Protein Quality Control along the Route to the Plant Vacuole

Giovinazzo, Giovanna; Bielli, Anna; Virgilio, Maddalena de; Frigerio, Lorenzo; Pesca, Michela; Faoro, Franco; Bollini, Roberto; Ceriotti, Aldo; Vitale, Alessandro

doi:10.2307/3870531

Cited by 59 publications

(132 citation statements)

References 15 publications

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“…4A). To monitor the efficacy of BFA treatment, we also followed the transport of the bean storage protein phaseolin, whose arrival in the vacuoles is followed by proteolytic processing and generation of a series of fragments in the 22-to 28-kDa range (21). Whereas BFA alone did not block the degradation of RTA during the chase (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells

Cola

Frigerio

Lord

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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When expressed in tobacco cells, the catalytic subunit of the dimeric ribosome inactivating protein, ricin, is first inserted into the endoplasmic reticulum (ER) and then degraded in a manner that can be partially inhibited by the proteasome inhibitor clastolactacystin ␤-lactone. Consistent with the implication of cytosolic proteasomes, degradation of ricin A chain is brefeldin A-insensitive and the polypeptides that accumulate in the presence of the proteasome inhibitor are not processed in a vacuole-specific fashion. Rather, these stabilized polypeptides are in part deglycosylated by a peptide:N-glycanase-like activity. Taken together, these results indicate that ricin A chain, albeit a structurally native protein, can behave as a substrate for ER to cytosol export, deglycosylation in the cytosol, and proteasomal degradation. Furthermore, retrotranslocation of this protein is not tightly coupled to proteasomal activity. These data are consistent with the hypothesis that ricin A chain can exploit the ER-associated protein degradation pathway to reach the cytosol. Although well characterized in mammalian and yeast cells, the operation of a similar pathway to the cytosol of plant cells has not previously been demonstrated.

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Section: Resultsmentioning

confidence: 99%

“…Frozen samples were homogenized by adding 2 vol of protoplast homogenization buffer supplemented with Complete protease inhibitor mixture (Roche) (21). Homogenates were used for immunoprecipitation with rabbit anti-RTA, anti-BiP (21), or anti-phaseolin antisera.…”

Section: Preparation Of Protein Extracts and Immunoprecipitationmentioning

confidence: 99%

Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells

Cola

Frigerio

Lord

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…The construction of pDHET343F has been described (15). This construct contains the complete coding sequence of a phaseolin variant in which the glycosylation site at position 341 has been destroyed.…”

Section: Methodsmentioning

confidence: 99%

Free Ricin A Chain, Proricin, and Native Toxin Have Different Cellular Fates When Expressed in Tobacco Protoplasts

Frigerio¹,

Vitale²,

Lord³

et al. 1998

Journal of Biological Chemistry

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The catalytic A subunit of ricin can inactivate eukaryotic ribosomes, including those of Ricinus communis where the toxin is naturally produced. How such plant cells avoid intoxication has remained an open question. Here we report the transient expression of a number of ricin A chain-encoding cDNA constructs in tobacco protoplasts. Ricin A chain entered the endoplasmic reticulum lumen, where it was efficiently glycosylated, but it was toxic to the cells and disappeared with time in a brefeldin A-insensitive manner, suggesting reverse translocation to the cytosol and eventual degradation. Proricin (the natural precursor form containing A and B chains joined together by a linker sequence) was glycosylated, transported to the vacuole, and processed to its mature form, but was not toxic. Free ricin A chain and proricin were not secreted, whereas free ricin B chain was found entirely in the extracellular medium. The coexpression of ricin A and B chains resulted in the formation of disulfide-linked, transport-competent heterodimers, which were secreted, with a concomitant reduction in the observed cytotoxicity. These results suggest that the production of ricin as a precursor is essential for its routing to the vacuole and for protection of ricin-producing cells.Ricin is a cytotoxin present in the endosperm of Ricinus communis seeds, where it accumulates in protein bodies (storage vacuoles) to 5% of the total particulate protein (1). Structurally, ricin is a heterodimeric glycoprotein comprising a ribosome-inactivating A chain (RTA) 1 and a galactose-binding B chain (RTB) covalently linked by a single disulfide bond. RTA (glycosylated molecular mass ϳof 32 kDa) catalyzes the removal of a single adenine from a highly conserved loop of 28 S/26 S/25 S rRNA within the context of a eukaryotic ribosome (2). Ribosomes depurinated in this manner are unable to bind the elongation factor-2⅐GTP complex, and protein synthesis is blocked at the translocation step of the elongation cycle (3). The precise activity of RTA varies depending on the source of ribosomes. Thus, a single A chain molecule can depurinate 1000 -2000 mammalian cell ribosomes/min under physiological conditions (2). This can be measured in vitro as a DC 50 (the concentration causing 50% depurination) of ϳ5 ng/ml under standard conditions. Although ricin A chain is certainly active against tobacco ribosomes, the DC 50 value is 650 ng/ml, showing that tobacco ribosomes are ϳ130-fold less sensitive than mammalian or salt-washed yeast ribosomes (4). Nevertheless, such is the potency of RTA that should it begin to accumulate within the cytosol of tobacco leaf protoplasts, the protein biosynthetic capacity of the expressing cells would be severely compromised.When heterodimeric ricin is presented to the surface of mammalian cells, RTB opportunistically binds to membrane components with exposed galactose residues. Toxin molecules are then endocytosed to a specific internal compartment from which RTA translocates to reach the cytosol, where the ribosomes are located. The...

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“…Expression constructs encoding ppRT, pRTA, pRTB, and phaseolin (pDHE-T343F) have been described previously (19,23). The ricin active site substitution E177D has also been previously documented (24).…”

Section: Methodsmentioning

confidence: 99%

The N-terminal Ricin Propeptide Influences the Fate of Ricin A-chain in Tobacco Protoplasts

Jolliffe

Cola

Marsden

et al. 2006

Journal of Biological Chemistry

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The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A-and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed.Ricin is a heterodimeric protein produced in the seeds of the castor oil plant Ricinus communis where it accumulates in the protein storage vacuoles (PSV) 3 of endosperm cells. Mature ricin consists of a ribosome-inactivating A-chain (RTA) linked by a disulfide bond and non-covalent interactions to a galactose binding B-chain (RTB). This heterodimer is toxic to mammalian cells because it can bind via RTB to a variety of galactosylated cell surface molecules and, following retrograde transport to the endoplasmic reticulum (ER) and delivery of RTA to the cytosol, irreversibly inactivate ribosomes. RTA is a potent N-glycosidase that depurinates 28 S/25 S/26 S ribosomal RNA (1, 2) at a site in the ribosome that is critical for binding elongation factor-2 ternary complexes (3, 4). This leads to a halt in protein synthesis and, ultimately, cell death.Although the ribosomes of Ricinus endosperm cells are susceptible to RTA-mediated depurination (5), intoxication of the producing tissue is avoided. Co-translational ER import is accompanied by N-glycosylation (6), disulfide bond formation (7), and proteolytic cleavage of the signal peptide (8), the first 26 residues of a 35-residue presequence at the N terminus of preproricin (9) (Fig. 1). Imported proricin consists of a 9-residue N-terminal propeptide, the mature RTA sequence, a 12-residue linker propeptide, and RTB. This precursor is catalytically inactive (10) because the RTB moiety sterically obstructs the substrate binding site of RTA, as it does in mature ricin heterodimers. In this form, proricin is delivered to PSV, and the mature RTA-RTB heterodimer is generated by the endoproteolytic removal of both the N-terminal and internal propeptides (7,(11)(12)(13)(14). Ricin holotoxin accumulates within the confines of the endosperm vacuoles to 5% of the total particulate protein (15, 16).The ricin precursor and its constituent subunits have been well studied in the heterologous system of tobacco protoplasts (17)(18)(19)(20). Although it is clear that th...

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Protein Quality Control along the Route to the Plant Vacuole

Cited by 59 publications

References 15 publications

Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells

Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells

Free Ricin A Chain, Proricin, and Native Toxin Have Different Cellular Fates When Expressed in Tobacco Protoplasts

The N-terminal Ricin Propeptide Influences the Fate of Ricin A-chain in Tobacco Protoplasts

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