Cloning and expression of pinB gene from Triticum monococum seeds.

Puroindolines (PIN) are low molecular weight, cysteine-rich, endosperm-specific, basic proteins with a unique tryptophan-rich domain found in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) as well as other members of Triticaceae. PINs appear to be involved in both flour softness as well as resistance against fungal diseases. These proteins are known to be the major components of 'friabilin' associated with the surface of water washed starch grains and possess lipid binding properties. Structural characterization of puroindolines from Triticum monococum was initiated by amplifying and subsequently cloning the corresponding pin gene into an expression vector, known as pET-32a(+). The protein contains five tryptophanin domains and ten cysteine residues. The pinB gene was fused with the 109aa Trx.Tag thioredoxin for a high-level expression. The cloning sites used for producing fusion proteins also contained cleavable His.Tag and S.tag sequences for detection and purification. After transformation of competent Origami cells, fusion protein expression was detected by growing a transformant in LB medium in the presence of 0.1 mM IPTG at room temperature for 6 hrs on a shaker. Both soluble and insoluble fusion proteins were extracted from Origami cells after sonication. Ni-NTA column (Qiagen) was used to extract and purify these fractions. Following an overnight digestion of the recombinant protein with enterokinase at room temperature, the corresponding fractions were electrophoresed in polyacrylamide gel, electroblotted onto a nitrocellulose membrane and cross-reacted with the anti-friabilin monoclonal antibody. We found that the recombinant PINB protein had a molecular weight of 16 kDa whereas TrxB was 21 kDa. Fusion protein ran at 34 kDa. PINB protein from wheat was shown to be immunologically related to a homologue, tryptophanin, in oat seed. Further study is currently underway to characterize these proteins structurally using NMR.

Genomic clones encoding 11S globulins in oats (Avena sativa L.).

We have isolated two complete genomic clones, Glav1 and Glav3, encoding 11S globulins (legumins) in oat. The structure of Glav1 deviates from that of the typical legumin gene. This clone possesses an extra intron and an extra exon that is composed entirely of repeats of sequences found elsewhere in the clone. If this exon is functional, the protein encoded by Glav1 will contain novel octapeptide and hendecapeptide repeats. The two Glav clones show stronger and more extensive homology with one another than with the two previously published genomic clones, OG1-E1 and ASglob5. This result suggests that the oat globulin gene family may be divided into distinct subfamilies or that there may be significant cultivar-specific differences among members of this gene family.

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons.

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of Mr=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (Mr=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.

Ultrastructure of the partially coated reticulum and dictyosomes during endocytosis by soybean protoplasts.

Individual and serial sections were used to obtain detailed information regarding the morphology and distribution of the partially coated reticulum (PCR) and to determine its relationship with dictyosomes in endocytotically active soybean (Glycine max. (L.) Merr.) protoplasts. The results confirm and extend the description of the PCR provided by T.C. Pesacreta and W.J. Lucas (1985, Protoplasma 125, 173-184) from whole cells of selected angiosperms. The PCR of soybean protoplasts consists of a set of interconnected tubular membranes bearing a clathrin-like coat over part of their cytoplasmic surface. A dilation, sometimes containing small vesicles, is frequently seen in this organelle. The PCR often appears associated with dictyosomes but also occurs independent of other cell organelles. Only one example of a direct connection between the PCR and dictyosomes was observed.Following adsorptive endocytosis of cationized ferritin, the label appears in the PCR within 2 min and accumulates with time. It is never observed in the membrane dilations. Serial sectioning established that dictyosomes are labelled with cationized ferritin around the periphery of several cisternae, including those on both sides of the same dictyosome.

Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts.

Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.