scholarly journals Role of the endoplasmic reticulum in the synthesis of reserve proteins and the kinetics of their transport to protein bodies in developing pea cotyledons.

1982 ◽  
Vol 93 (1) ◽  
pp. 5-14 ◽  
Author(s):  
M J Chrispeels ◽  
T J Higgins ◽  
S Craig ◽  
D Spencer
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Protein Body ◽  
Protein Glycosylation ◽  
Protein Bodies ◽  
Marker Enzyme ◽  
Tissue Extracts ◽  
Reserve Protein ◽  
Rough Er ◽  
Nadh Cytochrome

Developing pea (Pisum sativum L.) cotyledons were labeled with radioactive amino acids, glucosamine, and mannose in pulse an pulse-chase experiments to study the synthesis, glycosylation, and transport of the reserve proteins vicilin and legumin to the protein bodies. Tissue extracts were fractionated on sucrose gradients to isolate either the endoplasmic reticulum (ER) or the protein bodies. Immunoaffinity gels were used to determine radioactivity in the reserve proteins (legumin and vicilin). After pulse-labeling for 45 min with amino acids, about half the total incorporated radioactivity coincided closely with the position of the ER marker enzyme NADH-cytochrome c reductase at a density of 1.13 g . cm-3 on the sucrose gradient. Both radioactivity and enzyme activity shifted to a density of 1.18 g . cm-3 in the presence of 3 mM MgCl2 indicating that the radioactive proteins were associated with the rough ER. Approximately half of the incorporated radioactivity associated with the rough ER was in newly synthesized reserve protein and this accounted for 80% of the reserve protein synthesized in 45 min. Trypsin digestion experiments indicated that these proteins were sequestered within the ER. In pulse-chase experiments, the reserve proteins in the ER became radioactive without appreciable lag and radioactivity chased out of the ER with a half-life of 90 min. Radioactive reserve proteins became associated with a protein body-rich fraction 20-30 min after their synthesis and sequestration by the ER. Pulse-chase experiments with radioactive glucosamine and mannose in the presence and absence of tunicamycin indicated that glycosylation of vicilin occurs in the ER. However, glycosylation is not a prerequisite for transport of vicilin from ER to protein bodies. Examination of the reserve protein polypeptides by SDS PAGE followed by fluorography showed that isolated ER contained legumin precursors (Mr 60,000-65,000) but not the polypeptides present in mature legumin (Mr 40,000 and 19,000) as well as the higher molecular weight polypeptides of vicilin (Mr 75,000, 70,000, 50,000, and 49,000). The smaller polypeptides of vicilin present in vicilin extracted from protein bodies (Mr 12,000-34,000) were absent from the ER. The results show that newly synthesized reserve proteins are preferentially and transiently sequestered within the ER before they move to the protein bodies, and that the ER is the site of storage protein glycosylation.

The aspartic proteinase is expressed in Arabidopsis thaliana seeds and localized in the protein bodies

1999 ◽  
Vol 9 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Asuman Mutlu ◽  
Xia Chen ◽  
Sridhar M. Reddy ◽  
Susannah Gal
Keyword(s):  
Arabidopsis Thaliana ◽  
Seed Storage Protein ◽  
Protein Body ◽  
Seed Storage ◽  
Cell Types ◽  
Aspartic Proteinase ◽  
Protein Bodies ◽  
Marker Enzyme ◽  
Antigenic Peptides ◽  
2S Albumin

AbstractWe have been studying a seed aspartic proteinase, termed AtAP, from Arabidopsis thaliana. In previous work, we purified the proteinase, analysed its activity and isolated the cDNA sequence. In this paper, the expression of the mRNA for the aspartic proteinase was analysed in seed tissues both by Northern blots for overall regulation and by in situ hybridization to follow cell-specific localization of message. We found a 1.9 kb aspartic proteinase message in dry seeds and seed pods. This message was expressed in many different cell types of the mature dry seed. The localization of the protein within these cells was also determined. Antibodies were raised against the AtAP and purified using affinity chromatography on an AtAP–immobilized-pepstatin A–agarose column. This purified antibody recognized several AtAP peptides in seeds. To localize the enzyme in cells, we isolated protein bodies from the dry seeds of Arabidopsis using a non-aqueous isolation method. The AtAP activity and antigenic peptides were found to be highest in the protein body fraction and co-localized with the seed storage protein 2S albumin and the vacuolar marker enzyme α-mannosidase. This protein body localization of the AtAP was confirmed with immunocytochemical localization by electron microscopy and shows that the protein is not secreted by these cells.

Seasonally dependent formation of protein-storage vacuoles in the inner bark tissues of Salix microstachya

1990 ◽  
Vol 68 (8) ◽  
pp. 1747-1755 ◽  
Author(s):  
John S. Greenwood ◽  
Cobi Demmers ◽  
Suzanne Wetzel
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Protein Body ◽  
Protein Bodies ◽  
Electron Microscopic ◽  
Protein Storage ◽  
Inner Bark ◽  
Protein Storage Vacuoles ◽  
Body Development ◽  
Microscopic Studies

The inner bark tissues of temperate hardwoods often act in the temporary storage of reduced nitrogen as protein during the overwintering period. Electron microscopic studies reported here demonstrate the analogy between the protein-storage vacuoles of the inner bark tissues and protein bodies in seeds. Development of these organelles parallels that of protein body formation seen in many dicotyledonous seeds. Coincident with the synthesis and sequestering of specific proteins, the large central vacuoles of the phloem parenchyma cells are slowly replaced over a 3- to 4-week period with numerous smaller protein-storage vacuoles (protein bodies). These arise via the subdivision of the larger vacuole and subsequent filling of the smaller vacuoles with protein. During this process there is a proliferation of both free ribosomes and rough endoplasmic reticulum in the ground cytoplasm. Stacks of rough endoplasmic reticulum are present in the peripheral cytoplasm and surround the smaller vacuoles as proteinaceous material is deposited. Golgi complexes, although not numerous, are present in the ground cytoplasm during the filling of the protein storage vacuoles. Key words: protein-storage vacuoles, protein body development, Salix microstachya, hardening, nitrogen storage, dormancy onset.

scholarly journals In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

1983 ◽  
Vol 96 (4) ◽  
pp. 999-1007 ◽  
Author(s):  
R Bollini ◽  
A Vitale ◽  
MJ Chrispeels
Keyword(s):  
Endoplasmic Reticulum ◽  
In Vitro Translation ◽  
In Vitro Synthesis ◽  
Run Off ◽  
Reserve Protein ◽  
Rough Er ◽  
Pass Through ◽  
The One

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.

Ultrastructural and histochemical postfertilization megagametophyte and zygotic embryo development of white spruce (Picea glauca) emphasizing the deposition of seed storage products

1993 ◽  
Vol 71 (1) ◽  
pp. 98-112 ◽  
Author(s):  
Marek J. Krasowski ◽  
John N. Owens
Keyword(s):  
Endoplasmic Reticulum ◽  
Picea Glauca ◽  
Zygotic Embryo ◽  
Smooth Endoplasmic Reticulum ◽  
Protein Body ◽  
Seed Storage ◽  
White Spruce ◽  
Variable Number ◽  
Protein Bodies ◽  
Cytoplasmic Vesicles

Deposition of major storage substances in the megagametophyte and embryo of white spruce (Picea glauca (Moench) Voss) was studied ultrastructurally and histochemically during seed development. Lipid bodies appeared to be secreted by the smooth endoplasmic reticulum. In the megagametophyte they were deposited rapidly from the club-shaped embryo stage until the early organogenesis of the embryo. Major lipid accumulation in the embryo took place during rapid cotyledon development and simultaneously with the buildup of protein bodies. Formation of protein bodies in the megagametophyte and in the embryo was first detected approximately 6 and 29 days after fertilization, respectively. It is suggested that in the megagametophyte, this process proceeded by (i) deposition of amorphous protein clumps onto tonoplasts of subdividing vacuoles during early stages of protein body formation, (ii) fusion of small cytoplasmic vesicles possibly derived from the rough endoplasmic reticulum, and (iii) deposition of protein around dense, membrane-bound vesicles attached to tonoplasts. The third process was not observed in the embryo. During advanced formation, fusion of cytoplasmic vesicles into developing protein bodies was the only process observed in the megagametophyte and in the embryo. White spruce seed protein bodies contained a variable number of crystalloids and globoid cavities except in the embryo in which only single globoid cavities were observed. Key words: Picea, ultrastructure, histochemistry, megagametophyte, embryo, seed.

Immunocytochemical analysis of protein body formation in seeds of Sorghum bicolor

1989 ◽  
Vol 67 (10) ◽  
pp. 2850-2856 ◽  
Author(s):  
Hari B. Krishnan ◽  
Jerry A. White ◽  
Steven G. Pueppke
Keyword(s):  
Endoplasmic Reticulum ◽  
Sorghum Bicolor ◽  
Rough Endoplasmic Reticulum ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Protein Body ◽  
Electrophoretic Analysis ◽  
Protein Bodies ◽  
Aleurone Layer ◽  
Dark Staining

Electrophoretic analysis of sorghum (Sorghum bicolor (L.) Moench) seed prolamines in the presence of sodium dodecyl sulfate reveals major proteins of 27 and 25 kDa and two other proteins of 18 and 12 kDa. Antibodies were raised against this prolamine fraction and used to examine the subcellular distribution of the proteins in developing sorghum seeds. Protein bodies in the starchy endosperm and subaleurone cells usually are round in cross section and contain darkly staining materials arranged in concentric rings. Protein bodies in the first two layers beneath the aleurone layer are irregular in shape and contain discrete pockets of light and dark staining inclusions. Prolamines were detected in both types of protein bodies by immunolabeling. Other oganelles, including Golgi complexes, mitochondria, and amyloplasts, were not labeled. The protein bodies, which have ribosomes attached to their surfaces, are directly connected to the rough endoplasmic reticulum. In some instances, this endoplasmic reticulum was specifically labeled with protein A – gold particles. Based on these observations, we suggest that the rough endoplasmic reticulum serves as the site of both synthesis and accumulation of sorghum prolamines.

Endosperm Morphology and Protein Body Formation in Developing Wheat Grain

1981 ◽  
Vol 8 (1) ◽  
pp. 5 ◽  
Author(s):  
WP Campbell ◽  
JW Lee ◽  
TP O'brien ◽  
MG Smart
Keyword(s):  
Endoplasmic Reticulum ◽  
Culture Medium ◽  
Sucrose Concentration ◽  
Light And Electron Microscopy ◽  
Starch Synthesis ◽  
Protein Body ◽  
Protein Bodies ◽  
Wheat Grain ◽  
Body Formation ◽  
Intact Plants

The development of wheat grain from intact plants and from detached ears growing in culture has been studied by light and electron microscopy. Provided the sucrose concentration was at a level sufficient to maintain a normal rate of starch synthesis, the endosperm morphology of grain from cultured ears was essentially identical to that of endosperm from intact plants. If, however, sucrose concentration in the culture medium was very low (0.25%), some morphological abnormalities occurred in the endosperm near the crease and adjacent to the seed coat. The synthesis of storage protein in the endosperm is believed to occur largely on polyribosomes attached to endoplasmic reticulum even at the earliest stages of development. Protein bodies are always surrounded by a single membrane, the origin of which may vary. Some protein bodies arise by distention of the endoplasmic reticulum and clearly the membrane here represents the sac into which the protein is discharged after synthesis. In other cases the bounding membrane may be that of a true vacuole or it may be dictyosomal in origin. The methods by which it is suggested that protein bodies are formed in wheat endosperm have parallels in other seeds, although there are some significant differences.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Prevalence of the pit and antipit in the genus Glycine

1987 ◽  
Vol 45 ◽  
pp. 986-987
Author(s):  
R. W. Yaklich ◽  
E. L. Vigil ◽  
W. P. Wergin
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Seed Coat ◽  
Cell Types ◽  
Abaxial Surface ◽  
Legume Seed ◽  
Initial Report ◽  
Cone Cells ◽  
Glycine Max L

The legume seed coat is the site of sucrose unloading and the metabolism of imported ureides and synthesis of amino acids for the developing embryo. The cell types directly responsible for these functions in the seed coat are not known. We recently described a convex layer of tissue on the inside surface of the soybean (Glycine max L. Merr.) seed coat that was termed “antipit” because it was in direct opposition to the concave pit on the abaxial surface of the cotyledon. Cone cells of the antipit contained numerous hypertrophied Golgi apparatus and laminated rough endoplasmic reticulum common to actively secreting cells. The initial report by Dzikowski (1936) described the morphology of the pit and antipit in G. max and found these structures in only 68 of the 169 seed accessions examined.

Thin-section, freeze-fracture, and energy dispersive x-ray analysis studies of the protein bodies of tomato seeds

1980 ◽  
Vol 58 (6) ◽  
pp. 699-711 ◽  
Author(s):  
Ernest Spitzer ◽  
John N. A. Lott
Keyword(s):  
Freeze Fracture ◽  
Protein Body ◽  
Cell Types ◽  
Energy Dispersive ◽  
Protein Bodies ◽  
Elemental Content ◽  
Tomato Seed ◽  
Endosperm Tissue ◽  
X Ray ◽  
Edx Analysis

Protein bodies of dry seeds of tomato (Lycopersicon esculentum) from radicle, hypocotyl, cotyledon, and endosperm tissue were extensively studied using thin-sectioning, freeze-fracturing and energy dispersive x-ray (EDX) analysis. Protein bodies varied in size, were oval to circular in section, and generally consisted of a proteinaceous matrix, globoid crystal, and protein crystalloid components. Size, shape, and arrangements of globoid crystals and protein crystalloids varied even within the same cell. Globoid crystals were generally oval to circular in section. They were always surrounded by a proteinaceous matrix. In a given protein body the number present ranged from a few to numerous. A protein body generally contained only one protein crystalloid. In section, protein crystalloids were irregular or angular in shape. They were composed of substructural particles which formed lattice planes. EDX analysis of tomato seed globoid crystals revealed the presence of P, K, and Mg in all cases, a fact that is consistent with globoid crystals being phytin-rich. Rarely, small amounts of calcium were found along with P, K, and Mg in globoid crystals of each of the tissue regions considered. The distribution pattern of cells with Ca containing globoid crystals was random. Small amounts of Fe and Mn were also found in the globoid crystals of protein bodies from certain cell types. These two elements, unlike calcium, were specific in terms of their distribution. Globoid crystals from the protodermal cells often contained Mn and Fe. The globoid crystals from provascular tissue of radicle, hypocotyl, and cotyledon regions often contained Fe while globoid crystals in the first layer of large cells surrounding these provascular areas always contained Fe. Results from EDX analysis of the proteinaceous material from the protein bodies are presented and discussed as are variations in elemental content due to different fixations.

scholarly journals Analytical subcellular fractionation of needle-biopsy specimens from human liver

1978 ◽  
Vol 174 (2) ◽  
pp. 435-446 ◽  
Author(s):  
T J Peters ◽  
C A Seymour
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Needle Biopsy ◽  
Human Liver ◽  
Resolving Power ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Acid Hydrolases ◽  
Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

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