No.42 - è¾²æ¥­ç”Ÿç‰©è³‡æºç ”ç©¶æ‰€

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.