Purification of a seed glycoprotein: N-terminal and deglycosylation analysis of phaseolin

Paaren, Herbert E.; Slightom, Jerry L.; Hall, Timothy C.; Inglist, Adam S.; Blagrove, R.J.

doi:10.1016/s0031-9422(00)81410-7

Cited by 27 publications

(31 citation statements)

References 24 publications

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“…However, phaseolin accumulated in the storage vacuoles of bean cotyledonary cells lacks a few amino acids with respect to the ER-associated precursor (Bollini et al, 1982;D'Amico et al, 1992). The N-terminal sequence of phaseolin purified from bean cotyledons is the one predicted after signal peptide removal, indicating that trimming must be at the C terminus (Paaren et al, 1987). Therefore, the Ala-Phe-Val-Tyr sequence probably constitutes or is part of a short transient peptide and is most likely exposed on the surface of phaseolin trimers.…”

Section: The Sorting Signalmentioning

confidence: 97%

Sorting of Phaseolin to the Vacuole Is Saturable and Requires a Short C-Terminal Peptide

et al. 1998

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Phaseolin, one of the major legume proteins for human nutrition, is a trimeric glycoprotein of the 7S class that accumulates in the protein storage vacuoles of common bean. Phaseolin is cotranslationally introduced into the lumen of the endoplasmic reticulum; from there, it is transported through the Golgi complex to the storage vacuoles. Phaseolin is also transported to the vacuole in vegetative tissues of transgenic plants. By transient and permanent expression in tobacco leaf cells, we show here that vacuolar sorting of phaseolin is saturable and that saturation leads to Golgimediated secretion from the cell. A mutated phaseolin, in which the four C-terminal residues (Ala, Phe, Val, and Tyr) were deleted, efficiently formed trimers but was secreted entirely outside of the cells in transgenic tobacco leaves, indicating that the deleted sequence contains information necessary for interactions with the saturable vacuolar sorting machinery. In the apoplast, the secreted phaseolin remained intact; this is similar to what occurs to wild-type phaseolin in bean storage vacuoles, whereas in vegetative vacuoles of transgenic plants, the storage protein is fragmented. INTRODUCTIONPhaseolin is the major storage protein of common bean. Phaseolin is a member of the 7S vicilin class and one of the most important legume proteins for human nutrition; a number of efforts have been made to improve its nutritional value (Hoffman et al., 1988; Dyer et al., 1995). The structure, genetic makeup, cotranslational and post-translational modifications, and intracellular transport of phaseolin have been elucidated largely by numerous investigators (Bollini et al., 1982;Slightom et al., 1985;Sturm et al., 1987;Lawrence et al., 1994), but the mechanisms that allow correct intracellular targeting of phaseolin and the other 7S storage proteins have not been fully characterized.Phaseolin is a homotrimeric soluble protein that accumulates in the protein storage vacuoles of cotyledonary cells. Its synthesis, maturation, and intracellular targeting are mediated by the secretory pathway, which delivers proteins into the endoplasmic reticulum (ER) and from there to the cell surface or the vacuoles (Okita and Rogers, 1996). The Golgi complex as well as other intermediate compartments mediate this traffic.Protein constructs that have a transient signal peptide for cotranslational insertion into the ER, but no other specific sorting signal, are secreted from plant cells (Denecke et al., 1990;Hunt and Chrispeels, 1991). Soluble proteins destined for the different vacuoles are sorted from the proteins destined for the apoplast, probably at the exit of the Golgi complex (Ahmed et al., 1997;Paris et al., 1997). Sorting occurs because vacuolar proteins have structures, often identified as short stretches of amino acids in propeptides, that are not present in apoplastic proteins. The signals are variable, and different vacuolar sorting mechanisms must exist (Matsuoka et al., 1995;Kirsch et al., 1996). A putative integral membrane receptor that recognize...

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Section: The Sorting Signalmentioning

confidence: 97%

Sorting of Phaseolin to the Vacuole Is Saturable and Requires a Short C-Terminal Peptide

et al. 1998

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“…4). The additional polypeptide produced by the TFMSA procedure would not be detected on one-dimensional PAGE as was employed by Edge et al (6) and Paaren et al (25). The decrease in Mr for the deglycosylated membrane glycopolypeptide No.…”

Section: Discussionmentioning

confidence: 96%

“…For example, glycosylation may be required for proteins to assume the correct conformation for secretion since TM not only blocked invertase and acid phosphatase glycosylation but also prevented their secretion in yeast (24). Carbohydrate residues are putatively required for targeting delivery to specific organelles, since in the presence of TM, lysosomal enzymes of chick embryo fibroblasts were misdirected and secreted (25). Phytohemagglutinin, a plant storage protein, is synthesized and cotranslationally glycosylated on rough ER then further modified by the addition of fucose in the GA (3).…”

Section: Discussionmentioning

confidence: 99%

“…Chemical deglycosylation of proteins using TFMSA is a technique which avoids the problems of substrate specificity and steric hindrance inherent with an enzymic deglycosylation approach (6,25). Although TFMSA treatment of glycopolypeptide No.…”

Section: Discussionmentioning

confidence: 99%

“…The purified membrane glycopolypeptide 6 was deglycosylated as described by Paaren et al (25). The protein was precipitated with chloroform:methanol:H20 as described by Wessel and Flugge (32), dried, and dissolved in urea buffer (…”

Section: Chemical Deglycosylationmentioning

confidence: 99%

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Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

LaFayette¹,

Travis²

1989

Plant Physiol.

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Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosacchande moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [3H]glucosamine and [35S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.in the pathway of protein glycosylation is an effective strategy in elucidating the function of the glycan moiety of glycoproteins (7). One of the most specific inhibitors is TM,' a nucleoside antibiotic produced by Streptomyces lysosuperiflcus, which blocks the first step in the formation of the lipid-linked saccharide core, i.e. the transfer of GlcNAc-1-P from UDPGlcNAc to dolichyl-P to produce GlcNAc-PP-dolichyl (UDPGlcNAc:dolichyl-phosphate GlcNAc-1-P transferase) (30). In cultured soybean cells TM completely blocked the incorporation of [3H]mannose into lipid-linked oligosaccharides (14).

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Transformation of Plant Cells

Hall

DeRose

2007

Ciba Foundation Symposium 137 ‐ Applications of Plant Cell and Tissue Culture

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Purification of a seed glycoprotein: N-terminal and deglycosylation analysis of phaseolin

Cited by 27 publications

References 24 publications

Sorting of Phaseolin to the Vacuole Is Saturable and Requires a Short C-Terminal Peptide

Sorting of Phaseolin to the Vacuole Is Saturable and Requires a Short C-Terminal Peptide

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Transformation of Plant Cells

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