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. 2014 Nov 14;34(1):55–66. doi: 10.15252/embj.201488582

Figure 4. Processing of GRI-peptide by METACASPASE-9 is required for induction of elevated ion leakage.

Figure 4

  1. Recombinant AtMC9 (rAtMC9) cleaved GRI25–168 in vitro. Bacterially produced MBP-GRI25–168 (from 0 to 2 pmol left to right) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut (rAtMC9C147AC29A), and cleavage products were analyzed by Western blot with anti-MBP and anti-AtMC9 antibodies. Arrowheads in the anti-MBP blot (from top to bottom) indicate MBP-GRI25–168 (63.5 kDa), MBPMCS (53.5 kDa), rAtMC9-cleaved MBP-GRI25–168 (46 kDa). Arrowheads in the anti-AtMC9 blot indicate: 1: N-terminal domain + p20 + p10 subunits of AtMC9, 2: p20 + p10 subunits of AtMC9, 3: N-terminal domain + p20 subunit of AtMC9, 4: p20 subunit of AtMC9.
  2. Infiltration of wild-type, prk5-1 and atmc9-1 leaves with 37 nM GRIp31–96, GRIp65–84, GRIp68–78 or GST. GRIp65–84 and GRIp68–78 but not GRIp31–96 were able to induce elevated ion leakage in the atmc9-1 mutant.
  3. Schematic representation of the GRI and the cleavage sites for rAtMC9. The cleavage product detected in Western blot analysis with anti-MBP antibody is shown as black bar. Mass spectrometric analysis of MBP-GRI25–168 cleavage with rAtMC9 provided evidence for cleavage after SKTR and KANK; further analysis of GRIp31–96 cleavage by rAtMC9 provided evidence for cleavage after SK, SKTR and after KKIKK. The position of the resulting 11-aa-long peptide (68LLVSHYKKIKK78) is indicated by a white inset.
  4. Infiltration of Col-0 leaves with 37 nM of GRIp65–84, GRIp68–78, Y-GRIp68–78, GRIp31–96 or GST. Y-GRIp68–78 showed similar activity in cell death induction compared to the other GRI-derived peptides.
  5. 125I-labeled Y-GRIp68–78 (0.46 nM) bound specifically to Col-0 membrane fractions (light gray bars), the binding was significantly reduced in prk5-1 and prk5-2 plants. Excess of non-radioactive Y-GRIp65–84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of four samples, triangles show individual data points).
  6. Excess of GRIp68–78 and GRIp65–84 (10 μM) but not of GRIp31–96 or other peptides (GRIp31–51, GRIp47–68, GRIp80–96) competed the binding of 0.46 nM 125I-Y-GRIp68–78 to membrane extracts from Col-0 (all bars show average of four samples, triangles show individual data points).
  7. Saturation binding curve for 125I-Y-GRIp68–78 to Col-0 membrane extracts. Specific binding was calculated by subtracting non-specific binding from the total binding. The affinity of 125I-Y-GRIp68–78 to the receptor (Kd = 1.9 nM) was calculated by non-linear regression analysis. Scatchard plot is shown in Supplementary Fig S17.

Data information: Data in (B) and (D) are shown as average ± SD of four replicates consisting of four leaf disks each. Asterisks in (B) and (D) mark statistically significant differences from infiltration with GST according to Sidak’s test (P < 0.05). Asterisks in (E) and (F) mark statistically significant differences from peptide binding to Col-0 membrane fractions without competitor according to Sidak’s test (P < 0.01). All experiments were repeated three times with similar results. Source data are available online for this figure.