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Biomedical subjects

E M Herman

Publications and source records attributed to E M Herman.

At least 19 recordsLinked to original sources
PubMed2001

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies.

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.

Antigens, Plant↗
PubMed1998

Characterization of a maize tonoplast aquaporin expressed in zones of cell division and elongation.

We studied aquaporins in maize (Zea mays), an important crop in which numerous studies on plant water relations have been carried out. A maize cDNA, ZmTIP1, was isolated by reverse transcription-coupled PCR using conserved motifs from plant aquaporins. The derived amino acid sequence of ZmTIP1 shows 76% sequence identity with the tonoplast aquaporin gamma-TIP (tonoplast intrinsic protein) from Arabidopsis. Expression of ZmTIP1 in Xenopus laevis oocytes showed that it increased the osmotic water permeability of oocytes 5-fold; this water transport was inhibited by mercuric chloride. A cross-reacting antiserum made against bean alpha-TIP was used for immunocytochemical localization of ZmTIP1. These results indicate that this and/or other aquaporins is abundantly present in the small vacuoles of meristematic cells. Northern analysis demonstrated that ZmTIP1 is expressed in all plant organs. In situ hybridization showed a high ZmTIP1 expression in meristems and zones of cell enlargement: tips of primary and lateral roots, leaf primordia, and male and female inflorescence meristems. The high ZmTIP1 expression in meristems and expanding cells suggests that ZmTIP1 is needed (a) for vacuole biogenesis and (b) to support the rapid influx of water into vacuoles during cell expansion.

Amino Acid Sequence↗
PubMed1997

The upstream domain of soybean oleosin genes contains regulatory elements similar to those of legume storage proteins.

Seed reserve storage products consisting of proteins, oil and starch are accumulated in a developmentally coordinated pattern. The control of the vacuolar storage protein expression has been shown to be transcriptionally regulated and involves a series of positive and negative regulatory as well as enhancing gene elements. We have analyzed the upstream sequence of the genes encoding the soybean oleosins, the protein that encases the oil body. We have found that soybean oleosin genes possess regulatory elements in upstream domain that are similar to those found in vacuolar storage protein genes.

Amino Acid Sequence↗
PubMed1996

The maize gamma-zein sequesters alpha-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm.

Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, alpha, beta, gamma, and delta, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing alpha-zein, gamma-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas gamma-zein accumulated in seeds of these plants, stable accumulation of alpha-zein required simultaneous synthesis of gamma-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presence of gamma-zein, there was a turnover of alpha-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that gamma-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.

Enzyme-Linked Immunosorbent Assay↗
PubMed1995

Binding-protein expression is subject to temporal, developmental and stress-induced regulation in terminally differentiated soybean organs.

Binding protein (BiP) is a widely distributed and highly conserved endoplasmic-reticulum luminal protein that has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides. Analysis of cDNA sequences and genomic blots indicates that soybeans (Glycine max L. Merr.) possess a small gene family encoding BiP. The deduced sequence of BiP is very similar to that of other plant BiPs. We have examined the expression of BiP in several different terminally differentiated soybean organs including leaves, pods and seed cotyledons. Expression of BiP mRNA increases during leaf expansion while levels of BiP protein decrease. Leaf BiP mRNA is subject to temporal control, exhibiting a large difference in expression in a few hours between dusk and night. The expression of BiP mRNA varies in direct correlation with accumulation of seed storage proteins. The hybridization suggests that maturing-seed BiP is likely to be a different isoform from vegetative BiPs. Levels of BiP protein in maturing seeds vary with BiP mRNA. High levels of BiP mRNA are detected after 3 d of seedling growth. Little change in either BiP mRNA or protein levels was detected in maturing soybean pods, although BiP-protein levels decrease in fully mature pods. Persistent wounding of leaves by whiteflies induces massive overexpression of BiP mRNA while only slightly increasing BiP-protein levels. In contrast single-event puncture wounding only slightly induces additional BiP expression above the temporal variations. These observations indicate that BiP is not constitutively expressed in terminally differentiated plant organs. Expression of BiP is highest during the developmental stages of leaves, pods and seeds when their constituent cells are producing seed or vegetative storage proteins, and appears to be subject to complex regulation, including developmental, temporal and wounding.

Adaptation, Psychological↗
PubMed1995

Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole.

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seed-specific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope "himet" (Hoffman et al., 1988, Plant Mol. Biol. 11, 717-729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent.

Amino Acid Sequence↗
PubMed1994

Correct post-translational modification and stable vacuolar accumulation of phytohemagglutinin engineered to contain multiple methionine residues.

Most legume seed storage proteins are deficient in sulfur amino acids. In this study, we demonstrate that replacing specific amino acid residues of a seed protein with methionine residues at positions known to be occupied by methionine residues in homologous proteins, is an effective strategy to create methionine-enriched seed proteins. Mutant phytohemagglutinin polypeptides with three or four methionine residues were found to undergo correct post-translational modifications in transformed cultured tobacco cells and to accumulate stably in the protein storage vacuoles of transgenic tobacco seeds.

Amino Acid Sequence↗
PubMed1992

A soybean vacuolar protein (P34) related to thiol proteases is synthesized as a glycoprotein precursor during seed maturation.

We have examined the synthesis, posttranslational processing, and localization of soybean P34, a member of the papain superfamily. P34 has been identified as a constituent of oil storage organelles or oil bodies isolated from seed lysates and has been assumed to be one of the oil body proteins. Electron microscopic immunocytochemistry with a monoclonal antibody demonstrated that P34 is localized in the protein storage vacuoles but not in the oil bodies. Immunocytochemical observations of partially disrupted seed cells showed that the association of P34 with oil bodies appears to occur as a consequence of cell lysis. In vitro synthesis of P34 results in the formation of a 46-kDa polypeptide that increases to 47 kDa due to core glycosylation by canine microsomes. In vivo synthesis studies in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation, indicate that pro-P34 is 47 kDa. Since the cDNA sequence of prepro-P34 contains a single putative glycosylation site in the precursor domain, we conclude that P34, like a few other vacuolar proteins, is synthesized as a glycoprotein precursor. Pulse-chase experiments showed that the processing of pro-P34 to mature P34 occurs in a single step and that this posttranslational cleavage occurs on the carboxyl side of an Asn, which is typical of seed vacuolar proteins. Pro-P34 (47 kDa) is detected in immunoblots of maturing seeds. Analysis of RNA indicates that the P34 genes are expressed only during seed maturation and that the P34 mRNA is related to other thiol protease mRNAs detectable in other organs and plants. Unlike other seed thiol proteases that are synthesized only after seed germination, P34 accumulates during seed maturation.

Allergens↗
PubMed1992

Arabinogalactan-rich glycoproteins are localized on the cell surface and in intravacuolar multivesicular bodies.

We investigated the subcellular distribution of antigenic sites immunoreactive to the monoclonal antibody 16.4B4 (PM Norman, VPM Wingate, MS Fitter, CJ Lamb [1986] Planta 167: 452-459) in tobacco (Nicotiana tabacum) leaf cells. This antibody is directed against a glycan epitope in a family of plasma membrane arabinogalactan proteins of 135 to 180 kilodaltons, elaborated from a polypeptide of relative molecular mass 50 kilodaltons (PM Norman, P Kjellbom, DJ Bradley, MG Hahn, CJ Lamb [1990] Planta 181: 365-373). We demonstrated by immunogold electron microscopy that the epitope reactive with monoclonal antibody 16.4B4 is localized on the cell surface in the leaf parenchyma cell periplast. The 16.4B4 antigen is also localized in multivesicular invaginations of the plasma membrane also known as plasmalemmasomes, implying a biochemical and, hence, functional interrelationship between these structures. Monoclonal antibody 16.4B4 also labels intracellular multivesicular bodies that appear to represent internalized plasmalemmasomes. Antibody reactivity was also observed in partially degraded multivesicular bodies sequestered within the central vacuole. We propose that the subcellular distribution of the epitope reactive with monoclonal antibody 16.4B4 defines a plasmalemmasome (or multivesicular body-mediated) pathway for the internalization of the periplasmic matrix for vacuolar mediated disposal. The multivesicular bodies appear to be equivalent to the well-characterized endosomes and multivesicular bodies of animal cells involved in the internalization and lysosome-mediated degradation of extracellular materials.

Journal Article↗
PubMed1992

Vegetative and Seed-Specific Forms of Tonoplast Intrinsic Protein in the Vacuolar Membrane of Arabidopsis thaliana.

Reports from a number of laboratories describe the presence of a family of proteins (the major intrinsic protein family) in a variety of organisms. These proteins are postulated to form channels that function in metabolite transport. In plants, this family is represented by the product of NOD26, a nodulation gene in soybean that encodes a protein of the peribacteroid membrane, and tonoplast intrinsic protein (TIP), an abundant protein in the tonoplast of protein storage vacuoles of bean seeds (KD Johnson, H Höfte, MJ Chrispeels [1990] Plant Cell 2: 525-532). Other homologs that are induced by water stress in pea and in Arabidopsis thaliana and that are expressed in the roots of tobacco have been reported, but the location of the proteins they encode is not known. We now report the presence and derived amino acid sequences of two different TIP proteins in A. thaliana. alpha-TIP is a seed-specific protein that has 68% amino acid sequence identity with bean seed TIP; gamma-TIP is expressed in the entire vegetative body of A. thaliana and has 58% amino acid identity with bean seed TIP. Both proteins are associated with the tonoplast. Comparisons of the derived amino acid sequences of the seven known plant proteins in the major intrinsic protein family show that genes with similar expression patterns (e.g. water stress-induced or seed specific) are more closely related to each other than the three A. thaliana homologs are related. We propose that the nonoverlapping gene expression patterns reported here, and the evolutionary relationships indicated by the phylogenetic tree, suggest a functional specialization of these proteins.

Journal Article↗
PubMed1991

Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs.

We have characterized two cDNA clones for 24 kDa soybean oleosin, the seed oil body membrane protein. Differences in the predicted amino acid sequences of the two clones and the presence of a doublet on immunoblots indicate that 24 kDa oleosin exists in at least two isoforms in soybean. The predicted amino acid sequence also contains a unique carboxy terminal region that is dominated by a series of different tandem amino acid repeats.

Amino Acid Sequence↗
PubMed1991

The Purification, Properties, and Localization of an Abundant Legume Seed Lectin Cross-Reactive Material from Spartium junceum.

The seeds of Spartium junceum contained a large quantity of lectin-like protein that did not appear to be either a hemagglutinin or active lectin. The cross-reactive material (CRM), like most legume seed lectins, was a tetrameric glycoprotein of about 130,000 M(r). The singlesized subunits of about 33,000 M(r) were not covalently associated. The amino acid composition was typical of legume lectins and was rich in hydroxy-amino acids and poor in sulfur-containing amino acids. The Spartium CRM contained about 3.5% covalently associated carbohydrate, most likely of the high-mannose type, since the CRM was precipitated by concanavalin A. The CRM was localized by electron-microscopic immunocytochemistry and found to be exclusively in protein-filled vacuoles (protein bodies). Because this protein was so similar immunologically, structurally, and in its physiology, to classic legume seed lectins, it is most likely a lectin homolog. Similar seed lectin CRMs appear to be both common and widespread in the Leguminosae.

Journal Article↗
PubMed1990

Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family.

A 34,000-Da protein (P34) is one of the four major soybean oil body proteins observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated organic solvent-extracted oil bodies from mature seeds. P34 is processed during seedling growth to a 32,000-Da polypeptide (P32) by the removal of an amino-terminal decapeptide (Herman, E.M., Melroy, D.L., and Buckhout, T.J. (1990) Plant Physiol, in press). A soybean lambda ZAP II cDNA library constructed from RNA isolated from midmaturation seeds was screened with monoclonal antibodies directed against two different epitopes of P34. The isolated cDNA clone encoding P34 contains 1,350 base pairs terminating in a poly(A)+ tail and an open reading frame 1,137 base pairs in length. The open reading frame includes a deduced amino acid sequence which matches 23 of 25 amino-terminal amino acids determined by automated Edman degradation of P34 and P32. The cDNA predicts a mature protein of 257 amino acids and of 28,641 Da. The open reading frame extends 5' from the known amino terminus of P34 encoding a possible precursor and signal sequence segments with a combined additional 122 amino acids. Prepro-P34 is deduced to be a polypeptide of 42,714 Da, indicating that the cDNA clone apparently encodes a polypeptide of 379 amino acids. A comparison of the nucleotide and deduced amino acid sequences in the GenBank Data Bank with the sequence of P34 has shown considerable sequence similarity to the thiol proteases of the papain family. Southern blot analysis of genomic DNA indicated that the P34 gene has a low copy number.

Amino Acid Sequence↗
PubMed1990

Apparent processing of a soybean oil body protein accompanies the onset of oil mobilization.

The membrane surrounding the oil body contains several different specific polypeptides. To study the biosynthesis and posttranslational modification of these polypeptides we have prepared monoclonal antibodies against purified oil bodies of soybean (Glycine max). Three of the five monoclonals selected recognize a molecular mass 34 kilodalton protein (P34). Epitope mapping of CNBr and proteolytic fragments of P34 indicates that two of the anti-P34 monoclonal antibodies are directed at different epitopes. P34 is accumulated during seed maturation at the same time as the reserve proteins and oil. SDS/PAGE-immunoblots of germinating soybean seed cotyledons indicate that the protein is initially present as a molecular mass 34 kilodalton polypeptide and is processed to molecular mass 32 kilodalton on the fourth through sixth days of seedling growth simultaneously with the onset of oil mobilization. A comparison of reduced and carboxymethylated oil body proteins with nonreduced proteins by SDS/PAGE indicates that P34 exists in vivo as a dimer of molecular mass 58 kilodalton. Comparing the amino terminal sequences of P34 and P32 indicates that their difference is at least in part due to the removal of the amino terminus of P34. The amino terminal sequences of P34 and P32 were aligned to show that the transition of P34 to P32 was accompanied by the removal of a hydrophilic decapeptide (KKMKKEQYSC) at the amino terminus of P34. Hopp-Woods hydrophilicity analysis of the deleted amino terminus of P34 shows that it is more hydrophilic and charged than the sequence of the protein which immediately follows.

Journal Article↗
PubMed1989

Transport of proteins to the plant vacuole is not by bulk flow through the secretory system, and requires positive sorting information.

Plant cells, like other eukaryotic cells, use the secretory pathway to target proteins to the vacuolar/lysosomal compartment and to the extracellular space. We wished to determine whether the presence of a hydrophobic signal peptide would result in the transport of a reporter protein to vacuoles by bulk flow; to investigate this question, we expressed a chimeric gene in transgenic tobacco. The chimeric gene, Phalb, used for this study consists of the 1,188-bp 5' upstream sequence and the hydrophobic signal sequence of a vacuolar seed protein phytohemagglutinin, and the coding sequence of a cytosolic seed albumin (PA2). The chimeric protein PHALB cross-reacted with antibodies to PA2 and was found in the seeds of the transgenic plants (approximately 0.7% of total protein), but not in the leaves, roots, or flowers. Immunoblot analyses of seed extracts revealed four glycosylated polypeptides ranging in molecular weight from 29,000 to 32,000. The four polypeptides are glycoforms of a single polypeptide of Mr 27,000, and the heterogeneity is due to the presence of high mannose and endoglycosidase H-resistant glycans. The PHALB products reacted with an antiserum specific for complex plant glycans indicating that the glycans had been modified in the Golgi apparatus. Subcellular fractionation of glycerol extracts of mature seeds showed that only small amounts of PHALB accumulated in the protein storage vacuoles of the tobacco seeds. In homogenates made in an isotonic medium, very little PHALB was associated with the organelle fraction containing the endoplasmic reticulum and Golgi apparatus; most of it was in the soluble fraction. We conclude that PHALB passed through the Golgi apparatus, but did not arrive in the vacuoles. Transport to vacuoles is not by a bulk-flow mechanism, once proteins have entered the secretory system, and requires information beyond that provided by a hydrophobic signal peptide.

Amino Acid Sequence↗