Abstract
Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.
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References
Barbier-Brygoo H, Ephritikhine G, Klämbt D, Ghislain M, Guern J: Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts. Proc Natl Acad Sci USA 86: 891–895 (1989).
Barbier-Brygoo H, Ephritikhine G, Klämbt D, Maurel C, Palme K, Schell J, Guern J: Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts. Plant J 1: 83–93 (1991).
Brightman AO, Barr R, Crane FL, Morré DJ: Auxin-stimulated NADH oxidase purified from plasma membrane of soybean. Plant Physiol 86: 1264–1269 (1988).
Choi S: EMBL/GenBank/DDBJ databases accession number Z48451 (1995).
Diekmann W, Venis MA, Robinson DG: Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts. Proc Natl Acad Sci USA 92: 3425–3429 (1995).
Dohrmann U, Hertel R, Kowalik H: Properties of auxin binding sites in different subcellular fractions from maize coleoptiles. Planta 140: 97–106 (1978).
Gabathuler R, Cleland RE: Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles. Plant Physiol 79: 1080–1085 (1985).
Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269 (1983).
Hesse T, Feldwisch J, Balshüsemann D, Bauw G, Puype M, Vandekerckhove J, Löbler M, Klämbt D, Schell J, Palme K: Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin. EMBO J 8: 2453–2461 (1989).
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
Inohara N, Shimomura S, Fukui T, Futai M: Auxin-binding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure. Proc Natl Acad Sci USA 86: 3564–3568 (1989).
Jones AM: Auxin-binding protein. Annu Rev Plant Physiol Plant Mol Biol 45: 393–420 (1994).
Jones AM, Herman EM: KDEL-containing auxin-binding protein is secreted to the plasma membrane and cell wall. Plant Physiol 101: 595–606 (1993).
Jones AM, Lamerson P, Venis MA: Comparison of site I auxin binding and a 22-kilodalton auxin-binding protein in maize. Planta 179: 409–413 (1989).
Joshi CP: An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl Acids Res 15: 6643–6653 (1987).
Kornfeld R, Kornfeld S: Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54: 631–664 (1985).
Kunkel TA: Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82: 488–492 (1985).
Lazarus CM, Macdonald H: Characterization of a strawberry gene for auxin-binding protein, and its expression in insect cells. Plant Mol Biol 31: 267–277 (1996).
Leblanc N, Roux C, Pradier JM, Perrot-Rechenmann C: Characterization of two cDNAs encoding auxin-binding proteins in Nicotiana tabacum. Plant Mol Biol 33: 679–689 (1997).
Löbler M, Klämbt D: Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization. J Biol Chem 260: 9848–9853 (1985).
Löbler M, Klämbt D: Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). II. Localization of a putative auxin receptor. J Biol Chem 260: 9854–9859 (1985).
Munro S, Pelham, HRB: AC-terminal signal prevents secretion of luminal ER proteins. Cell 48: 899–907 (1987).
Nakamura C, Ono H: Solubilization and characterization of a membrane-bound auxin-binding protein from cell suspension cultures of Nicotiana tabacum. Plant Physiol 88: 685–689 (1988).
Napier RM, Fowke LC, Hawes C, Lewis M, Pelhem HRB: Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J Cell Sci 102: 261–271 (1992).
NiBharlo G, Gallagher TF: EMBL/GenBank/DDBJ databases accession number U77952 (1996).
Palme K, Hesse T, Campos N, Garbers C, Yanofsky MF, Schell J: Molecular analysis of an auxin-binding protein gene located on chromosome 4 of Arabidopsis. Plant Cell 4: 193–201 (1992).
Pelham HRB: The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 15: 483–486 (1990).
Ray PM: Auxin-binding sites ofmaize coleoptiles are localized on membranes of the endoplasmic reticulum. Plant Physiol 59: 594–599 (1977).
Ray PM, Dohrmann U, Hertel R: Specificity of auxin-binding sites on maize coleoptile membranes as possible receptor sites for auxin action. Plant Physiol 60: 585–591 (1977).
Rück A, Palme K, Venis MA, Napier RM, Felle HH: Patchclamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts. Plant J 4: 41–46 (1993).
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA: Primer-directed enzymatic ampli-fication ofDNAwith a thermostableDNApolymerase. Science 239: 487–491 (1988).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanger F, Nicklen S, Loulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Santoni V, Vansuyt G, Rossignol M: The changing sensitivity to auxin of the plasma-membrane H+-ATPase: relationship between plant development andATPase content ofmembranes. Planta 185: 227–232 (1991).
Schwob E, Choi SY, Simmons C, Migliaccio F, Ilag L, Hesse T, Palme K, Söll D: Molecular analysis of three maize 22 kDa auxin-binding protein genes: transient promoter expression and regulatory regions. Plant J 4: 423–432 (1993).
Shimomura S, Inohara N, Fukui T, Futai M: Different properties of two types of auxin-binding sites in membranes from maize coleoptiles. Planta 175: 558–566 (1988).
Shimomura S, Liu W, Inohara N, Watanabe S, Futai M: Structure of the gene for an auxin-binding protein and a gene for 7SL RNA from Arabidopsis thaliana. Plant Cell Physiol 34: 633–637 (1993).
Shimomura S, Sotobayashi T, Futai M, Fukui T: Purification and properties of an auxin-binding protein from maize shoot membranes. J Biochem 99: 1513–1524 (1986).
Simpson CG, Leader DJ, Brown JWS: Plant intron sequences. In: Croy RRD (ed) Plant Molecular Biology Labfax, pp. 183–251. Bios Scientific Publishers, Oxford (1993).
Thiel G, Blatt MR, Fricker MD, White IR, Millner P: Modulation of K+ channels in Vicia stomatal guard cells by peptide homologs to the auxin-binding protein C terminus. Proc Natl Acad Sci USA 90: 11493–11497 (1993).
Tillmann U, Viola G, Kayser B, Siemeister G, Hesse T, Palme K, Löbler M, Klämbt D: cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): isolation and characterization by immunologicalmethods. EMBO J 8: 2463–2467 (1989).
Venis MA, Napier RM: Auxin receptors and auxin binding proteins. Crit Rev Plant Sci 14: 27–47 (1995).
Venis MA, Napier RM, Barbier-Brygoo H, Maurel C, Perrot-Rechenmann C, Guern J: Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity. Proc Natl Acad Sci USA 89: 7208–7212 (1992).
Venis MA, Thomas EW, Barbier-Brygoo H, Ephritikhine G, Guern J: Impermeant auxin analogues have auxin activity. Planta 182: 232–235 (1990).
Vreugdenhil D, Harkes PAA, Libbenga KR: Auxin-binding by particulate fractions from tobacco leaf protoplasts. Planta 150: 9–12 (1980).
Walton JD, Ray PM: Evidence for receptor function of auxin binding sites in maize. Red light inhibition of mesocotyl elongation and auxin binding. Plant Physiol 68: 1334–1338 (1981).
Yu LX, Lazarus CM: Structure and sequence of an auxin-binding protein gene from maize (Zea mays L.) Plant Mol Biol 16: 925–930 (1991).
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Watanabe, S., Shimomura, S. Cloning and expression of two genes encoding auxin-binding proteins from tobacco. Plant Mol Biol 36, 63–74 (1998). https://doi.org/10.1023/A:1005999821066
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DOI: https://doi.org/10.1023/A:1005999821066