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PubMed2005

Subcellular localization of neural-specific NPDC-1 protein.

NPDC-1 is a gene specifically expressed in neural cells when they stop to divide and begin to differentiate. Immunocytochemical study analysis of differentiated PC12 cells transfected with NPDC-tag vectors showed that NPDC-1 is transported in vesicles from the Golgi apparatus to the cell membrane and is then likely internalized into endosomes. The protein colocalized, at least partially, with synaptic vesicle proteins: synaptophysin, synaptobrevin 2, and Rab3 GEP (Rab3 GTP/GDP exchange protein). Moreover, subcellular fractionation of rat brain showed that crude synaptic membrane and crude synaptic vesicle fractions were enriched in NPDC-1. Although NPDC-1 bound Rab3 GEP in vitro, it seems unlikely to be involved in Ca2+-dependent exocytosis and, thus, in synaptic vesicle trafficking.

Animals↗
PubMed2001

Cytoplasmic gamma actin as a Z-disc protein.

To investigate the precise localization of cytoplasmic gamma actin in skeletal muscle and the relationship to dystrophin molecules, we designed an antibody against the N-terminal peptide of cytoplasmic gamma actin. Western blot analysis using SDS-PAGE and isoelectric focusing (IEF) gel revealed that the antibody reacted only with the actin isoforms having gamma motility, confirming that the antibody is specific to the cytoplasmic (nonmuscle) gamma actin. Immunohistochemical analysis of the skeletal muscle of the adult mouse revealed a dot-like staining pattern of the antibody in transverse sections and a striated staining pattern in longitudinal sections. The double immunostaining technique revealed the colocalization of cytoplasmic gamma actin with alpha-actinin, implying the localization of the actin on the Z-disc. Contrary to previous findings (1), we did not detect the colocalization of cytochrome oxidase, a mitochondria marker, with this actin.

Actins↗
PubMed1994

Vitronectin in colorectal adenocarcinoma--synthesis by stromal cells in culture.

We have investigated the expression and cellular source of vitronectin in colorectal adenocarcinoma. Immunofluorescence staining of tissue sections revealed the presence of vitronectin in the stroma of the 11 tumors studied, but not in adjacent normal colon. A method was devised for the isolation from colorectal adenocarcinomas of fibroblast-like cells that stained positive for vimentin but negative for cytokeratin. These tumor-derived stromal cells synthesized and secreted vitronectin, as revealed by metabolic labeling and immunoprecipitation. This was confirmed by Southern blot analysis of polymerase chain reaction amplification products from reverse-transcribed RNA. Normal skin fibroblasts did not synthesize vitronectin. Immunofluorescence staining showed vitronectin deposited at focal contact sites in the tumor-derived cells, where it colocalized with vinculin and the alpha v integrin subunit. The deposition of vitronectin into focal contact sites was not dependent on the presence of serum. The finding that vitronectin can be synthesized and secreted by tumor-derived fibroblast-like cells in culture indicates that vitronectin expression can be promoted by as yet unknown signals provided in disease states, such as cancer.

Adenocarcinoma↗
PubMed1993

Colocalization of retinal dystrophin and actin in postsynaptic dendrites of rod and cone photoreceptor synapses.

In this paper we demonstrate immunostaining specific for dystrophin in photoreceptor synapses of human, bovine and rat retinas. Cryosections of retinas incubated with dystrophin-specific monoclonal antibodies displayed a punctuate staining pattern in the outer plexiform layer. This pattern resulted from binding of the antibodies to synaptic complexes of both rods and cones, shown by double-labelling with antibodies to either synaptophysin or actin. Confocal laser fluorescence microscopy demonstrated that dystrophin staining colocalized predominantly with actin, which is concentrated in the postsynaptic portions of the synaptic complex. No significant dystrophin immunolabel was seen in the presynaptic terminals labelled with antibodies to synaptophysin, a marker of synaptic vesicles. Immunoblot analysis confirmed the presence of approximately 420 kDa and approximately 360 kDa dystrophin-like polypeptide bands associated with membranes of the bovine retina. We speculate that retinal dystrophin is involved in the linkage of actin filaments to the postsynaptic plasma membrane. Such a linkage may be important for the generation of synaptic microdomains and for certain phenomena of synaptic plasticity. The absence of dystrophin in patients suffering from Duchenne's muscular dystrophy is accompanied by visual problems and abnormalities of the electroretinogram. Therefore it is likely that retinal dystrophin plays a role in certain stages of synaptic transmission between photoreceptors and the postsynaptic dendritic complex formed by horizontal and bipolar cells.

Actins↗
PubMed1994

Pathological alterations of the cerebral microvasculature in Alzheimer's disease and related dementing disorders.

Alterations of the cerebral microvasculature have been reported in aging and in neurodegenerative disorders such as Alzheimer's disease. However, the exact role of microvascular alterations in the pathogenesis of neurodegeneration remains unknown. In the present report, the cerebral cortex microvasculature was studied by immunohistochemistry using a monoclonal antibody against vascular heparan sulfate proteoglycan protein core in normal aging controls. Alzheimer's disease, Down syndrome, Guam amyotrophic lateral sclerosis/parkinsonian dementia complex, Pick's disease and dementia pugilistica. In all dementing illnesses, increased microvascular pathology was evident compared to normal controls. Decreased microvascular density and numerous atrophic vessels were the primary abnormalities observed in all dementing disorders. These microvascular abnormalities demonstrated regional and laminar selectivity, and were primarily found in layers III and V of frontal and temporal cortex. Quantitative analysis employing computer-assisted microscopy demonstrated that the decrease in microvascular density in Alzheimer's disease was statistically significant compared to age-matched controls. In addition, extracellular heparan sulfate proteoglycan deposits were observed which colocalized with thioflavine S-positive senile plaques in Alzheimer's disease, Down syndrome and selected Guam dementia cases. In some cases, heparan sulfate proteoglycan was seen in senile plaques that appeared to be diffuse or primitive plaques that stained weakly with thioflavine. Heparan sulfate proteoglycan-containing neurons were also observed in Alzheimer's disease, as well as in Down syndrome and Guam cases. Glial staining for heparan sulfate proteoglycan was never observed. Our data support previous observations that microvascular pathology is found in aging and in Alzheimer's disease. The changes in Alzheimer's disease exceed those found in normal aging controls. We also found microvascular pathology in all other dementing disorders studied. Our studies further demonstrated that the microvascular pathology displays regional and laminar patterns which parallel patterns of neuronal loss. Finally, we also found that heparan sulfate proteoglycan is present in senile plaques and neurons not only as previously reported in Alzheimer's disease, but also in Down syndrome and Guam cases. Heparan sulfate proteoglycan in senile plaques may be derived from either the degenerating microvasculature or from degenerating neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗
PubMed1996

Co-localization of the ketohexokinase and glucokinase regulator genes to a 500-kb region of chromosome 2p23.

The glucokinase regulator (GCKR) is a 65-kDa protein that inhibits glucokinase (hexokinase IV) in liver and pancreatic islet. The role of glucokinase (GCK) as pancreatic beta cell glucose sensor and the finding of GCK mutations in maturity onset diabetes of the young (MODY) suggest GCKR as a further candidate gene for type 2 diabetes. The inhibition of GCK by GCKR is relieved by the binding of fructose-1-phosphate (F-1-P) to GCKR. F-1-P is the end product of ketohexokinase (KHK, fructokinase), which, like GCK and GCKR, is present in both liver and pancreatic islet. KHK is the first enzyme of the specialized pathway that catabolizes dietary fructose. We have isolated genomic clones containing the human GCKR and KHK genes. By fluorescent in situ hybridization (FISH), KHK maps to Chromosome (Chr) 2p23.2-23.3, a new assignment corroborated by somatic cell hybrid analysis. The localization of GCKR, originally reported by others as 2p22.3, has been reassessed by high-resolution FISH, indicating that, like KHK, GCKR maps to 2p23.2-23.3. The proximity of GCKR and KHK was further demonstrated both by two-color interphase FISH, which suggests that the two genes lie within 500 kb of each other, and by analysis of overlapping YAC and P1 clones spanning the interval between GCKR and KHK. A new microsatellite polymorphism was used to place the GCKR-KHK locus between D2S305 and D2S165 on the genetic map. The colocalization of these two metabolically connected genes has implications for the interpretation of linkage or allele association studies in type 2 diabetes. It also raises the possibility of coordinate regulation of GCKR and KHK by common cis-acting regulatory elements.

Base Sequence↗
PubMed1993

Localization of transforming growth factor beta isoforms in the normal murine small intestine and colon.

BACKGROUND: The transforming growth factor beta (TGF-beta) proteins are key regulators of cellular growth and differentiation. Previous studies have shown that TGF-beta 1 is a potent growth inhibitor of cultured jejunal epithelial cells. The reported distribution of TGF-beta 1 messenger RNA (mRNA) expression along the intestinal villus has been controversial. The purpose of the current study is to determine the loci of TGF-beta protein expression in the normal small intestine and colon. METHODS: Intestinal localization of TGF-beta isoform mRNA and protein was examined by Northern blot analysis and immunohistochemistry using isoform specific reagents. RESULTS: TGF-beta 1, TGF-beta 2, and TGF-beta 3 mRNA were found in homogenates from the intact mouse jejunum and colon. The three isoforms colocalized in these tissues. Expression in the small intestinal epithelium was most prominent in cells located on the villus tip, and no staining was detected in the crypt. Occasional lymphocytes in the lamina propria were immunopositive, and all layers of the muscularis were moderately stained. This pattern was seen in all regions of the small intestine. The surface epithelium of the colon was intensely immunopositive, whereas cells in the glands were only weakly stained. CONCLUSIONS: TGF-beta molecules may serve overlapping functions in the intestinal tract, and expression in the epithelium may function to arrest growth of cells emerging from the crypt and induce or maintain the terminally differentiated state.

Animals↗
PubMed2006

The Drosophila microtubule associated protein Futsch is phosphorylated by Shaggy/Zeste-white 3 at an homologous GSK3beta phosphorylation site in MAP1B.

The Drosophila homologue of the microtubule associated protein MAP1B is encoded by the futsch locus. The deduced protein Futsch is about twice the size of MAP1B and shows high homology in the N- and C-terminal domains. The central part of Futsch is characterized by a highly repetitive structure based on a 37 amino acid motif. Futsch, like MAP1B, colocalizes with microtubules and is necessary for the organization of the microtubule cytoskeleton during axonal growth and synaptogenesis. To further analyze the functional relevance of Futsch as a MAP1B-like protein, we performed a molecular analysis of the conserved protein domains. Using a number of antisera, we show that, unlike the MAP1B polyprotein, which is cleaved to generate a heavy and light chain, Futsch is expressed as a single protein. The function of MAP1B is in part regulated by phosphorylation mediated by kinases that include casein kinase 2 and glycogen synthase kinase 3beta (GSK3beta). We show here that at least one GSK3beta phosphorylation site of MAP1B is conserved in Futsch and that this site can be phosphorylated by GSK3beta and its Drosophila homologue, Shaggy/Zeste-white 3. To test the functional relevance of these findings we generated a number of minigenes and assayed their ability to rescue the phenotype of futsch mutants. Our data highlight some differences between MAP1B and Futsch but demonstrate that important structural and functional aspects are conserved between fly and vertebrate members of this protein family.

Amino Acid Sequence↗
PubMed2004

Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson's disease.

The mechanisms underlying Parkinson's disease (PD) and Lewy body (LB) formation, a pathological hallmark of PD, are incompletely understood; however, mitochondrial dysfunction is likely to be at least partially responsible. To study the processes that might be related to nigral neurodegeneration and LB formation, we employed nonbiased quantitative proteomics with isotope-coded affinity tag (ICAT) to compare the mitochondrial protein profiles in the substantia nigra (SN) between controls and mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a potent mitochondrial toxicant, and an adjuvant, probenecid (prob), for 5 weeks, which produced selective nigrostriatal neurodegeneration with formation of LB-like cytoplasmic inclusions in the remaining nigral neurons. This method identified a total of more than 300 proteins; of these proteins, more than 100 displayed significant changes in relative abundance in the MPTP/prob-treated mice compared to the controls. We validated one of these proteins, DJ-1, whose mutation has been implicated in familial PD, with Western blot analysis, followed by immunohistochemical studies of its distribution in the SN in relation to cytoplasmic inclusions in mice, as well as in classical LBs in PD patients. The results demonstrated that DJ-1 was not only colocalized with alpha-synuclein in dopaminergic neurons but also to cytoplasmic inclusions in mice treated with MPTP/prob. In addition, DJ-1 was present in the halo but not in the core of classical LBs in patients with PD. Our findings suggested that DJ-1 might play an important role in mitochondrial dysfunction, as well as LB formation in PD.

Animals↗
PubMed1997

Expression of macrophage migration inhibitory factor in rat retina and its immunohistochemical localization.

Macrophage migration inhibitory factor (MIF) plays an important regulatory role for the T-cell activation induced by mitogenic or antigenic stimuli. We examined expression of MIF in rat retina. Reverse transcription-polymerase chain reaction analysis of a retinal tissue homogenate revealed that MIF mRNA was constitutively expressed. A single band specific for MIF protein was also observed by Western blot analysis. Immunohistochemistry using frozen sections of retinal tissues reacted with an anti-rat MIF antibody showed that MIF was localized in astrocytes. Müller cells and pigment epithelial (RPE) cells. This topological finding was confirmed by colocalization of MIF protein with glial fibrillary acidic protein and vimentin, which are putative immunohistochemical markers for astrocytes and Müller cells. It is well known that the retinal glial cells, as well as RPE cells, play an active role in inflammatory and immunological responses in the retina. Considering these facts, constitutive expression of MIF in these cells suggested the possibility that the protein contributes to regulation of retinal tissue inflammation as well as its local immunity.

Animals↗
PubMed2003

Syndecan-1 accumulates in lysosomes of poorly differentiated breast carcinoma cells.

Expression patterns of syndecan-1, the cell surface heparan sulfate proteoglycan (HSPG) predominant on epithelial cells, were analyzed in tissue samples from 30 infiltrating human breast carcinomas and in 9 human breast carcinoma cell lines. Immunohistochemical staining demonstrates that while a subset of the breast carcinomas lose syndecan-1, this proteoglycan is expressed or overexpressed in a majority of the cases. Interestingly, cells in poor grade tumors contain intracellular syndecan-1, an observation that has not been previously described and was thus further investigated. Examination of cultured breast carcinoma cell lines indicates that they also display the phenotype of the syndecan-1 positive tumors and thereby provide a model system for analysis of intracellular syndecan-1. All cell lines examined express syndecan-1, and poorly differentiated lines such as BT549 cells internalize the proteoglycan from the cell surface where it accumulates as intact HSPG in intracellular vesicles. Colocalization studies using fluorescent markers identify these to be lysosomes. This finding is unexpected, as the accepted mechanism for degradation of syndecan HSPG following endocytosis is fragmentation of the protein core and glycosaminoglycan chains in endosomes, followed by delivery of the fragments to lysosomes. Lysosomal inactivation using ammonium chloride demonstrates that well-differentiated lines such as T47D and MCF-7 cells, which maintain the majority of syndecan-1 on their cell surfaces, also target intact constitutively endocytosed syndecan-1 to lysosomes. Taken together, these results suggest that mammary epithelial cells utilize a previously uncharacterized mechanism for syndecan-1 catabolism. In this pathway the proteoglycan remains intact as it passes through the endosomal system, prior to arriving at its site of intracellular degradation in lysosomes.

Animals↗
PubMed2002

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species.

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.

Chromosome Banding↗
PubMed2000

Altered natural killer cell differentiation in CD34+ progenitors from chronic myeloid leukemia patients.

IL-15 and SCF fail to induce NK differentiation and proliferation of CD34+ hematopoietic progenitors from chronic myeloid leukemia patients in contrast to normal stem cells although, both normal and leukemic CD34+ cells display comparable expression of c-kit or IL-15 receptor subunits. Interestingly, confocal microscopy analysis revealed that leukemic and most normal CD34+ cells produce and secrete IL-15, as shown by its trafficking through the Golgi apparatus and early endosomes. However, only leukemic progenitors express the membrane bound IL-15. Colocalization and internalization of IL-15Rbeta/gammac and IL-15Ralpha/gammac complexes indicated that IL-15 was specifically uptaken by leukemic progenitors. We also demonstrated that in both normal and leukemic progenitors, the signaling kinase Jak3 is constitutively pre-associated with the gammac chain. Anti-IL-15 neutralizing mAb treatment resulted in down-regulation of gammac chain and disruption of gammac/Jak3 interaction in normal but had no effect in leukemic progenitors. Our results suggest the existence in both normal and leukemic CD34+ cells of a constitutive production of a bioactive IL-15 that does not lead to NK differentiation and further indicate that membrane bound IL-15 and constitutive activation of gammac are hallmarks of leukemic progenitors. Oncogene (2000).

Antigens, CD34↗
PubMed1998

Synaptic vesicle protein SV2B, but not SV2A, is predominantly expressed and associated with microvesicles in rat pinealocytes.

Microvesicles are endocrine counterparts of neuronal synaptic vesicles, and accumulate and secrete classic neurotransmitters. In mammalian pinealocytes, microvesicles accumulate L-glutamate through a vesicular glutamate transporter and secrete it through exocytosis. To characterize the molecular organization of microvesicles in more detail, we investigated in this study the expression and localization of synaptic vesicle protein 2 (SV2) in rat pinealocytes. RT-PCR analysis indicated that transcripts specific for two isoforms, SV2A, a ubiquitous form present in neuronal and endocrine cells, and SV2B, a neuron-specific form, are amplified in pineal RNAs. Northern blotting with specific transcripts indicated that the mRNA for SV2B is predominantly expressed, whereas that for SV2A is below the detection limit. Site-specific antibodies against SV2B recognized a single 72-kDa polypeptide in the pineal membrane fraction, whereas anti-SV2A antibodies did not recognize any polypeptides. Immunohistochemical analysis of cultured cells indicated that SV2B is expressed in pinealocytes but not in other types of cells. SV2B is present in somata and is especially rich in processes, which are filled with microvesicles. SV2B is colocalized with synaptophysin and synaptotagmin, markers for microvesicles. Immunoelectron microscopy indicated that SV2B is associated with microvesicles. These results indicated that SV2B, but not SV2A, is expressed in rat pinealocytes and associated with microvesicles. As SV2B is also expressed in cultured alphaTC6 clonal pancreatic a cells, SV2B is not a protein specific for neurons.

Animals↗
PubMed1996

Specific interaction between human kinetochore protein CENP-C and a nucleolar transcriptional regulator.

CENP-C is a human kinetochore protein that was originally identified as a chromosomal autoantigen in patients with scleroderma spectrum disease. To begin to establish a comprehensive protein map of the human centromere, affinity chromatography was used to identify nuclear proteins that specifically interact with CENP-C. Whereas a number of polypeptides appeared to interact with the full-length CENP-C protein, only a pair of similarly sized proteins of approximately 100 kDa specifically interacted with the isolated carboxyl-terminal third of the CENP-C protein. Neither protein of the doublet bound to control affinity columns. Affinity purification and microsequence analysis of the proteins in the doublet identified them as the two highly related nucleolar transcription factors, UBF1 and UBF2 (also known as the nucleolar autoantigen NOR-90). Immunoblot analysis confirmed that both proteins also interacted with the full-length CENP-C polypeptide with similar affinities. Double indirect immunofluorescence using monospecific antibodies demonstrated that a subset of CENP-C and UBF/NOR-90 is colocalized at nucleoli of interphase HeLa cells, suggesting that the in vitro interaction detected by affinity chromatography may reflect an interaction that occurs in vivo. We discuss the implications of these findings in terms of the properties of interphase centromeres and the role of the nucleolus in scleroderma autoimmunity.

Amino Acid Sequence↗
PubMed1996

Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology.

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.

Animals↗
PubMed1999

A cell-free system to study regulation of focal adhesions and of the connected actin cytoskeleton.

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of beta1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of beta1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated beta1 receptors show that the cytoplasmic portion of beta1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified alpha-actinin colocalizes and redistributes with beta1 receptors on ventral plasma membranes depleted of actin, implicating binding of alpha-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

Actinin↗
PubMed2001

Elicitor-induced association of isoflavone O-methyltransferase with endomembranes prevents the formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesis.

The bioactive isoflavonoids of the Leguminosae often are methylated on the 4'-position of their B-rings. Paradoxically, reverse genetic evidence implicates alfalfa isoflavone O-methyltransferase (IOMT) in the biosynthesis of 4'-O-methylated isoflavonoids such as the phytoalexin medicarpin in vivo, whereas biochemical studies indicate that IOMT has strict specificity for methylation of the A-ring 7-hydroxyl of daidzein, the presumed substrate for O-methylation, in vitro. Radiolabeling and isotope dilution studies now confirm that daidzein is not an intermediate in isoflavonoid phytoalexin biosynthesis in alfalfa. Furthermore, protein gel blot analysis and confocal microscopy of a transiently expressed IOMT-green fluorescent protein fusion in alfalfa leaves show that the operationally soluble IOMT localizes to endomembranes after elicitation of the isoflavonoid pathway. We propose that IOMT colocalizes with the endoplasmic reticulum-associated isoflavone synthase cytochrome P450 to ensure rapid B-ring methylation of the unstable 2,4',7-trihydroxyisoflavanone product of isoflavone synthase, thereby preventing its dehydration to daidzein and subsequent A-ring methylation by free IOMT. In this way, metabolic channeling at the entry point into isoflavonoid phytoalexin biosynthesis protects an unstable intermediate from an unproductive metabolic conversion.

Benzopyrans↗