol- ysis during seedling growth (2, 39). Alternatively, the two domains may have evolved such that the KD 18 and KD 16 perform different functions due to variations in the two domains. The situation may be analogous to the different types of the mammalian apolipoproteins (31,32). Second, the close similarity between the amino acid sequences of the hairpin oc-helices of KD 18 and KD 16 reflects a severe structural/functional constraint imposed by the hydrophobic core of the oil body. Although the maize oleosins are synthe- sized in the rough endoplasmic reticulum, they do not have an N-terminal signal sequence that is processed co- or post- translationally (5). The signal residing in the oleosins, which is required for subcelIuIar protein trafficking, may be provided by the very hydrophobic a-helix hairpin domain. In the two maize oleosins, each of the proposed cY-helices of the hairpin has 33 amino acid residues, which would form a 5nm a-helix. This length, plus an additional but unknown value contrib- uted by the middle polypeptide loop, makes the hairpin longer than the thickness of the acyl portion (4.5 nm) of a bilayer phospholipid membrane. Therefore, the hairpin structure may be stable in the monolayer phospholipid surface of an oil body but not in the bilayer phospholipid of a normal cell membrane. We propose that the hairpin domain provides a recognition signal for oleosins in subcellular protein trafficking as well as a strong anchorage for the protein on the oil body surface. The instability of the hairpin domain in a normal bilayer phospholipid membrane may explain the selective removal of the maize oleosins from the membrane after lipolysis in postgermination (39, 40).

Acknowledgments-We thank Drs. K. Lai, L. L. Walling, and Y. C. Chang for excellent advice on our work.

1.

2.

3.

4. 5.

6.

REFERENCES

Huang, A. H. C. (1985) in Modern Methods of Plant Analysis (Linskins, H. F., and Jackson, J. F., eds) Vol. 1, pp. 145-151, Springer, Verlag, Berlin

Huang, A. H. C., Qu, R., Wang, S. M., Vance, V. B., Cao, Y. Z., and Lin, Y. H. (1987) in The Metabolism, Structure, and Function of Plant Lipids (Stumpf, P. K., Mudd, J. B., and Nes, W. D., eds) pp. 239-246, Plenum Publishing Co., New York

Stymme, S., and Stobard, A. K. (1987) in The Biochemistry of Plants (Stumpf, P. K., and Conn, E. E., eds) Vol. 10, pp. 175- 214, Academic Press, New York

Yatsu, L. Y., and Jacks, T. L. (1972) Plant Physiol. 49, 937-943 Qu, R., Wang, S-m., Lin, Y-h., Vance, V. B., and Huang, A. H.

C. (1986) &o&em. J. 235, 57-65 Vance, V. B., and Huang, A. H. C. (1987) J. Biol. Chem. 262,

11275-11279

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ber 3, 2020http://w

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.jbc.org/D

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2242 Maize Oil Body Proteins

7. Herman, E. M. (1987) Plunta 172, 336-345 8. Murphy, D. J., Cummins, I., and Kang, A. S. (1989) Biochem. J.

258,285-293 9. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038

10. Taylor, B., and Powell, A. (1982) Focus (Idaho) 4, 4-6 11. Sanger, F., Nicklen, S., and Colson, A. R. (1977) Proc. N&l. Acad.

Sci. Cr. S. A. 74,5463-5467 12. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

13. Chang, Y. C. (1989) Developmental Regulation of Soybean Chlo- rophyll a/b Binding Protein Gene Expression. Ph.D. disserta- tion, University of California, Riverside

14. von Heijne, G. (1987) Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit, Academic Press, San Diego

15. Heidecker, G., and Messing, J. (1986) Annu. Reu. Plant Physiol. 37,439-466

16. Lutecke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., Kern, H. F., and Scheele, G. A. (1987) EMBO J. 6,43-48

17. Messing, J., Geraghty, D., Heidecker, G., Hu, N.-T., Kridl, J., and Rubenstein, I. (1983) in Genetic Engineering of Plants, An Agricultural Perspective (Kosuge, T., and Meredith, C. P., eds), pp. 211-227, Plenum Publishing Co., New York

18. Hoffman, L. M., and Donaldson, D. D. (1985) EMBO J. 4, 883- 889

19. Dickenson, C. D., Evans, R. P., and Nielsen, N. C. (1988) Nucleic Acids. Res. 16, 371

20. Forde, B. G., Heyworth, A., Pywell, J., and Kreis, M. (1985) Nucleic Acids. Res. 13, 7327-7339

21. Goldberg, R. B. (1986) Phil. Trans. Roy. Sci. (London) B314, 343-353

22. Capaldi, R. A., and Vanderkooi, G. (1972) Proc. N&l. Acud. Sci. U. S. A. 69,930-932

23. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132

24. Chou, P. Y., and Fasman, G. D. (1978) Annu. Reu. Biochem. 47, 251-276

25. Green, N. M., and Flanangan, M. T. (1976) Biochem. J. 153, 729-732

26. MacLennan, D. H., Brandl, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 696-700

27. Engleman, D. M., and Steize, T. A. (1981) Cell 23, 411-422 28. Schiffer, M., and Edmundson, A. B. (1967) Biophys. J. 7, 121-

125 29. Taylor, J. W., Miller, R. J., and Kaiser, E. T. (1982) Mol.

Pharmucol. 22,657-666 30. Nemethy, G., Leach, S. J., and Scheraga, H. A. (1966) J. Phys.

Chem. 70,998-1008 31. Driscoll, D. M., and Getz, G. S. (1986) Methods Enzymol. 128,

41-70 32. Luo, C.-C., Li, W.-H., Moore, M. N., and Chan, L. (1986) J. Mol.

Biol. 187,325-340 33. Dayhoff, M. O., Eck, R. V. and Park, C. M. (1972) in Atlas of

Protein Sequence and Structure 1972 (Dayhoff, M. O., ed) Vol. 5, pp. 88-99, National Biomedical Research Foundation, Wash- ington, D. C.

34. Smith, T. F., and Waterman, M. S. (1981) Adu. Appl. Math. 2, 482-489

35. Doolittle, R. F. (1986) Of URFs and ORFs: A Primer on How to Analvze Derived Amino Acid Seouences. PP. 12-15, University Science Books, Mill Valley, CA . - _

36. Davhoff. M. 0.. Barker. W. C., and Hunt, L. T. (1983) Methods &zytkol. 9 1; 524-545

37. Maizel, J. V., and Lenk, R. P. (1981) Proc. N&l. Acad. Sci. U. S. A. 78, 7665-7669

38. Vance, V. B., and Huang, A. H. C. (1988) J. Biol. Chem. 263, 1476-1481

39. Wang, S., and Huang, A. H. C. (1987) J. Biol. Chem. 262,2270- 2274

40. Fernandez, D. E., Qu, R., Huang, A. H. C., and Staehelin, L. A. (1988) Plant Physiol. 86, 270-274

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R D Qu and A H Huangand the deduced protein structure.

Oleosin KD 18 on the surface of oil bodies in maize. Genomic and cDNA sequences

1990, 265:2238-2243.J. Biol. Chem. 

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