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Y Matsubayashi

Publications and source records attributed to Y Matsubayashi.

At least 19 recordsLinked to original sources
PubMed2001

Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells.

The classical mitogen-activated protein kinase (MAPK, also known as ERK) pathway is widely involved in eukaryotic signal transductions. In response to extracellular stimuli, MAPK becomes activated and translocates from the cytoplasm to the nucleus. At least two pathways for the nuclear import of MAPK are shown to exist; passive diffusion of a monomer and Ran-dependent active transport of a dimer, the detailed molecular mechanism of which is unknown. In this study, we have reconstituted nuclear import of MAPK in vitro by using digitonin-permeabilized cells with GFP-fused MAPK protein (GFP-MAPK), which is too large to pass through the nuclear pore by passive diffusion. GFP-MAPK was able to accumulate in the nucleus irrespective of its phosphorylation state. This import of GFP-MAPK occurred even in the absence of any soluble cytosolic factors or ATP but was inhibited by wheat germ agglutinin or an excess amount of importin-beta or at low temperatures. Moreover, MAPK directly bound to an FG repeat region of nucleoporin CAN/Nup214 in vitro. Taken together, these results suggest the third pathway for nuclear import of MAPK, in which MAPK passes through the nuclear pore by directly interacting with the nuclear pore complex.

Biological Transport↗
PubMed2001

Peptide signals and their receptors in higher plants.

At least four peptides play a vital role in plant cell-cell communication by means of their specific receptors. Two of these receptors have been identified as receptor kinases, which form a large family of receptor molecules in plants. These findings highlight the significance of receptor-mediated peptide signaling in various physiological events in plants, and predict the existence of further peptide-signal-interacting receptor kinases. Some candidates have been found in plant genomes. Here, we outline recent progress and future challenges in the signaling peptide analysis, which began with systemin, phytosulfokine, CLAVATA3 and S-locus cysteine-rich protein (also called S-locus protein 11).

Arabidopsis↗
PubMed2001

Diversity of Arabidopsis genes encoding precursors for phytosulfokine, a peptide growth factor.

Phytosulfokine-alpha (PSK-alpha), a unique plant peptide growth factor, was originally isolated from conditioned medium of asparagus (Asparagus officinalis) mesophyll cell cultures. PSK-alpha has several biological activities including promoting plant cell proliferation. Four genes that encode precursors of PSK-alpha have been identified from Arabidopsis. Analysis of cDNAs for two of these, AtPSK2 and AtPSK3, shows that both of these genes consist of two exons and one intron. The predicted precursors have N-terminal signal peptides and only a single PSK-alpha sequence located close to their carboxyl termini. Both precursors contain dibasic processing sites flanking PSK, analogous to animal and yeast prohormones. Although the PSK domain including the sequence of PSK-alpha and three amino acids preceding it are perfectly conserved, the precursors bear very limited similarity among Arabidopsis and rice (Oryza sativa), suggesting a new level of diversity among polypeptides that are processed into the same signaling molecule in plants, a scenario not found in animals and yeast. Unnatural [serine-4]PSK-beta was found to be secreted by transgenic Arabidopsis cells expressing a mutant of either AtPSK2 or AtPSK3 cDNAs, suggesting that both AtPSK2 and AtPSK3 encode PSK-alpha precursors. AtPSK2 and AtPSK3 were expressed demonstrably not only in cultured cells but also in intact plants, suggesting that PSK-alpha may be essential for plant cell proliferation in vivo as well as in vitro. Overexpression of either precursor gene allowed the transgenic calli to grow twice as large as the controls. However, the transgenic cells expressing either antisense cDNA did not dramatically decrease mitogenic activity, suggesting that these two genes may act redundantly.

Amino Acid Sequence↗
PubMed2000

120- and 160-kDa receptors for endogenous mitogenic peptide, phytosulfokine-alpha, in rice plasma membranes.

Plant cells in culture secrete a sulfated peptide named phytosulfokine-alpha (PSK-alpha), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable (125)I-labeled PSK-alpha analog, [N(epsilon)-(4-azidosalicyl)Lys(5)]PSK-alpha (AS-PSK-alpha), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands was completely inhibited by unlabeled PSK-alpha and partially decreased by PSK-alpha analogs possessing moderate binding activities. In contrast, PSK-alpha analogs that have no biological activity showed no competition for (125)I-AS-PSK-alpha binding, confirming the specificity of binding proteins. Analysis of the affinity of (125)I incorporation into the protein by ligand saturation experiments gave apparent K(d) values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of (125)I-AS-PSK-alpha cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of (125)I-AS-PSK-alpha was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data suggest that the 120- and 160-kDa proteins are PSK-alpha receptors that mediate the biological activities of PSK-alpha.

Affinity Labels↗
PubMed2000

Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro.

An in vitro assay system to detect tyrosylprotein sulfotransferase (TPST) activity of higher plant cells was established, using synthetic oligopeptides based on the deduced amino acid sequence of a phytosulfokine-alpha (PSK-alpha) precursor. TPST activity was found in microsomal membrane fractions of rice, asparagus and carrot cells and it was confirmed that acidic amino acid residues adjacent to the tyrosine residues of acceptor peptides were essential to the sulfation reaction. The asparagus TPST exhibited a broad pH optimum of 7.0-8.5, required manganese ions for maximal activity and appeared to be a membrane-bound protein localized in the Golgi apparatus. These enzymes should be defined as a new class of plant sulfotransferases that catalyze tyrosine O-sulfation of a PSK-alpha precursor and other unknown proteins.

Amino Acid Sequence↗
PubMed2000

Peptide growth factor phytosulfokine-alpha contributes to the pollen population effect.

Density-dependent pollen germination and tube growth in vitro is a well-documented phenomenon, termed the pollen population effect, but far less is known about its molecular basis. We present evidence to support phytosulfokine-alpha [Y(SO3H)IY(SO3H)TQ; PSK-alpha] as a native bioactive factor contributing to this effect. Mature pollen grains of Nicotiana tabacum L. var. macrophylla were incubated in liquid medium for 2 h. Pollen germination frequency increased in a density-dependent manner from 625 to 46,000 grains/ml. Conditioned medium, obtained from the medium of pollen cultured at a density of 10,000 pollen grains/ml for 12 h, promoted the germination of pollen cultured at a low density (625 grains/ml). A rabbit antiserum against PSK-alpha specifically inhibited the promotive effect of conditioned medium. Quantification by enzyme-linked immunosorbent assay showed that the conditioned medium contained 0.4 nM of PSK-alpha. Exogenous PSK-alpha also stimulated pollen germination in the low-density culture. These results indicate that PSK-alpha is an important regulator involved in the pollen population effect.

Cells, Cultured↗
PubMed2000

Molecular cloning and characterization of OsPSK, a gene encoding a precursor for phytosulfokine-alpha, required for rice cell proliferation.

We previously characterized an OsPSK cDNA encoding a precursor of phytosulfokine-alpha (PSK-alpha), a peptide plant growth factor. Southern blot analysis suggested that OsPSK is a single-copy gene in rice, which we have isolated and characterized. The OsPSK gene consists of one large intron and two exons. The 5-amino acid PSK-alpha sequence located close to the COOH-terminus of the precursor is encoded in the second exon. A putative TATA box was found at position -68 with respect to the transcription initiation site. Upstream of this sequence, several potential regulatory elements, including one CAAT-box, three CCAAT-boxes, one enhancer core-like sequence, and three E-boxes could be identified. By constructing plasmids with various lengths of the 5'-upstream regions of the OsPSK gene fused to the coding sequence for bacterial beta-glucuronidase (GUS), we demonstrated a region 1.9 kb upstream of the transcription initiation point, which contains most of the putative 5'-regulatory elements, to be sufficient for maximal-level GUS expression in transformed rice Oc cells. The promoter of the OsPSK gene gave significantly higher levels of GUS expression than the CaMV 35S promoter. These results suggest that the OsPSK promoter could be useful for the constitutive expression of a foreign gene at high levels in transformed rice culture cells. Northern blot analyses suggest that the expression of OsPSK is reinforced by auxin and cytokinin.

Amino Acid Sequence↗
PubMed2000

A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation.

Somatic embryogenesis of the carrot (Daucus carota L.) depends on a set of factors, some of which accumulate in culture medium (conditioned medium, CM). When embryogenic cell clusters were transferred to an embryo-inducing medium, addition of CM derived from somatic embryo culture markedly stimulated somatic embryo formation. The active principles were purified using a simple bioassay system and identified to be phytosulfokines (PSKs), sulfated oligopeptide growth factors originally isolated from a CM derived from asparagus (Asparagus officinalis L.) mesophyll culture. Quantification studies using a competition ELISA system employing an anti-PSK-alpha polyclonal antibody showed that PSK production might be related to growth of cells, rather than development of somatic embryos. Thus the stimulatory effect of PSK on somatic embryo formation might be due to promotion of cell proliferation.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2000

A rapid and efficient system of Agrobacterium infection-mediated transient gene expression in rice Oc cells and its application for analysis of the expression and antisense suppression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene.

A rapid and efficient system for Agrobacterium infection-mediated transient gene expression in rice has been developed. Using this system, transient expression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene was analyzed. The results suggest that the Agrobacterium infection-mediated transient gene expression system is as efficient in rice Oc cells as in tobacco BY-2 cells and might be useful for rapid analysis not only of foreign gene expression, but also of antisense gene suppression.

Actins↗
PubMed2000

Phytosulfokine-alpha, a peptide growth factor found in higher plants: its structure, functions, precursor and receptors.

Phytosulfokine-alpha, a sulfated pentapeptide growth factor universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. It is similar to animal polypeptide hormones in that it is processed from a larger precursor, preprophytosulfokine, although the putative processing sites do not conform to consensus sequences for endoproteolytic processing sites flanking animal prohormones. Like the animal preprohormones, preprophytosulfokine also has a signal peptide at the N-terminus for targeting to secretory pathways. The preprophytosulfokine gene has been confirmed to be expressed in vivo as well as in vitro.

Amino Acid Sequence↗
PubMed1999

Oryza sativa PSK gene encodes a precursor of phytosulfokine-alpha, a sulfated peptide growth factor found in plants.

Phytosulfokine-alpha [PSK-alpha, Tyr(SO(3)H)-Ile-Tyr(SO(3)H)-Thr-Gln], a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at very low concentrations. Oryza sativa PSK (OsPSK) cDNA encoding a PSK-alpha precursor has been isolated. The cDNA is 725 base pairs long, and the 89-aa product, preprophytosulfokine, has a 22-aa hydrophobic region that resembles a cleavable leader peptide at its NH(2) terminus. The PSK-alpha sequence occurs only once within the precursor, close to the COOH terminus. [Ser(4)]PSK-alpha was secreted by transgenic rice Oc cells harboring a mutated OsPSK cDNA, suggesting proteolytic processing from the larger precursor, a feature commonly found in animal systems. Whereas PSK-alpha in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. Preprophytosulfokine mRNA was detected at an elevated constitutive level in rice Oc culture cells on RNA blot analysis. Although PSK-alpha molecules have never been identified in any intact plant, reverse transcription-PCR analysis demonstrated that OsPSK is expressed in rice seedlings, indicating that PSK-alpha may be important for plant cell proliferation both in vitro and in vivo. DNA blot analysis demonstrated that OsPSK homologs may occur in dicot as well as monocot plants.

Amino Acid Sequence↗
PubMed1999

Characterization of specific binding sites for a mitogenic sulfated peptide, phytosulfokine-alpha, in the plasma-membrane fraction derived from Oryza sativa L.

Treatment of rice cells with an endogenous mitogenic peptide, phytosulfokine-alpha (PSK-alpha), results in cell proliferation. In the present study, [3H]PSK-alpha prepared by catalytic reduction of a PSK-alpha analog containing tetradehydroisoleucine was employed to identify putative PSK-alpha target molecules on rice plasma membranes. Membrane binding of the ligand was found to be saturable, reversible and pH dependent. Scatchard analysis demonstrated the existence of both high- and low-affinity binding sites with Kd values of 1.4 nM and 27 nM, respectively. Competition studies with [3H]PSK-alpha and several PSK-alpha analogs showed that displacing activity closely corresponds to the ability to induce cell proliferation. The properties of the binding sites distributed on plasma membranes are consistent with the function of PSK-alpha receptors in activating a cascade of molecular events involved in plant cell proliferation.

Binding Sites↗
PubMed1999

The endogenous sulfated pentapeptide phytosulfokine-alpha stimulates tracheary element differentiation of isolated mesophyll cells of zinnia

Dispersed zinnia (Zinnia elegans) mesophyll cells cannot differentiate into tracheary elements (TEs) at low cell density conditions even if auxin and cytokinin are present in the medium, indicating the involvement of intercellular interactions during the initiation and/or subsequent progresses in TE differentiation. When zinnia cells were incubated at a low density (2.5 x 10(4) cells mL(-1)) in TE-inductive medium in the presence of various concentrations of phytosulfokine (PSK)-alpha, which was originally identified as an intercellular signal peptide involved in cell proliferation, TE differentiation was strongly stimulated in a dose-dependent fashion; more than 35% of the living cells differentiated into TEs by 5 d of culture in the presence of 10 nM PSK-alpha. Enzyme-linked immunosorbent assay and mass spectroscopy confirmed that cultured zinnia cells produce nanomolar levels of PSKs under inductive conditions. These results suggest that PSK-alpha is a factor responsible for TE differentiation of zinnia mesophyll cells.

Journal Article↗
PubMed1999

Promotive effects of the peptidyl plant growth factor, phytosulfokine-alpha, on the growth and chlorophyll content of Arabidopsis seedlings under high night-time temperature conditions.

In order to investigate the function of the peptidyl plant growth factor, phytosulfokine-alpha (PSK-alpha), in plants, we examined the effect of PSK-alpha on the growth and chlorophyll content of Arabidopsis seedlings under high night-time temperature conditions. Although exposure to high night-time temperatures markedly reduced the fresh weight and chlorophyll content of the seedlings, these parameters in the plants supplied with PSK-alpha remained at the same levels as those of non-treated controls. These effects were not apparent when [2-5]PSK, Tyr-SO3H and kinetin were similarly supplied. The results suggest that PSK-alpha not only promotes cell proliferation, but may aid plants in their tolerance of heat stress.

Adenine↗