Homodimerization of pokeweed antiviral protein as a mechanism to limit depurination of pokeweed ribosomes

Abstract

Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from depurination during the synthesis of these enzymes. For example, pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.

Figure 1

Expression of monomeric and complexed pokeweed antiviral protein (PAP) in pokeweed leaves. (A) Proteins (4 µg) of total cell lysate, apoplastic fluid or protoplasts were separated by 12% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to nitrocellulose and probed with a polyclonal antibody specific to PAP (1:5000). (B) Total protoplast protein (8 µg) or proteins from the microsomal membrane (MM) fraction of protoplasts (8 µg) were also probed for the presence of PAP as described in (A). Samples that were boiled prior to separation are labelled B, and NB indicates samples that were not boiled. Molecular weights of monomeric (29 kDa) and complexed (58 kDa) PAP are indicated.

Figure 2

Separation of monomer from complexed pokeweed antiviral protein (PAP) by ion exchange chromatography. PAP was purified from the total cell lysate of pokeweed leaves by ion exchange. Positively charged proteins were eluted from the anionic column using an NaCl step gradient. Aliquots (10 µL) from each fraction were separated by 12% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) without prior boiling, transferred to nitrocellulose and probed with a polyclonal antibody specific to PAP (1:5000). Purified monomeric PAP was loaded (20 ng) as a standard. Molecular weights of monomeric (29 kDa) and complexed (58 kDa) PAP are indicated.

Figure 3

Mass spectrometric analysis of complexed pokeweed antiviral protein (PAP). (A) Partial peptide spectra of monomeric PAP (i, ii) and complexed PAP (iii, iv) following trypsin digestion. Peak 837.4 corresponds to peptide YPTLESK observed from the digestion of monomeric PAP, but not observed in the spectra of complexed PAP. (B) Molecular model of PAP; β‐strands and α‐helices are indicated, together with the helix containing the heptad YPTLESK (coloured blue). Amino acids essential for enzyme activity are coloured pink.

Figure 4

Bimolecular fluorescence complementation of pokeweed antiviral protein (PAP) dimerization in pokeweed protoplasts. Protoplasts were transfected with plasmids encoding the N‐ or C‐terminus of green fluorescent protein (GFP) fused to PAP or PAP_Y123A. As a negative control for bimolecular interaction, cells were transfected with plasmids encoding only the N‐ or C‐terminus of GFP. As a positive control for fluorescence, protoplasts were transfected with a plasmid encoding intact GFP. Transfected protoplasts were examined by fluorescence or bright field laser scanning microscopy. Percentage values indicate the mean numbers of fluorescent protoplasts relative to the total number of cells ± standard error from five fields of view. The scale bar indicates 50 µm. (B) Expression of individual constructs, PAP–GFP_N, PAP–GFP_C, PAP_Y123A–GFP_N and PAP_Y123A–GFP_C, following transfection into pokeweed protoplasts. Lysates of protoplasts (10 µg) were separated by 12% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to nitrocellulose and probed with a polyclonal antibody specific to GFP (1:5000). The molecular weights of fusion proteins containing the N‐terminus of GFP (47 kDa) and the C‐terminus of GFP (39 kDa) are indicated. (C) In vitro chemical cross‐linking. Recombinant wild‐type PAP and PAP_Y123A (125 ng) were incubated with or without (+, −) 0.05% glutaraldehyde and separated by 12% SDS‐PAGE with prior boiling. Proteins were transferred to nitrocellulose and probed with a polyclonal antibody specific to PAP (1:5000). Molecular weights of monomeric (29 kDa) and complexed (58 kDa) PAP are indicated.

Figure 5

Depurination of rRNA by monomeric and complexed pokeweed antiviral protein (PAP). Isolated ribosomes of pokeweed and lysate of rabbit reticulocytes were incubated with monomeric or complexed PAP (5 nm) or buffer alone (–PAP). rRNA was isolated and primer extension analysis was performed using two end‐labelled primers, one annealing downstream of the expected depurination site (Dep’n) and the other annealing downstream of the 5′ end of 28S rRNA. cDNA products were separated on a 7 m urea/6% acrylamide gel, and extension patterns were visualized with a phosphorimager. –rRNA indicates extension reactions without rRNA template. The percentage depurination was estimated by the densitometry of band intensities of the Dep’n cDNA product relative to the 28S ribosomal cDNA product. Values represent means ± standard error for three independent experiments.

Figure 6

Translation inhibition by monomeric and complexed pokeweed antiviral protein (PAP). Poly(U) RNA was translated in rabbit reticulocyte lysate in the presence of increasing molar amounts of monomeric or complexed PAP and [³H]‐phenylalanine. –RNA indicates a translation reaction without mRNA and –PAP indicates a reaction without PAP. The protein products were precipitated in trichloroacetic acid (TCA) and quantified by liquid scintillation counting. Percentage incorporation refers to the amount of [³H]‐phenylalanine incorporated into protein relative to the total amount in the translation mix. Values represent means ± standard error for three independent experiments.