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PEPTIDE PLANT HORMONE, PHYTOSULFOKINE 73 asparagus and carrot. Among these, the greatest abundance of binding sites was found in the carrot membranes: approximately 150 fmol per mg microsomal proteins, with an estimated dissociation constant (Kd) of 4.2 nM. This PSK receptor protein was visualized by photoaffinity labeling of carrot plasma membrane fractions using the photoactivable 125 I-labeled PSK analog [N -(4-azidosalicyl) Lys 5 ]PSK (Fig. 5), the binding activity of which are ≈ 10% of unmodified PSK.(Matsubayashi Y & Sakagami Y. 2000) SDS-PAGE analysis of the labeled proteins indicated that a 120-kD protein and a minor 150-kD protein specifically interact with PSK. Both proteins contain approximately 10 kD of N- linked oligosaccharide chains that can be cleaved by treatment with peptide N-glycosidase F. We purified these PSK-binding proteins from microsomal fractions of carrot cells by Triton X- 100 solubilization and specific ligand-based affinity chromatography using a [Lys 5 ]PSK-Sepharose column containing a long spacer chain between ligand and matrix. (Matsubayashi Y. et al. 2002) SDS-PAGE of the proteins in the fractions eluted by PSK showed specific recovery of a major 120- kD protein and a minor 150-kD protein. Both proteins were absent in the fractions eluted by an inactive PSK analogs; this indicates that binding of the proteins is specific. Several independent purifications were performed, yielding 50 µg of the major 120-kD protein with 96,000-fold purification from 4,800 mg of microsomal proteins (corresponds to 24 L of suspension-cultured cells), with an overall recovery rate of 40%. (Matsubayashi Y. et al. 2002) Based on the internal sequence of this protein, a 3.5-kb cDNA clone was isolated from the carrot cDNA library. The cDNA encoded a 1021-amino-acid protein with a deduced molecular mass of 112 kD, with features found in several hormone receptors in plants and animals (Fig. 5). It contained an N-terminal hydrophobic signal sequence, extracellular leucine-rich repeats (LRRs), a transmembrane domain, and a cytoplasmic kinase domain. Northern blot analysis showed that mRNA of this protein accumulated ubiquitously in leaf, apical meristem, hypocotyl and root of carrot seedlings, although its expression level was far lower than in cultured carrot cells. The major extracellular domain of this protein contained 21 tandem copies of a 24-amino-acid LRR; it has been suggested that this string of LRRs plays a key role in protein-protein interactions. (Kobe B & Deisenhofer J. 1994) In addition, a 36-amino-acid island was detected in the 18 th LRR. An island domain has also been found among the extracellular LRRs of the brassinosteroid receptor BRI1, and has been shown to be critical for its function. (Li J & Chory J. 1997) Transgenic carrot cells overexpressing the cDNA of the major 120-kD protein showed a significant increase in PSK binding sites in the membrane fractions and accelerated growth in response to PSK, compared with control cells. Photoaffinity cross-linking and immunoprecipitation analysis of the membrane proteins derived from the transformants revealed expression of the 150- kD protein in addition to the 120-kD protein, indicating that both proteins are encoded by a single gene. In contrast, expression of antisense mRNA of the major 120-kD protein significantly inhibited callus growth. These findings strongly suggest that this receptor kinase is a component of a functional PSK receptor that directly interacts with PSK.
74 Yoshikatsu MATSUBAYASHI Fig. 6. Schematic of the 120-kD PSK receptor. The diagram shows the signal peptide (SP, red), extracellular leucine-rich repeats (LRRs, yellow), a 36-amino-acid island, a transmembrane domain (TM, blue), and a cytoplasmic kinase domain (green). Future perspectives Now that in vitro function of PSK and the molecular basis of ligand-receptor interaction in PSK signaling have been established, the next phase of research is characterization of the in vivo role of PSK and its downstream signaling pathway in plants. The carrot PSK receptor exhibits a high percentage of amino acid identity with several LRR receptor-like kinases found in Arabidopsis. The sequencing of the Arabidopsis genome is now complete, and large collections of gene-disruption lines are available. Once PSK-binding activities of these LRR-RLKs are confirmed, direct clues to in vivo function of PSK will be provided by phenotypes of knockout mutants. References 1. Beisswanger R, Corbeil D, Vannier C, Thiele C, Dohrmann U, Kellner R, Ashman K, Niehrs C, Huttner WB. (1998) Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2. Proc Natl Acad Sci USA 95: 11134-11139. 2. Bundgaard JR, Vuust J, Rehfeld JF. (1997) New consensus features for tyrosine O-sulfation determined by mutational analysis. J Biol Chem 272: 21700-21705. 3. Fukuda H, Komamine A. (1980) Establishment of an experimental system for the tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans. Plant Physiol 65: 57-60. 4. Hanai H, Matsuno T, Yamamoto M, Matsubayashi Y, Kobayashi T, Kamada H, Sakagami Y. (2000) A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation. Plant Cell Physiol 41: 27-32. 5. Hanai H, Nakayama D, Yang H, Matsubayashi Y, Hirota Y, Sakagami Y. (2000) Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro. FEBS Lett 470: 97-101. 6. Harris RB. (1989) Processing of pro-hormone precursor proteins. Arch Biochem Biophys 275: 315-333. 7. Hazum E. (1983) Photoaffinity labeling of peptide hormone receptors. Endocr Rev 4: 352-362. 8. Huttner WB. (1982) Sulphation of tyrosine residues, a widespread modification of proteins. Nature 299: 273-276. 9. Huttner WB. (1988) Tyrosine sulfation and the secretory pathway. Annu Rev Physiol 50: 363-376. 10. Kobayashi T, Eun C-H, Hanai H, Matsubayashi Y, Sakagami Y, Kamada H. (1999) Phytosulfokine-, a peptidyl plant growth factor, stimulates somatic embryogenesis in carrot. J Exp Botany 50:1123-1128. 11. Kobe B, Deisenhofer J. (1994) The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 19: 415-421.
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ISSN 0435-1096 Gamma Field Symposia
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The lecturers and the members of th
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KANEKO, T. KARITA, E. KASHIMA, M. K
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TANO, S. TSUCHIDA, Y. TSUTSUMI, N.
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The Symposium Committee Shigeki NAG
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CONTENTS Y. KAMIYA Biosyntheses and
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2 Yuji KAMIYA Fig. 1. Gibberellin b
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4 Yuji KAMIYA using the receptor pr
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6 Yuji KAMIYA Fig. 3. Origin of sid
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8 Yuji KAMIYA References AKIYOSHI,
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10 Yuji KAMIYA SHINOMURA, T. (1997)
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12 Yuji KAMIYA 8’ 8’ 273
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14 Motoyuki ASHIKARI, Hironori ITOH
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Page 31 and 32: 20 Motoyuki ASHIKARI, Hironori ITOH
Page 37 and 38: 26 Atsuhiro OKA and how their signa
Page 39 and 40: 28 Atsuhiro OKA At almost the same
Page 41 and 42: 30 Atsuhiro OKA division for xylem
Page 43 and 44: 32 Atsuhiro OKA the RR domain in th
Page 45 and 46: 34 Atsuhiro OKA Fig. 4. Cross-talk
Page 47 and 48: 36 Atsuhiro OKA OKA, A., SAKAI, H.
Page 49 and 50: 38 Atsuhiro OKA ARR4 PHYB PHYBP
Page 51 and 52: Gamma Field Symposia, No. 42, 2003
Page 53 and 54: ETHYLENE-SIGNALING PATHWAY 43 signa
Page 55 and 56: ETHYLENE-SIGNALING PATHWAY 45 The f
Page 57 and 58: ETHYLENE-SIGNALING PATHWAY 47 Snf3p
Page 59 and 60: ETHYLENE-SIGNALING PATHWAY 49 In sp
Page 61 and 62: ETHYLENE-SIGNALING PATHWAY 51
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Page 65 and 66: MECHANISM OF BRASSINOSTEROID SIGNAL
Page 77 and 78: 68 Yoshikatsu MATSUBAYASHI are grow
Page 79 and 80: 70 Yoshikatsu MATSUBAYASHI Fig. 3.
Page 81: 72 Yoshikatsu MATSUBAYASHI Fig. 5.
Page 85 and 86: 76 Yoshikatsu MATSUBAYASHI 33. Yang
Page 87 and 88: 78 Yoshikatsu MATSUBAYASHI GUS
Page 89 and 90: 80 BRI1
Page 91 and 92: 82 BRI 10
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Page 95 and 96: 86 PSK 23 BIL1/CFP
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