The Role of CDC48 in the Retro-translocation of Non-ubiquitinated Toxin Substrates in Plant Cells
2008; Elsevier BV; Volume: 283; Issue: 23 Linguagem: Inglês
10.1074/jbc.m709316200
ISSN1083-351X
AutoresRichard S. Marshall, Nicholas A. Jolliffe, Aldo Ceriotti, Christopher J. Snowden, Janet M. Lord, Lorenzo Frigerio, Lynne M. Roberts,
Tópico(s)Transgenic Plants and Applications
ResumoWhen the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterize the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that although RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesized RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated. When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterize the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that although RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesized RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated. As in mammalian and yeast cells, the plant cell endoplasmic reticulum (ER) 3The abbreviations used are: ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; RTA, ricin toxin A chain; RCA A, Ricinus communis agglutinin A chain; Endo H, endoglycosidase H; RFP, red fluorescent protein; UPR, unfolded protein response; YFP, yellow fluorescent protein; MES, 2-N-morpholinoethanesulfonic acid. 3The abbreviations used are: ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; RTA, ricin toxin A chain; RCA A, Ricinus communis agglutinin A chain; Endo H, endoglycosidase H; RFP, red fluorescent protein; UPR, unfolded protein response; YFP, yellow fluorescent protein; MES, 2-N-morpholinoethanesulfonic acid. is a major protein folding compartment. Newly synthesized soluble and membrane proteins destined to remain in the ER, or to be transported to the Golgi complex, vacuoles, or apoplast, are scrutinized by a stringent quality control system (1Vitale A. Plant Cell. 2001; 13: 1260-1262PubMed Google Scholar). Such surveillance ensures that newly synthesized proteins assume their native conformation and, where appropriate, assemble into oligomers. In mammalian and yeast cells, proteins that fail to fold and/or assemble correctly are usually retro-translocated to the cytosol and degraded by cytosolic proteasomes, in a tightly coupled pathway referred to as ER-associated protein degradation (ERAD) (2Romisch K. Annu. Rev. Cell Dev. Biol. 2005; 21: 435-456Crossref PubMed Scopus (276) Google Scholar, 3Meusser B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (999) Google Scholar). For the majority of misfolded or unassembled substrates, extraction from the ER membrane requires a cytosolic ubiquitin-interacting AAA-ATPase complex designated CDC48 in yeast (p97, also known as valosin-containing protein, in mammalian cells), in association with the adaptor proteins Ufd1p and Npl4p (4Braun S. Matuschewski K. Rape M. Thoms S. Jentsch S. EMBO J. 2002; 21: 615-621Crossref PubMed Scopus (290) Google Scholar, 5Ye Y. Meyer H.H. Rapoport T.A. Nature. 2001; 414: 652-656Crossref PubMed Scopus (897) Google Scholar, 6Rabinovich E. Kerem A. Frohlich K.-U. Diamant N. Bar-Nun S. Mol. Cell. Biol. 2002; 22: 626-634Crossref PubMed Scopus (470) Google Scholar, 7Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (438) Google Scholar, 8Ye Y. Meyer H.H. Rapoport T.A. J. Cell Biol. 2003; 162: 71-84Crossref PubMed Scopus (501) Google Scholar). This complex is believed to use its ATPase activity to segregate ubiquitinated ERAD substrates (7Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (438) Google Scholar, 9Rape M. Hoppe T. Gorr I. Kalocay M. Richly H. Jentsch S. Cell. 2001; 107: 667-677Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), and/or mechanically extract them from the ER membrane in readiness for delivery to proteasomes (5Ye Y. Meyer H.H. Rapoport T.A. Nature. 2001; 414: 652-656Crossref PubMed Scopus (897) Google Scholar, 8Ye Y. Meyer H.H. Rapoport T.A. J. Cell Biol. 2003; 162: 71-84Crossref PubMed Scopus (501) Google Scholar). However, not all ERAD substrates are modified by ubiquitin and extracted by this ATPase (8Ye Y. Meyer H.H. Rapoport T.A. J. Cell Biol. 2003; 162: 71-84Crossref PubMed Scopus (501) Google Scholar, 10Lee R.J. Liu C.W. Harty C. McCracken A.A. Latterich M. Romisch K. DeMartino G.N. Thomas P.J. 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A pathway closely resembling ERAD also operates in plant cells but, by comparison, relatively little is known about substrate recognition, extraction from the ER membrane, or the pathway(s) and machineries for degradation (13Ceriotti, A., and Roberts, L. M. (2006) in Plant Cell Monographs (Robinson, D. G., ed) Vol. 4, pp. 75-98, Springer Verlag, Berlin/HeidelbergGoogle Scholar). The Arabidopsis thaliana genome contains sequences for three CDC48-like proteins, one of which has been functionally shown to complement a yeast CDC48 mutant (14Feiler H.S. Desprez T. Santoni V. Kronenberger J. Caboche M. Traas J. EMBO J. 1995; 14: 5626-5637Crossref PubMed Scopus (100) Google Scholar, 15Rancour D.M. Dickey C.E. Park S. Bednarek S.Y. Plant Physiol. 2002; 130: 1241-1253Crossref PubMed Scopus (90) Google Scholar). This AAA-ATPase has been found to play a role in the quality control of a mutant polytopic membrane protein, barley (Hordeum vulgare) powdery mildew resistance protein (MLO), but its involvement in ERAD is thus far limited to this one example (16Muller J. Piffanelli P. Devoto A. Miklis M. Elliott C. Ortmann B. Schulze-Lefert P. Panstruga R. Plant Cell. 2005; 17: 149-163Crossref PubMed Scopus (90) Google Scholar). The process of retro-translocation, deglycosylation, and proteasomal degradation in plant cells was first characterized by studying the biosynthesis of the catalytic A subunit (RTA) of the A-B plant toxin ricin (17Di Cola A. Frigerio L. Lord J.M. Ceriotti A. Roberts L.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14726-14731Crossref PubMed Scopus (84) Google Scholar). This orphan polypeptide, once delivered into the ER by its signal peptide, was shown to retro-translocate in a manner unrelated to its propensity for ubiquitination (18Di Cola A. Frigerio L. Lord J.M. Roberts L.M. Ceriotti A. Plant Physiol. 2005; 137: 287-296Crossref PubMed Scopus (40) Google Scholar). Although it was shown that ubiquitination of internal lysine residues is not a strict requirement for ER to cytosol transport of this protein, the involvement or otherwise of CDC48 was not investigated. Here we have studied the effects of a dominant negative form of Arabidopsis CDC48 on the export of the orphan catalytic polypeptides of ricin and its close relative, Ricinus communis agglutinin, in tobacco protoplasts. The data presented clearly demonstrate that the retro-translocation of these proteins requires the participation of CDC48, and is irrespective of their glycosylation or ubiquitination status. Recombinant DNA—All DNA constructs were generated in the CaMV 35S-promoter-driven expression vectors pDHA (see Ref. 19Tabe L.M. Wardley-Richardson T. Ceriotti A. Aryan A. McNabb W. Moore A. Higgins T.J. J. Anim. Sci. 1995; 73: 2752-2759Crossref PubMed Scopus (130) Google Scholar, for toxin- and phaseolin-based constructs) and pamPAT-MCS (GenBank™ accession number AY436765 (16Muller J. Piffanelli P. Devoto A. Miklis M. Elliott C. Ortmann B. Schulze-Lefert P. Panstruga R. Plant Cell. 2005; 17: 149-163Crossref PubMed Scopus (90) Google Scholar)) or pGreenII-0029 (see Ref. 20Hellens R.P. Edwards E.A. Leyland N.R. Bean S. Mullineaux P.M. Plant Mol. Biol. 2000; 42: 819-832Crossref PubMed Scopus (1224) Google Scholar, for CDC48- or fluorescent protein-based constructs). Expression constructs encoding RTA, phaseolin (pDHE-T343F), CDC48, and cytosolic YFP have been described previously (16Muller J. Piffanelli P. Devoto A. Miklis M. Elliott C. Ortmann B. Schulze-Lefert P. Panstruga R. Plant Cell. 2005; 17: 149-163Crossref PubMed Scopus (90) Google Scholar, 21Frigerio L. Vitale A. Lord J.M. Ceriotti A. Roberts L.M. J. Biol. Chem. 1998; 273: 14194-14199Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 22Pedrazzini E. Giovinazzo G. Bielli A. de Virgilio M. Frigerio L. Pesca M. Faoro F. Bollini R. Ceriotti A. Vitale A. Plant Cell. 1997; 9: 1869-1880Crossref PubMed Scopus (147) Google Scholar, 23Hunter P.R. Craddock C.P. Di Benedetto S. Roberts L.M. Frigerio L. Plant Physiol. 2007; 145: 1371-1382Crossref PubMed Scopus (163) Google Scholar). The ricin active site substitution E177D, lysine substitutions K4R and K239R, and the removal of the proricin signal peptide and N-terminal propeptide have also been previously documented (18Di Cola A. Frigerio L. Lord J.M. Roberts L.M. Ceriotti A. Plant Physiol. 2005; 137: 287-296Crossref PubMed Scopus (40) Google Scholar, 24Chaddock J.A. Roberts L.M. Protein Eng. 1993; 6: 425-431Crossref PubMed Scopus (46) Google Scholar, 25Jolliffe N.A. Di Cola A. Marsden C.J. Lord J.M. Ceriotti A. Frigerio L. Roberts L.M. J. Biol. Chem. 2006; 281: 23377-23385Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). All derivative constructs used in this work were generated using the QuikChange™ in vitro mutagenesis system (Stratagene, La Jolla, CA) using the following mutagenic primers (and their reverse complements, not shown): a stop codon was introduced into the prepro-RCA sequence (26Roberts L.M. Lamb F.I. Pappin D.J. Lord J.M. J. Biol. Chem. 1985; 260: 15682-15686Abstract Full Text PDF PubMed Google Scholar) immediately after the open reading frame of the A chain to generate pRCA A using 5′-CCTCCACCGTCGTCAGAGTTTTAGTTGCTTATAAGGCCAGTGGTGCC-3′, and the equivalent active site substitution E176D was introduced into pRCA A using 5′-GGTTTGCATCCAAATGATTTCAGACGCAGCAAGATTCCAGTACATTG-3′. Sites of amino acid mutations are underlined. wtCDC48 and CDC48QQ were cloned into the HindIII-SmaI sites of the CaMV 35S-cassette using primers 5′-ATATATATAAGCTTATGTCTACCCCAGCTG-3′ and 5′-AACGAAGCCCGGGCTAATTGTAGAGATC-3′, for subsequent insertion into EcoRV-cut pGreenII-0029, used for tobacco leaf infiltration. Restriction enzyme sites are underlined. To generate cytosolic RFP, the monomeric RFP1 coding region was amplified by PCR from pcDNA1-mRFP (27Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (2004) Google Scholar) and cloned into the XbaI-SacI sites of the CaMV 35S-cassette using primers 5′-GCGCGCGTCTAGAATGGCCTCCTCCGAGGAC-3′ and 5′-TAATGATGGAGCTCTTAGGCGCCGGTGGAGTGGC-3′, again for subsequent insertion into EcoRV-cut pGreenII-0029. Restriction enzyme sites are underlined. Transient Transfection of Tobacco Leaf Protoplasts and Pulse-Chase Experiments—Protoplasts were prepared from axenic leaves (4 to 7 cm long) of Nicotiana tabacum cv. Petit Havana SR1 (28Maliga P. Sz-Breznovits A. Marton L. Nat. New Biol. 1973; 244: 29-30Crossref PubMed Scopus (339) Google Scholar), and subjected to polyethylene glycol-mediated transfection with one or more plasmids as previously described (22Pedrazzini E. Giovinazzo G. Bielli A. de Virgilio M. Frigerio L. Pesca M. Faoro F. Bollini R. Ceriotti A. Vitale A. Plant Cell. 1997; 9: 1869-1880Crossref PubMed Scopus (147) Google Scholar). Cells were radiolabeled with Pro-Mix (a mixture of [35S]cysteine and [35S]methionine (GE Healthcare)), and chased for the times indicated in the figures, as previously described (21Frigerio L. Vitale A. Lord J.M. Ceriotti A. Roberts L.M. J. Biol. Chem. 1998; 273: 14194-14199Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In some experiments, before radioactive labeling, protoplasts were incubated for 1 h at 25 °C in K3 medium (3.78 g/liter Gamborgs B5 basal medium with minimal organics, 750 mg/liter CaCl2·2H2O, 250 mg/liter NH4NO3, 136.2 g/liter sucrose, 250 mg/liter xylose, 1 mg/liter 6-benzylaminopurine, 1 mg/liter α-naphthalenacetic acid) supplemented with either 50 μg/ml tunicamycin (Sigma; 5 mg/ml stock in 10 mm NaOH) or 5 mm 1-deoxymannojirimycin (Sigma; 0.2 m stock in sterile H2O). When indicated, clasto-lactacystin β-lactone (Calbiochem, San Diego, CA; 20 mm stock in dimethyl sulfoxide) was added to a concentration of 80 μm at the beginning of the labeling period. At the desired time points, 3 volumes of cold W5 medium (9 g/liter NaCl, 0.37 g/liter KCl, 18.37 g/liter CaCl2·2H2O, 0.9 g/liter glucose) were added and protoplasts were pelleted by centrifugation at 60 × g for 10 min at 4 °C. Separated cell and media samples were frozen on dry ice and stored at -80 °C, unless further manipulations were to be performed as below. Tobacco Leaf Infiltration—The lower epidermis of 3 to 5-cm long tobacco leaves from 3 to 4-week-old plants were pressure infiltrated with a culture of Agrobacterium tumefaciens (transformed with empty vector, pGreenII-0029, or with either wild type or mutant CDC48) diluted to an A600 of 0.1 in infiltration media (50 mm MES, pH 5.6, 0.5% (w/v) glucose, 2 mm Na3PO4, 100 μm acetosyringone (10 mm stock in EtOH)). The plant was then incubated in greenhouse conditions for a further 3 days before preparation of protoplasts. Protoplast Fractionation—Protoplast pellets (from 500,000 cells) were resuspended in 140 μl of 12% sucrose buffer (100 mm Tris-HCl, pH 7.6, 10 mm KCl, 1 mm EDTA, 12% (w/w) sucrose, supplemented immediately before use with Complete™ protease inhibitor mixture (Roche Applied Science)) and homogenized by pipetting 50 times with a Gilson-type micropipette through a 200-μl tip. Intact cells and debris were removed by centrifugation at 500 × g for 5 min at 4 °C. 130 μl was loaded onto a 17% (w/w) sucrose pad and centrifuged at 100,000 × g for 30 min at 4 °C. Pellets (microsomes) and supernatants (soluble proteins) were frozen on dry ice and stored at -80 °C. Protease Protection Assay—Protoplast pellets (from 500,000 cells) were homogenized in 12% sucrose buffer as described above, this time omitting protease inhibitors, and cell debris was removed by centrifugation at 500 × g for 5 min at 4 °C. Supernatants were divided into three aliquots and incubated for 30 min at 25 °C with either buffer (negative control) or proteinase K (5 mg/ml stock in 50 mm Tris-HCl, pH 8.0, 1 mm CaCl2) at a final concentration of 75 μg/ml in the presence or absence of 1% Triton X-100. Phenylmethylsulfonyl fluoride was added to a final concentration of 20 mm to inhibit proteinase K before immunoprecipitation. Samples were frozen on dry ice and stored at -80 °C. Preparation of Protein Extracts and Immunoprecipitation— Frozen samples were homogenized by adding 2 volumes of cold protoplast homogenization buffer (150 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1.5 mm EDTA, 1.5% (w/v) Triton X-100, supplemented immediately before use with Complete™ protease inhibitor mixture). Homogenates were used for immunoprecipitation with polyclonal rabbit anti-RTA, anti-BiP (22Pedrazzini E. Giovinazzo G. Bielli A. de Virgilio M. Frigerio L. Pesca M. Faoro F. Bollini R. Ceriotti A. Vitale A. Plant Cell. 1997; 9: 1869-1880Crossref PubMed Scopus (147) Google Scholar), anti-phaseolin (22Pedrazzini E. Giovinazzo G. Bielli A. de Virgilio M. Frigerio L. Pesca M. Faoro F. Bollini R. Ceriotti A. Vitale A. Plant Cell. 1997; 9: 1869-1880Crossref PubMed Scopus (147) Google Scholar), anti-calreticulin (29Denecke J. Carlsson L.E. Vidal S. Hoglund A.S. Ek B. van Zeijl M.J. Sinjorgo K.M. Palva E.T. Plant Cell. 1995; 7: 391-406Crossref PubMed Scopus (184) Google Scholar), or anti-GRP94 (30Klein E.M. Mascheroni L. Pompa A. Ragni L. Weimar T. Lilley K.S. Dupree P. Vitale A. Plant J. 2006; 48: 657-673Crossref PubMed Scopus (44) Google Scholar) antisera. Immunoselected polypeptides were analyzed by 15% SDS-PAGE. Gels were fixed, treated with Amplify™ (GE Healthcare), and radioactive polypeptides revealed by fluorography. Band intensity was determined using TotalLab 2003 software (Non-linear Dynamics, Newcastle-upon-Tyne, UK). Toxicity Measurements—Triplicate aliquots of 330,000 protoplasts were co-transfected with a toxin-encoding plasmid or empty vector (pDHA), a CDC48-encoding plasmid or empty vector (pamPAT-MCS), and the phaseolin-encoding plasmid (pDHE-T343F). After 16 h of recovery, protoplasts were pulse-labeled for 1 h before being pelleted as described above. Polypeptides immunoselected from homogenates using anti-phaseolin antiserum were separated by SDS-PAGE, before fluorography and densitometry as before. Toxicity of the various constructs was expressed as a percentage of phaseolin synthesis with respect to protoplasts co-transfected with empty vector instead of toxin. Endoglycosidase H Treatment—Protein A-Sepharose beads carrying immunoprecipitated protein were resuspended in 20 μl of sodium citrate buffer (0.25 m sodium citrate, pH 5.5, 0.2% (w/v) SDS) and boiled for 5 min. Supernatants were treated with 10 milliunits of endoglycosidase H (Roche Applied Science; 5 milliunits/μl of stock) at 37 °C for 16 h. Confocal Microscopy—Transfected protoplasts were mounted in K3 and imaged with a Leica TCS SP5 confocal laser-scanning microscope, using a ×40 oil immersion objective lens (NA 1.25). YFP was excited at 514 nm and detected in the 525 to 583 nm range. RFP was excited at 561 nm and detected in the 592 to 635 nm range. Simultaneous detection of YFP or RFP was performed by combining the settings indicated above in the sequential scanning facility of the microscope, as instructed by the manufacturer. Statistical Analysis—Each experiment was repeated 3 or 4 times (see individual figures) and data groups were analyzed using one-way analysis of variance. When a significant effect between sample groups was detected, the groups were compared using Tukey post hoc tests. Statistical analysis was performed using SPSS version 14.0. Dominant Negative CDC48QQ Induces an Up-regulation of ER Chaperones—We rationalized that the presence of a dominant negative CDC48 would lead to the accumulation of aberrant proteins within the ER lumen as a prelude to an up-regulation of ER chaperones as part of the unfolded protein response (UPR). Such a phenotype would confirm the efficacy of the expressed CDC48 mutant. We therefore prepared protoplasts from sections of leaf tissue that had been subjected to Agrobacterium-mediated infiltration. The transformed protoplasts expressed empty vector, wild-type CDC48, or a mutant CDC48 in which the conserved glutamate residues of the Walker B motifs (Glu308 and Glu581) of the two ATPase domains had been replaced by glutamine (denoted CDC48QQ) (16Muller J. Piffanelli P. Devoto A. Miklis M. Elliott C. Ortmann B. Schulze-Lefert P. Panstruga R. Plant Cell. 2005; 17: 149-163Crossref PubMed Scopus (90) Google Scholar). After 3 days, the levels of BiP, calreticulin, and GRP94 were increased in cells that were expressing CDC48QQ (Fig. 1A). The quantitation of these data is shown in Fig. 1B, where it can be seen that a statistically significant difference is observed in chaperone levels when CDC48QQ is expressed. It should be noted that the up-regulation observed is likely to be an underestimate because of the agrobacterial infiltration method used in these experiments, although more efficient that other methods of plant cell transformation, is unlikely to be 100% efficient. ER chaperones will therefore not be induced in a proportion of the cells taken for analysis. Expression of Dominant Negative CDC48 Increases the Stability of ER-sequestered RTA—We then followed the fate of ER-sequestered RTA in tobacco protoplasts 16 h after PEG-mediated co-transfection of plasmids encoding toxin together with either wild-type CDC48 or mutant CDC48, or in the presence of the glycosylation inhibitor (and UPR inducer) tunicamycin (31Martinez I.M. Chrispeels M.J. Plant Cell. 2003; 15: 561-576Crossref PubMed Scopus (335) Google Scholar). To minimize inhibition of protein synthesis and thereby maximize the amount of newly made toxin that could be visualized, we used in these (and some of the subsequent) experiments a catalytic point mutant, RTAE177D, which has been shown to have virtually native structure (32Allen S.C.H. Moore K.A.H. Marsden C.J. Fulop V. Moffat K.G. Lord J.M. Ladds G. Roberts L.M. FEBS J. 2007; 274: 5586-5599Crossref PubMed Scopus (10) Google Scholar) but a 70-fold reduced potency to ribosomes (24Chaddock J.A. Roberts L.M. Protein Eng. 1993; 6: 425-431Crossref PubMed Scopus (46) Google Scholar). As shown from the representative pulse-chase experiment in Fig. 2A, the glycosylated RTA made in a 1-h pulse with radiolabeled cysteine and methionine was degraded with a half-life of ∼3.5 h in cells expressing wild-type CDC48. In cells expressing CDC48QQ, the rate of RTA degradation was significantly and reproducibly reduced as shown by quantification and statistical analysis of bands taken from four independent repeats of this experiment (Fig. 2B). Although the expression of CDC48QQ was in itself toxic to cells, such that transfected cells synthesized only 40% as much RTA as cells expressing wild-type CDC48, the RTA that was made was more stable (Fig. 2), with a half-life of greater than 5 h (data not shown). The slightly increased gel mobility of the A chain bands during the chase (Fig. 2A, lanes 6–8 and 10–12) most likely represents the emergence of a mannose-trimmed species. Fig. 2B quantifies the kinetics of degradation averaged from four separate experiments. It is noticeable that, unlike CDC48QQ, tunicamycin treatment to block glycosylation and promote a generalized UPR does not impede the disappearance of RTA (Fig. 2A, lanes 13–16). Expression of Mutant CDC48 Hampers the Retro-translocation of Toxin Subunits—We have previously shown that a proportion of retro-translocated RTA uncouples from the degradation pathway and refolds to inactivate ribosomes (18Di Cola A. Frigerio L. Lord J.M. Roberts L.M. Ceriotti A. Plant Physiol. 2005; 137: 287-296Crossref PubMed Scopus (40) Google Scholar). A reduction in the protein biosynthetic capacity of these cells is therefore indicative of retro-translocation activity. To monitor protein synthesis in the transfected cell population alone we quantified, in triplicate, the levels of a reporter protein (phaseolin) encoded by a plasmid that was co-transfected (in a triple transfection) into protoplasts along with a plasmid encoding a cytosolic RTA, an ER-targeted RTA (RTA), or the nonrecombinant vector pDHA (no toxin) together with one of the CDC48 constructs (or vector alone (pamPAT-MCS)). Following overnight expression and a 1-h pulse with radiolabeled cysteine and methionine, the triplicate phaseolin immunoprecipitates were quantified from gels. As shown in Fig. 3A, protein synthesis in cells making ER-targeted RTA (with and without the wild-type CDC48 construct) is reduced by ∼60%. In contrast, protein synthesis in cells expressing RTA and the dominant negative CDC48QQ was reduced by only 30%, a statistically significant difference (p < 0.05). That such rescue was a general consequence of expressing CDC48QQ was ruled out from controls that show a consistent inhibition of protein synthesis (by more than 80%) when ricin A chain was deliberately expressed in the cytosol without a signal peptide (cytosolic RTA), either in the presence or absence of wild-type or mutant CDC48 (Fig. 3A). The significant rescue of protein synthesis shown in Fig. 3 therefore occurred only when RTA was initially targeted into the ER lumen in the presence of CDC48QQ. This would suggest that CDC48QQ mitigates the toxic effect of RTA by impeding its retro-translocation to the cytosol. Because this experiment relies on efficient co-transfection, we checked the ability of protoplasts to take up multiple plasmids. Fig. 3B is a representative set of images showing that protoplasts competent to take up a YFP expressing plasmid in all cases concomitantly co-express an RFP plasmid. To confirm toxin retro-translocation, we determined the location of the stabilized RTA. Cells expressing ER-targeted RTAE177D alone or together with either wild-type or mutant CDC48 were pulsed with 35S-labeled amino acids before being fractionated into membranes (M) and cytosol (C). As shown in Fig. 4A (lane 4), a significant amount of the RTAE177D synthesized was recovered in the cytosol fraction under these conditions. By contrast, virtually no toxin accumulated in the cytosol of cells expressing CDC48QQ (Fig. 4A, lane 8). BiP immunoprecipitates from the same samples indicate the integrity of the membrane fractions and show that the cytosolic RTA was unlikely to result from membrane leakage during cell lysis and membrane preparation. It is noticeable that the form accumulating in the cytosol was equivalent to the slowest migrating species observed in the membrane fractions. This higher RTA band is reminiscent of the mannose-untrimmed species seen in previous pulse-chase experiments (18Di Cola A. Frigerio L. Lord J.M. Roberts L.M. Ceriotti A. Plant Physiol. 2005; 137: 287-296Crossref PubMed Scopus (40) Google Scholar). Regardless of the precise glycan forms, however, it is clear that co-expression of mutant CDC48QQ impaired the retro-translocation of RTA to the cytosol. To investigate whether RTA was retained inside the membranes we carried out a protease protection experiment using cell extracts containing ER microsomes (25Jolliffe N.A. Di Cola A. Marsden C.J. Lord J.M. Ceriotti A. Frigerio L. Roberts L.M. J. Biol. Chem. 2006; 281: 23377-23385Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Accordingly, at the 3-h chase point, when RTA was largely sensitive to proteinase K under normal conditions (Fig. 4B, lane 5), a proportion of this protein was clearly protected from protease when made in the presence of CDC48QQ (Fig. 4B, lane 11, and quantified from three independent repeats in Fig. 4C). Based on the statistical analysis, there is significantly more protease-protected RTA in the cells expressing CDC48QQ (Fig. 4C). The faster migrating species, visualized upon incubation of the samples with protease, most likely represents a membrane-protected remnant of the fraction of RTA that was in the process of being retro-translocated at the time of cell lysis and that may have been partially exposed to the cytosol. Consistent with this, we observed the complete disappearance of this fragment when the membranes were treated with protease concomitant with membrane solubilization (Fig. 4B, lanes 3, 6, 9, and 12). Toxins Can Be Retro-translocated in Mannose-trimmed and Untrimmed Forms, but Only When CDC48 Is Active—We next used cells expressing either ER-targeted RTAE177D or a comparable inactive version of its relative, R. communis agglutinin (RCA AE176D (33Roberts L.M. Lord J.M. Eur. J. Biochem. 1981; 119: 31-41Crossref PubMed Scopus (59) Google Scholar)), and analyzed the presence or absence of these orphan proteins in the cytosol and membrane fractions over time. It was noticeable that RTA, but not RCA A, underwent a slight shift in size during pulse-chase experiments (Fig. 5, top panel). This slight downsizing of RTA can be blocked using the ER mannosidase inhibitor 1-deoxymannojirimycin (Fig. 5, second panel) to generate a species that is now comparable with the sharper bands observed for RCA A. Clearly, the appearance of RTA in the cytosol does not critically depend on prior mannose-trimming events. Taken together, these observations suggest that the core glycan of RCA A may not normally be a substrate for extensive mannosidase action, and that the retro-translocation machinery does not differentiate between the different glycosylated forms presented by RTA and RCA A. As observed before for RTA (17Di Cola A. Frigerio L. Lord J.M. Ceriotti A. Roberts L.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14726-14731Crossref PubMed Scopus (84) Google Scholar, 18Di Cola A. Frigerio L. Lord J.M. Roberts L.M. Ceriotti A. Plant Physiol. 2005; 137: 287-296Crossref PubMed Scopus (40) Google Scholar), there is a qualitative loss of both toxin chains from membrane fractions with time (Fig.
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