Protein Gel Blot Research Articles

The sea urchin egg yolk granule is a storage compartment for HCL-32, an extracellular matrix protein

We have utilized protein gel blot analysis and immunogold labelling to define the intracellular storage compartment for HCL-32, a 32-kDa protein component of the sea urchin embryonic extracellular matrices, the hyaline layer and basal lamina. Anti-HCL-32 antiserum specifically labelled yolk granules in unfertilized eggs. Cortical granules, mitochondria, sparse granules, and lipid vacuoles were not labelled. Label continued to be detected in the yolk granules through to the blastula stage of development. However, by the gastrula stage no labelling was detected in the yolk granules. In protein gel blot analysis HCL-32 was detected in yolk granules prepared from unfertilized eggs. These results clearly define the yolk granule as a storage compartment for HCL-32, an extracellular matrix protein.Key words: embryo, yolk granule, extracellular matrix.

The sea urchin egg yolk granule is a storage compartment for HCL-32, an extracellular matrix protein

We have utilized protein gel blot analysis and immunogold labelling to define the intracellular storage compartment for HCL-32, a 32-kDa protein component of the sea urchin embryonic extracellular matrices, the hyaline layer and basal lamina. Anti-HCL-32 antiserum specifically labelled yolk granules in unfertilized eggs. Cortical granules, mitochondria, sparse granules, and lipid vacuoles were not labelled. Label continued to be detected in the yolk granules through to the blastula stage of development. However, by the gastrula stage no labelling was detected in the yolk granules. In protein gel blot analysis HCL-32 was detected in yolk granules prepared from unfertilized eggs. These results clearly define the yolk granule as a storage compartment for HCL-32, an extracellular matrix protein.

Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants.

To enhance the disease resistance of plants expressing a foreign peptide, the gene for sarcotoxin IA, which is an antimicrobial peptide from an insect consisting of 39 amino acid residues, was introduced into tobacco (Nicotiana tabacum) under the control of a high expression promotor via Agrobacterium-mediated transformation. In transgenic plants, sarcotoxin IA mRNA accumulated to detectable levels, however, the amount of the peptide produced was so small that we could scarcely detect it by protein gel blot analysis, probably because of the instability of short peptides in plant cells. To improve the expression efficiency, genes for four types of amino-terminal and carboxyl-terminal translational fusions of sarcotoxin IA together with the GUS gene were introduced into tobacco. In all four types of transgenic tobacco plants, high level transcripts similar to that in the direct expression sarcotoxin IA construct were found. Protein gel blot analysis with both anti-sarcotoxin IA and GUS antibodies showed production of high levels of fusion protein in all transgenic plants. Among them, three types had abnormal membranes and phenotypes, although no such abnormalities were found in transgenic plants in which only sarcotoxin IA was expressed in a secretable form. All together, these results indicated that, for stable and effective expression of a foreign short peptide in transgenic plants, expression as a fusion protein is useful and that secretion of sarcotoxin IA outside of cells is necessary for generation of useful antimicrobial transgenic plants.

The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.

A novel protein with DNA binding activity from tobacco chloroplast nucleoids.

A 41-kD DNA binding protein with a basic pl was purified from chloroplast nucleoids in photomixotrophically cultured tobacco cells, and its amino acid sequence was determined. Using this sequence information, its cDNA (CND41) was isolated, and its nucleotide sequence was determined. The predicted amino acid sequence of CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids. A distinctive helix-turn-helix motif in the lysine-rich N-terminal region of the mature protein and an aspartyl protease active site motif were predicted. Expression of a series of truncated CND41 proteins in Escherichia coli indicated that the lysine-rich region is essential for DNA binding and that CND41 nonspecifically binds chloroplast DNA. Protein gel blot analyses showed CND41 mainly in cells and/or tissues containing nonphotosynthesizing, actively growing plastids. In addition, the accumulation of chloroplast transcripts in these cells and/or tissues (e.g., transcripts for QB binding protein of photosystem II [psbA] and large subunit of ribulose bisphosphate carboxylase [rbcL]) was negatively correlated with the accumulation of CND41. Analyses of cultured cells of transgenic tobacco with reduced CND41 levels showed a higher level of expression of chloroplast genes compared with that of the wild type. We discuss the possible function of CND41 as a negative regulator of chloroplast gene expression.

High-Efficiency Blotting of Proteins of Diverse Sizes Following Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

High-molecular-weight proteins often blot onto nitrocellulose membranes poorly following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, resulting in low levels of detection on immunoblots, and hence difficulty in analyzing rare proteins. Moreover, optimizing conditions for the transfer of high-molecular-weight proteins to nitrocellulose frequently results in the inefficient transfer or loss of lower molecular weight proteins. This problem is particularly vexing during the analysis of large proteins which may be processed to one or more smaller biologically active forms. Using radiolabeled protein standards and phosphorimaging technology, we have quantitated the efficacy of many different protein gel electrophoresis and blotting protocols. Here we report novel gel and blotting conditions which significantly improve the transfer and retention of high-molecular-weight proteins, without sacrificing the efficient transfer of lower molecular weight proteins. Using this newly described procedure, we have detected a rare 500-kDa protein in immunoblots which was previously not detectable with any of the commonly used blotting procedures. Since the improved conditions offer increased sensitivity across a spectrum of protein sizes, they should be widely applicable.

Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue.

The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

In the maize endosperm, the Opaque2 (O2) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. Immunodetection of wild-type or mutant O2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68- to 72-kD range, whereas by using isoelectric focusing, seven to nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns to a single band corresponding to the nonphosphorylated component. In vivo and in vitro labeling confirmed that O2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated O2 polypeptides were able to bind an oligonucleotide containing the O2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime to nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that O2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that O2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears to be influenced by environmental conditions.

Localization of a Rho GTPase Implies a Role in Tip Growth and Movement of the Generative Cell in Pollen Tubes.

The Rho family GTPases function as key molecular switches, controlling a variety of actin-dependent cellular processes, such as the establishment of cell polarity, cell morphogenesis, and movement in diverse eukaryotic organisms. A novel subfamily of Rho GTPases, Rop, has been identified in plants. Protein gel blot and RNA gel blot hybridization analyses indicated that one of these plant Rho GTPases, Rop1, is expressed predominantly in the male gametophyte (pollen and pollen tubes). Cell fractionation analysis of pollen tubes showed that Rop is partitioned into soluble and particulate fractions. The particulate Rop could be solubilized with detergents but not with salts, indicating that it is tightly bound to membranes. The membrane association appears to result from membrane anchoring via a geranylgeranyl group because an in vitro isoprenylation assay demonstrated that Rop1Ps is geranylgeranylated. Subcellular localization, using indirect immunofluorescence and confocal microscopy, showed that Rop is highly concentrated in the cortical region of the tube apex and in the periphery of the generative cell. The cortical Rop protein at the apex forms a gradient with decreasing concentration from tip to base and appears to be associated with the plasma membrane. These results suggest that the apical Rop GTPase may be involved in the signaling mechanism that controls the actin-dependent tip growth of pollen tubes. Localization of the Rop GTPase to the periphery of the generative cell is analogous to that of myosin, suggesting that the Rop GTPase plays an important role in the modulation of an actomyosin motor system involved in the movement of the generative cell.

Organ-specific expression of the stress-related anionic peroxidases in cucumber flowers

The study revealed a marked qualitative and quantitative differences in the pattern of expression of three stress-related cucumber (Cucumis sativus L., cv. Laura) isoperoxidases. Activity staining, as well as the protein gel blot (Western blotting) confirmed that the proteins studied are differentially expressed in both male and female sepals, in male but not in female pedicels and in, pistil. By using antibodies specific to three stress-related peroxidases, one serologically related pistil-specific anionic peroxidase was detected. This specific band had never been observed in other flower organs. Differential appearance of the stress-related peroxidase isoenzymes in both male and female flowers demonstrates that these proteins are developmentally regulated, showing an organ-specific expression.

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes.

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.

Stable expression of vicilin fromVicia faba with eight additional single methionine residues but failure of accumulation of legumin with an attached peptide segment in tobacco seeds

Two different attempts have been undertaken to improve the amino acid composition of storage proteins from field bean (Vicia faba) by genetic engineering. First, legumin was modified to generate a new peptide sequence at the C-terminus containing 4 methionine residues. Second, vicilin was modified by generating 8 single methionine residues distributed over the peptide sequence. The genes were expressed in different systems includingin vitro transcription and translation and stable transformation into tobacco. The modified legumin was found to be unstable when expressed in tobacco seeds. Although specific mRNA was detected on RNA gel blots, no protein could be found by using protein gel blotting and ELISA. Furthermore, a protease preparation able to process the original legumin precursorin vitro degraded the modified legumin precursor. Contrary, the modified vicilin was accumulated in seeds of tobacco transformed with the gene under the control of the seed specific USP promoter. Both the original and the modified vicilin could be detected on protein gel blots at the expected position. Two-dimensional electrophoresis was employed to analyse the expression of original vicilin. Three vicilin-specific products of almost equal size were observed, indicating a slight modification leading to a change of pI. Quantitative determination using competitive ELISA showed that there is no significant difference in accumulation between original and modified vicilin. In both cases, three plants were found with vicilin amounts in the range of 1–3% of total globulin.

Identification of a component of the sea urchin hyaline layer, HLC-175, which undergoes proteolytic processing during development

To define the role(s) played by the sea urchin extraembryonic matrix, the hyaline layer, we have previously purified and characterized a number of the protein components of this structure. We are currently studying the timing and significance of the proteolytic processing of these species. The localization of HLC-175 in the egg and 1-hr-old embryo was determined by indirect immunofluorescence analysis. The relationship between HLC-175 and the 109- and 81 kDa species was determined by a combination of native gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and protein gel blot analyses using the anti-175, -109 and 81 kDa antisera. Using gel exclusion chromatography we have fractionated a mixture of proteins extracted from the surface of 1-hr-old sea urchin embryos. A set of fractions eluting from the column contained three species of apparent molecular masses 175-, 109- and 81 K. These species comigrated on analysis by either non-reducing SDS-PAGE or native gel electrophoresis. Inclusion of the reducing agent, dithiothreitol, in the solubilizing solutions abolished comigration of these polypeptides. When polyclonal antisera were prepared against each of these antigens cross-reactivity between the 175- and 109 kDa species and between the 175- and 81 kDa species was detected. Developmental protein gel blot analyses revealed a precursor-product relationship between the 175- and the 109- and 81 kDa polypeptides. Indirect immunofluorescence analysis confirmed the localization of HLC-175 to the hyaline layer. The results reported here clearly identify HLC-175 as a component of the hyaline layer. In addition, we have shown that between the early- and mid-blastula stages of development HLC-175 is processed into two smaller species. This reaction will serve as a useful probe to identify the putative cell surface remodeling activity(ies).

The maize RNA‐binding protein, MA16, is a nucleolar protein located in the dense fibrillar component

SummaryA developmentally and environmentally regulated gene in maize, MA16, encoding an RNA‐binding protein that binds preferentially to uridine and guanosine‐rich RNAs has previously been described. To gain some insight into the function of MA16 the distribution of MA16 mRNA and protein during maize development was investigated using in situ hybridization, RNA and protein gel blot analysis and immunocytochemistry. The results show that MA16 is expressed throughout development of the embryo and seedling in different tissues and at different levels. The level of MA16 mRNA is higher in developing and expanding structures such as the root elongation zone and young leaves. After stress treatment MA16 mRNA increases in total and polysomal RNA, but no significant change in the level of the protein was detected. MA16 is a non‐ribosomal nucleolar protein. Using immunoelectron microscopy the MA16 protein has been located in the dense fibrillar component and to a lesser extent in the granular component of the nucleolus. It was found that MA16 contains the conserved sequence motifs R(G)nY(G)nR and RR(E/D)(G)nY(G)n repeated in the C‐terminal of the molecule that conforms imperfectly to the GAR motif proposed for nucleolar proteins. In light of these results the stress regulation of MA16 and a likely role for this protein in pre‐rRNA processing and/or ribosome assembly is discussed.

Cellular Concentrations and Uniformity of Cell-Type Accumulation of Two Lea Proteins in Cotton Embryos.

The levels and cell-type distribution of late embryogenesis abundant (Lea) proteins D-7 and D-113 have been determined in mature cotton embryos by immunochemical methods. The two proteins were expressed in and purified from Escherichia coli and utilized for antibody production in rabbits. The antiserum to each protein was found to interact with all members of each protein family in cotton extracts by protein gel blotting. Using these antibodies in quantitative "rocket" immunoelectrophoreses, D-7 proteins were found to accumulate to ~8 x 1015 molecules per embryo, which is equivalent to ~109 molecules per "average cell." D-113 proteins accumulate to ~1016 molecules per embryo, which equates to ~1.3 x 109 molecules per average cell. These values calculate to concentrations of about 226 and 283 [mu]M, respectively, in the cell aqueous phase immediately prior to seed desiccation. In immunocytochemical studies using the fluorophor rhodamine linked to the secondary antibody, both proteins appeared to be evenly present in the cytosol of all cell types present in the embryo, including both cotyledon and axis epidermal cells. Thus, their function does not appear related to unique functions of specific cell or tissue types. The very high molar concentrations of the two proteins, coupled with their unusual predicted structure and their cytosol location, would seem to reduce the number of their conceivable functions.

The use of antisense mRNA to inhibit the tonoplast H+ ATPase in carrot.

Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35S promoter. Bafilomycin-sensitive ATPase, H(+)-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of the A subunit was inhibited in the tonoplast fraction, but not in the Golgi fraction. Two-dimensional protein gel blots of total microsomes of wild-type and control transformant cell lines revealed two major immunoreactive polypeptides in the acidic pI range. In contrast, highly purified tonoplast membranes contained only the less acidic polypeptide. Because the less acidic polypeptide was preferentially diminished in the two antisense cell lines, we infer that the antisense constructs specifically blocked expression of a tonoplast-specific isoform of the V-ATPase A subunit in carrot. Regenerated plants containing the antisense constructs exhibited altered leaf morphologies and reduced cell expansion. The altered phenotype was correlated with the presence of the antisense construct.

Protein-protein interactions and structural entities within the sea urchin extraembryonic matrix, the hyaline layer

We have investigated the effects of a variety of experimental conditions on the structural integrity of the sea urchin extraembryonic matrix, the hyaline layer. Removal of Ca 2+ resulted in the quantitative release of hyalin from isolated layers. Protein gel blot analyses indicated that, in the absence of Ca 2+, hyalin was also quantitatively released from the layers surrounding 1-h-old embryos. However, no other polypeptides of the hyaline layer were released in significant amounts. The layers remaining after removal of hyalin were refractory to digestion with proteinase K and dissociated only in the presence of chaotropes. These results provide insights into the structural organization within the hyaline layer as well as the role of the embryonic cell surface in maintaining the structural integrity of this extraembryonic matrix.

Cell-specific expression of the carrot EP2 lipid transfer protein gene.

A cDNA corresponding to a 10-kD protein, designated extracellular protein 2 (EP2), that is secreted by embryogenic cell cultures of carrot was obtained by expression screening. The derived protein sequence and antisera against heterologous plant lipid transfer proteins identified the EP2 protein as a lipid transfer protein. Protein gel blot analysis showed that the EP2 protein is present in cell walls and conditioned medium of cell cultures. RNA gel blot analysis revealed that the EP2 gene is expressed in embryogenic cell cultures, the shoot apex of seedlings, developing flowers, and maturing seeds. In situ hybridization showed expression of the EP2 gene in protoderm cells of somatic and zygotic embryos and transient expression in epidermis cells of leaf primordia and all flower organs. In the shoot apical meristem, expression is found in the tunica and lateral zone. In maturing seeds, the EP2 gene is expressed in the outer epidermis of the integument, the seed coat, and the pericarp epidermis, as well as transiently in between both mericarps. Based on the extracellular location of the EP2 protein and the expression pattern of the encoding gene, we propose a role for plant lipid transfer proteins in the transport of cutin monomers through the extracellular matrix to sites of cutin synthesis.

Photoperiod Control of Poplar Bark Storage Protein Accumulation

Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.

Tissue-specific expression of divergent actins in soybean root.

It has been proposed that the evolution of distinct classes of genes encoding the kappa-, lambda-, and mu-actins in soybean is the result of an ancient divergence in patterns of actin gene expression. In this study, antisera against a family of synthetic actin peptides from a divergent region within the predicted actin polypeptide sequences have been used to explore the differential expression of plant actins. Antiserum elicited against a 16-residue synthetic lambda-actin peptide SAc4:257 reacted with a 46-kilodalton protein in soybean extracts, showed specificity for the lambda-peptide over the divergent kappa- and mu-actin peptides in enzyme-linked immunosorbent assays, and reacted strongly and preferentially with root protoderm in apical roots and in lateral root primordia. Antiserum elicited against the synthetic kappa-actin peptide SAc1:257 reacted with 46-kilodalton protein on protein gel blots, showed partial specificity toward the immunogenic kappa-peptide over the divergent lambda- and mu-peptides, and reacted strongly with all root tissues with the exception of root cap. These data support the hypothesis that ancient classes of plant actin genes may have been preserved because of their role in developmentally controlled differences in tissue-specific actin expression and/or function. The possibility that other diverse actin classes have unique patterns of regulation is discussed.