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C D Dickinson

Publications and source records attributed to C D Dickinson.

24 records · Page 2Linked to original sources
PubMed1990

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.

Base Sequence↗
PubMed1990

A short domain of the plant vacuolar protein phytohemagglutinin targets invertase to the yeast vacuole.

Phytohemagglutinin (PHA), the seed lectin of the common bean, accumulates in protein storage vacuoles of storage parenchyma cells in cotyledons. When expressed in yeast, PHA is efficiently targeted to the yeast vacuole [Tague and Chrispeels (1987). J. Cell Biol. 105, 1971-1979]. To identify vacuolar sorting information in PHA, a series of 3' deletions of the PHA gene were fused in-frame to a truncated yeast invertase gene. An amino-terminal portion of PHA composed of a 20-residue signal sequence and 43 residues of the mature protein efficiently targeted invertase to the yeast vacuole. Internal deletions in a short PHA-invertase fusion showed that targeting information exists between residues 14 and 23 of mature PHA. Based on examinations of three-dimensional structures of related lectins, only a portion of these residues would be available on the surface of PHA for interaction with a putative receptor. Amino acid replacements at these positions in a PHA-invertase hybrid caused secretion of the invertase. The results indicate the presence of a vacuolar targeting domain in PHA that is centered at position 19 of the mature protein. This sequence of PHA also shows sequence identity to a vacuolar sorting domain characterized in yeast carboxypeptidase Y. Single amino acid alterations in a short PHA-invertase hybrid protein that caused the highest levels of secretion introduced a glycosylation site at position 21 of PHA. This observation suggests that glycan addition may interfere with recognition of a sorting determinant. These same amino acid changes did not dramatically increase secretion in a long PHA-invertase fusion or in PHA itself. Thus, a second domain of PHA may function in concert with the first one to bring about correct targeting of PHA.

Amino Acid Sequence↗
PubMed1989

Characterization of the glycinin gene family in soybean.

We characterized the structure, organization, and expression of genes that encode the soybean glycinins, a family of storage proteins synthesized exclusively in seeds during embryogenesis. Five genes encode the predominant glycinin subunits found in soybeans, and they have each been cloned, sequenced, and compared. The five genes have diverged into two subfamilies that are designated as Group-I and Group-II glycinin genes. Each glycinin gene contains four exons and three introns like genes that encode related proteins in other legumes. Two other genes have been identified and designated as "glycinin-related" because they hybridize weakly with the five glycinin genes. Although not yet characterized, glycinin-related genes could encode other glycinin subunit families whose members accumulate in minor amounts in seeds. The three Group-I glycinin genes are organized into two chromosomal domains, each about 45 kilobase pairs in length. The two domains have a high degree of homology, and contain at least five genes each that are expressed either in embryos or in mature plant leaves. Gel blot studies with embryo mRNA, as well as transcription studies with 32P-RNA synthesized in vitro from purified embryo nuclei, indicate that glycinin and glycinin-related genes become transcriptionally activated in a coordinated fashion early in embryogenesis, and are repressed coordinately late in seed development. In addition to transcriptional control processes, posttranscriptional events also are involved in regulating glycinin and glycinin-related mRNA levels during embryogenesis.

Amino Acid Sequence↗
PubMed1989

Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.

Base Sequence↗
PubMed1987

Self-assembly of proglycinin and hybrid proglycinin synthesized in vitro from cDNA.

An in vitro system was developed that results in the self-assembly of subunit precursors into complexes that resemble those found naturally in the endoplasmic reticulum. Subunits of glycinin, the predominant seed protein of soybeans, were synthesized from modified cDNAs using a combination of the SP6 transcription and the rabbit reticulocyte translation systems. Subunits produced from plasmid constructions that encoded either Gy4 or Gy5 gene products, but modified such that their signal sequences were absent, self-assembled into trimers equivalent in size to those precursors found in the endoplasmic reticulum. In contrast, proteins synthesized in vitro from Gy4 constructs failed to self-assemble when the signal sequence was left intact (e.g., preproglycinin) or when the coding sequence was modified to remove 27 amino acids from an internal hydrophobic region, which is highly conserved among the glycinin subunits. Various hybrid subunits were also produced by trading portions of Gy4 and Gy5 cDNAs and all self-assembled in our system. The in vitro assembly system provides an opportunity to study the self-assembly of precursors and to probe for regions important for assembly. It will also be helpful in attempts to engineer beneficial nutritional changes into this important food protein.

Journal Article↗