Proc. Natl. Acad. Sci. USA Vol. 94, pp. 3783–3788, April 1997 Cell Biology Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol (ricinytoxinytranslocationyretrograde transportysulfation) ANDRZEJ RAPAK,PÅL Ø. FALNES, AND SJUR OLSNES* Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway Communicated by R. John Collier, Harvard Medical School, Boston, MA, January 30, 1997 (received for review November 25, 1996) ABSTRACT Translocation of ricin A chain to the cytosol (20). Because this labeling takes place in the Golgi apparatus, only has been proposed to take place from the endoplasmic reticulum molecules that have already been transported retrograde to the (ER), but attempts to visualize ricin in this organelle have failed. Golgi complex will be labeled. Furthermore, because only the A Here we modified ricin A chain to contain a tyrosine sulfation chain carrying the sulfation site will be labeled, the B chain, which site alone or in combination with N-glycosylation sites. When migrates at almost the same rate as the modified A chain, will not reconstituted with ricin B chain and incubated with cells in the complicate the interpretation of the data. We here present 35 presence of Na2 SO4, the modified A chains were labeled. The evidence that sulfated ricin A chain is transported retrograde to labeling was prevented by brefeldin A and ilimaquinone, and it the ER and then translocated to the cytosol. appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been MATERIALS AND METHODS 35 transported retrograde to this organelle. A chain containing Materials and Buffers. Na2 SO4 was from Amersham. Rat C-terminal N-glycosylation sites became core glycosylated, indi- monoclonal (9G10) anti-glucose regulated protein 94 (GRP94) cating retrograde transport to the ER. In part of the toxin was from StressGen Biotechnologies (Victoria, Canada). Affin- molecules, the A chain was released from the B chain and ity-purified rabbit anti-BIP, was obtained from Linda Hendershot translocated to the cytosol. The finding that glycosylated A chain (St. Jude’s Children Hospital, Memphis, TN). This antibody was was present in the cytosol indicates that translocation takes place found to cross-react with cytosolic heat shock protein 70 (HSP70). after transport of the toxin to the ER. Mouse monoclonal (1D3) anti-protein disulfate isomerase (PDI) was obtained from Steven Fuller (European Molecular Biology Ricin is a plant toxin that is extensively used in the construction Laboratory, Heidelberg, Germany). Anti-p58 was obtained from of immunotoxins for targeted therapy of cancer and other dis- Jaakko Saraste (University of Bergen, Bergen, Norway). Anti- eases (1–3). The toxin consists of an enzymatically active A chain rab5 was obtained from Harald Stenmark (Institute for Cancer and a B chain with lectin properties (4). The A chain enters the Research, Oslo). Hepes medium contained bicarbonate-free cytosol and inactivates ribosomes by depurination of a single Eagle’s minimum essential medium buffered with 20 mM Hepes adenosine residue in 28S ribosomal RNA (5, 6). The B chain (N-2-hydroxethylpiperazine-N9-2-ethanesulfonic acid) to pH 7.4. binds to galactose-containing surface receptors (7). The surface- PBS contained 140 mM NaCl and 10 mM Na2HPO4 (pH 7.2). bound toxin is slowly endocytosed (8). Part of the internalized Lysis buffer consisted of PBS (pH 7.2) containing 1 mM EDTA, ricin is transported back to the cell surface, part is delivered to 1% Triton X-100, 200 unitsyml aprotinin, and 1 mM phenyl- lysosomes and degraded, and part is transported to the trans- methylsulfonyl fluoride (PMSF). Rabbit anti-ricin was obtained Golgi network (9). Translocation of ricin A chain to the cytosol by standard immunization. Endoglycosidase H (endo H) and has been proposed to take place from the endoplasmic reticulum endoglycosidase F (PNGase F) were from New England Biolabs. (ER; refs. 10–12). In fact, Shigella toxin, which has the same Cell Culture. Cells were maintained and propagated under intracellular target as ricin (13, 14), has been visualized in the ER standard conditions (5% CO2 in Eagle’s minimal essential (15), and the related cholera and Escherichia coli heat labile medium containing 5% fetal calf serum). Two days before the toxins have C-terminal (KyH)DEL sequences that could act as experiment, the cells were seeded into 12- or 24-well Costar 5 4 ER retrieval signals (16). Pseudomonas aeruginosa exotoxin A has plates at a density of 10 and 5 3 10 cells per well, respectively. a C terminus that, after removal of the C-terminal lysine residue, Plasmids. To form ricin A-sulf-1, a C-terminal extension was ends in RDEL (16). Ricin does not contain such a sequence. introduced by PCR using wild-type ricin A chain cDNA (21) However, addition of a KDEL sequence to the C terminus of ricin as template and 59-GGATCCTATCAAGATGGGTATTCG- A chain was found to increase the toxic effect of whole ricin and, TAGTCTTCTGCGCTGCTTGGTGGAGGTGCGCA- in particular, that of the free A chain (10, 12, 18). TCTATACACC-39 as the reverse primer. To form ricin A-sulf-2, To test if ricin is transported retrograde from the trans-Golgi ricin A-sulf-1 was used as template, and 59-CGTCGGATC- network to the ER, we decided to use transfer of N-linked CCGGGCTATCACTGGGATGTGTTATTTTTG- oligosaccharides onto free glycosylation sites in ricin A chain as GTGCCGTTAGATGGGTATTCGTAGTCTTCTGCGC-39 a test for the presence of toxin in the ER. It has been found that was used as the reverse primer. In both cases 59-GCAAT- only '5% of endocytosed ricin is located in the trans-Golgi CACTCATCTTTTCACTGATGTTC-39 was used as the for- network (19). It follows that if labeled toxin is added to the cells, ward primer. The product was cut with BglII and BamHI and there will be a serious background problem when one looks for cloned into pRA (21) that had been cut with the same enzymes toxin in the Golgi apparatus and in the ER. To reduce this and dephosphorylated. From the plasmids obtained, cDNA for background, we used tyrosine sulfation as a labeling procedure the modified A chains were cut out with NcoI and EcoRI and cloned into pMAL-cN which had been cut partially with NcoI The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: ER, endoplasmic reticulum; SLO, streptolysin O; PDI, accordance with 18 U.S.C. §1734 solely to indicate this fact. protein disulfate isomerase; MBP, maltose-binding protein; endo H, endoglycosidase H; PNGase F, endoglycosidase F; HSP70, heat shock Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA protein 70; GRP94, glucose regulated protein 94. 0027-8424y97y943783-6$2.00y0 *To whom reprint requests should be addressed. e-mail: olsnes@ PNAS is available online at http:yywww.pnas.org. radium.uio.no. 3783 Downloaded by guest on September 25, 2021 3784 Cell Biology: Rapak et al. Proc. Natl. Acad. Sci. USA 94 (1997) and to completion with EcoRI. pMAL-cN was obtained by with 0.5% Triton X-100. The lysate and cytosol were exposed cutting pMAL-c (New England Biolabs) with StuI and ligating to immobilized anti-ricin antibodies, and the adsorbed mate- in an NcoI linker: 59-GCCATGGC-39. rial was subjected to nonreducing SDSyPAGE. Measurement of Cytotoxicity of Endocytosed Toxin. Vero SDSyPAGE. SDSyPAGE was carried out as described (22) cells were incubated for4hinHepes medium without leucine in the absence or presence of 2-mercaptoethanol, as indicated. with increasing amounts of toxin. The cells were then trans- The gels were fixed in 4% acetic acidy27% methanol for 30 min 3 35 ferred to Hepes medium containing 1 mCiyml [ H]leucine (1 and then, in the case of proteins labeled with Na2 SO4, treated Ci 5 37 GBq) and no unlabeled leucine and were incubated for with 1 M sodium salicylate (pH 5.8) in 2% glycerol for 30 min. 20 min at 378C. The cells were then extracted with 5% Dried gels were exposed to Kodak XAR-5 films in the absence trichloroacetic acid for 10 min, followed by a brief wash in 5% of intensifying screens at 2808C for fluorography. trichloroacetic acid, and subsequently dissolved in 0.1 M KOH, Immunoblots. The samples were subjected to reducing SDSy and the cell-associated radioactivity was measured. PAGE in a 12% gel, and proteins were transferred to a poly(vi- Glycosidase Treatment. Vero cells were incubated with ricin nylidene difluoride) membrane (Immobilon-P, Millipore). After containing A-sulf-2, lysed, and immunoprecipitated with im- blocking with 5% milk in PBS for 1 h, the membranes were mobilized anti-ricin. The beads were heated at 1008C for 10 incubated for1hwithrabbit anti-Rab 5 antisera (1:1000), rabbit min in denaturing buffer (0.5% SDSy1% mercaptoethanol). anti-BIP antibody (1 mgyml), rabbit anti-p58 antisera (1:500), rat Digestion was carried out with Endo H or PNGase F for 2 h monoclonal anti-GRP94 (1:1000), or mouse monoclonal anti- at 378C. The reaction was terminated by the addition of PDI antibody (1:10). Then the blots were incubated with a second concentrated SDS sample buffer and boiling for 5 min. antibody labeled with alkaline phosphatase (Promega), and pro- Purification of Recombinant Proteins and Reconstitution with teins were visualized using 5-bromo-4-chloro-3-indolyl phos- Ricin B Chain. After induction with isopropyl b-D-thiogalacto- phateynitro blue tetrazolium solution according to the manufac- side, bacteria were harvested, washed with PBS, resuspended in turer’s instruction. column buffer (20 mM TriszCl, pH 7.5y200 mM NaCly0.1 mM PMSF) and sonicated.