Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5053-5056, July 1986 Biochemistry Extracellular localization of pokeweed antiviral protein MICHAEL P. READY*, DENNIS T. BROWNt, AND JON D. ROBERTUS** *Clayton Foundation Biochemical Institute, Department of Chemistry, and tCell Research Institute and Department of Microbiology, University of Texas, Austin, TX 78712 Communicated by Esmond E. Snell, March 24, 1986 ABSTRACT Pokeweed antiviral protein is an enzyme of molecule may well be inactive until it is processed and Mr 29,000 known to inactivate a wide variety of eukaryotic packaged in the seed. ribosomes. We have used electron microscopy to show that the However, the case is not so clear for proteins such as the antibody specific for the protein is bound within the cell wall pokeweed enzyme, which are not cytotoxic to animals. In matrix of leaf mesophyll cells from Phytolacca americana. Any addition, reports have suggested that pokeweed antiviral penetration or breakage of the cell wall and membrane could protein does not inhibit protein synthesis on pokeweed allow the enzyme to enter the cytoplasm, where it is likely to ribosomes (19, 20). If this were true, it would mean that inhibit protein synthesis in the damaged cell. We speculate that pokeweed could not shut down its own ribosomes if they pokeweed antiviral protein is a defensive agent whose principal were usurped by an invading virus. Recently we speculated 'function is probably antiviral. (5) that this state of affairs is unlikely; a protein that makes up as much as 0.5% of the plant's soluble protein and that Many higher plants contain proteins that enzymatically attacks ribosomes with a Kcat of 400 mol/mol per min must inhibit protein synthesis on eukaryotic ribosomes. These exist to inhibit protein synthesis and probably inhibits its own proteins fall into two main classes. One class contains protein synthesis under some conditions. In this communi- heterodimers of the form AB, where the B chain recognizes cation we show that pokeweed antiviral protein is heavily cell-surface receptors and mediates protein uptake and the A sequestered in the cell wall of pokeweed cells. The isolation chain attacks and inactivates large ribosomal subunits. Ex- of the enzyme outside the cytoplasm is consistent with the amples of these true cytotoxins are ricin (1), abrin (2), and antiviral hypothesis for its action. A breach in the cell, as modeccin (3). required for many virus infections, could allow entrance of The second major class of ribosome-inhibiting proteins the protein into the cytoplasm. Ifpokeweed antiviral protein contains enzyme monomers ofMr -30,000. Their mechanism is capable of inhibiting pokeweed ribosomes, this inhibition ofaction appears to be very similar to that ofthe heterodimer would block viral replication. A chains; lacking a B-chain carrier, however, this second group of proteins is only slightly cytotoxic. Examples of this MATERIALS AND METHODS class are pokeweed antiviral protein (4), dodecandrin (5), and gelonin (6). Pokeweed Antiviral Protein-Sepharose 4B Afflnity Column. Recently we have shown that single-chain proteins such as Five grams of CNBr-activated Sepharose 4B (Sigma) was pokeweed antiviral protein are evolutionarily related to the A swollen on a sintered glass filter with five 200-ml aliquots of chains ofricin and modeccin (7). This comparison was based 1 mM HCL. The gel was then rinsed with 85 ml of coupling largely on amino-terminal sequence homologies but was also buffer (10 mM NaHCO3, pH 8.3/500 mM NaCl) and trans- consistent with kinetic comparisons (8). The relative degree ferred to a flask containing 100 mg of pokeweed antiviral of sequence similarity was also consistent with the presence, protein in coupling buffer. The flask was incubated overnight degree, or absence of immunological crossreactivity (5, at 40C with shaking. The gel was recovered on a sintered glass 9-11). We have also shown that the toxin B chain has had a filter and washed with 40 ml of coupling buffer and then separate evolutionary history (12) and was subsequently transferred to a flask containing 100 ml of 0.2 M glycine (pH joined to an inhibitor gene to produce the class of 8.0) and shaken overnight at 40C to block unoccupied heterodimeric cytotoxins. coupling sites. The physiological role of these proteins is still somewhat The gel was again recovered on sintered glass and washed controversial, although the heterodimeric proteins are prob- four times with alternating 75-ml aliquots of coupling buffer ably defensive. Several laboratories have shown that ricin, and 100 mM NaOAc, pH 4.0/500 mM NaCl. The gel was then although a powerful inhibitor of animal ribosomes, is a resuspended in 10 mM Hepes/NaOH, pH 7.7/150 mM NaCl, relatively weak inhibitor of plant ribosomes (13, 14). Harley packed in a Pharmacia K9/30 column, and washed with and Beevers (14) demonstrated that ricin is roughly 20,000 several changes of the same buffer. times more active against rat liver ribosomes than against Antibody Preparation. Whole IgG was prepared from the ribosomes from five plants, including castor bean (Ricinus serum of pokeweed antiviral protein-inoculated rabbits by communis) from which the toxin is derived. This is consistent the method of ref. 8. Two hundred fifty milligrams of with the notion that ricin is a defensive agent against animal antibody, dialyzed into 10 mM Hepes/NaOH, pH 7.7/150 seed predators. Ricin is synthesized as a single polypeptide, mM NaCl, was applied to the Sepharose 4B column equili- cleaved by posttranslational processing (15), and then pack- brated in the same buffer. The column was washed with aged in the endosperm (16, 17). Processing may play a role in buffer until the A2,0 of the effluent returned to the baseline; preventing self-inhibition. Indeed, although ricin is far less the column was then eluted with 10 mM sodium formate (pH active toward plant than animal ribosomes, physiologically 2.5). Three-milliliter fractions were collected in tubes con- reasonable levels of purified soluble enzyme do severely taining 1 ml of 0.5 M Hepes/KOH at pH 8.0. Twenty-nine inhibit growth of castor seedlings (18). Thus, the precursor milligrams of protein was recovered. IgG was precipitated from the eluate by addition of The publication costs ofthis article were defrayed in part by page charge ammonium sulfate to 37.5% saturation and pelleted by payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5053 Downloaded by guest on October 1, 2021 5054 Biochemistry: Ready et al. Proc. Natl. Acad. Sci. USA 83 (1986) V 4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~O tz;~~~~~- ,- a ~~~~*;^ ^; o¢ ; g¢02;l§;V.¢. ., t%!A W-t, . t8isi . *' zS 6nsAnXX 8 ~ -w~wf ,*"V|^,s - e- 4. PI #. FIG. 1. Localization of pokeweed antiviral protein in thin sections of pokeweed mesophyll cells. Sections of cells were stained with rabbit anti-pokeweed antiviral protein, followed by ferritin-conjugated goat anti-rabbit IgG. V, vacuole; C, cytoplasm; Ch, chloroplast; W, cell wall; I, calcium oxalate inclusion. Note heavy ferritin binding in the wall matrix. The low contrast seen in these sections (here and in Fig. 2) is a result of the fact that these tissues were not treated with osmium tetroxide prior to embedding and are not poststained with lead or uranium salts. centrifugation. The pellet was resuspended in and exhaus- merized by UV exposure at 40C. The resulting blocks were tively dialyzed against phosphate-buffered saline/1 mM trimmed so that thin cross sections of the leaves could be cut MgOAc2 for a final concentration of 9.4 mg/ml. from areas close to the center ofthe disc. Unstained sections Immunoelectrophoresis (21) of the purified antibody were first floated on a solution of 1% bovine serum albumin against pokeweed extract exhibited a single precipitin band. in distilled water (to reduce nonspecific binding) and all Purity was further tested by immunoblot analysis, modified subsequent treatments were done with reagents containing from the method of Barinaga et al. (22). Varying levels of 1% bovine serum albumin. Sections were next floated for 30 pokeweed extract (10-200 pug of protein) were subjected to min on a 1:100 dilution of anti-pokeweed antiviral protein NaDodSO4/PAGE and then transferred nonelectrophoreti- prepared as described above, washed with 1% bovine serum cally to nitrocellulose by using 25 mM Tris/192 mM albumin, and floated on a 1:200 dilution offerritin-conjugated glycine/20% methanol, pH 8.3, as the transfer buffer. The goat anti-rabbit IgG (Cappel Laboratories, Cochranville, PA) blot was blocked with bovine serum albumin as in ref. 22 and for 30 were then extensively washed with 1% then incubated with affinity-purified rabbit anti-pokeweed min. They antiviral protein followed by incubation with fluorescein bovine serum albumin and finally with distilled water prior to isothiocyanate-labeled goat anti-rabbit IgG. Examination of electron microscopy. Control sections were processed in an the blot under long-wave UV light showed a single band in identical manner, except that they were not exposed to each lane ofpokeweed extract in the same position as control anti-pokeweed antiviral protein. All specimens were photo- lanes of purified enzyme. graphed without heavy metal staining in a JOEL 100 CX Electron Microscopy. Four-milliliter discs were removed electron microscope. from the center of the youngest leaves of a pokeweed plant by using a cork borer and were deposited directly into cold RESULTS AND DISCUSSION phosphate-buffered saline, pH 7.2/1.5% gluteraldehyde and submerged in the fixative for 2 hr. The tissues were washed Fig. 1 is an electron micrograph of thin-sectioned pokeweed in distilled water, transferred through a graded alcohol series mesophyll cells that have been treated with rabbit anti- consisting of 20%, 40%, 60%, 80%, 90%, and 95% ethanol at pokeweed antiviral protein and ferritin-labeled goat anti- 40C, and finally dehydrated in 100% ethanol.