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. 2021 Oct 17;18(6):1350–1366. doi: 10.1080/15548627.2021.1976965

Figure 5.

Figure 5.

Distinct interaction surfaces in ATG8 for binding with SH3P2-SH3 and NBR1-UBA. (A) 10 μM wild-type or variants of GST-ATG8f was mixed with 10 μM SH3P2-SH3 (left panel) or NBR1-UBA (right panel) respectively, and incubated with glutathione resins in 1X PBS, 5 mM DTT. The bound proteins were eluted with glutathione and analyzed on SDS-PGAE with Coomassie Brilliant Blue staining. (B) YFP-ATG8f or its variants was co-expressed with HA-tagged NBR1 or HA-tagged SH3P2 in Arabidopsis protoplasts respectively. Lysates were immunoprecipitated by GFP-Trap method and detected by anti-HA or anti-GFP antibodies. (C) YFP-NBR1 was transiently co-expressed with wild-type or mutants of CNX-mCherry-ATG8f in Arabidopsis protoplasts. YFP-NBR1 was overlapped with CNX-mCherry-ATG8f, but not with the other two variants (Y26E R29E and Y50A L51A), suggesting these mutations are essential for the interaction between NBR1 and ATG8f in Arabidopsis protoplasts. Similar results were obtained from three different independent experiments. The linear Pearson correlation coefficient (rp) and the nonlinear Spearman correlation coefficient (rs) indicate the extent of colocalization with the value of +1.0 for complete colocalization. (D) SH3P2-GFP was transiently co-expressed with wild-type or mutants CNX-mCherry-ATG8f in Arabidopsis protoplast. SH3P2-GFP was recruited by wild-type or the Y26E R29E variant of CNX-mCherry-ATG8f, but not by the Y50A L51A variant. Similar results were obtained from three different independent experiments. The linear Pearson correlation coefficient (rp) and the nonlinear Spearman correlation coefficient (rs) indicate the extent of colocalization with the value of +1.0 for complete colocalization. (E) YFP-ATG8f, its variants (Y26E R29E, Y50A L51A) and GFP were co-expressed together with 5HA-NBR1 and SH3P2-5Flag in Arabidopsis protoplasts for 12 h respectively, and were subjected to GFP trap assay. The resulting immunoprecipitation (IP) and cell lysate were analyzed by immunoblotting (IB) using anti-Flag, anti-HA or anti-GFP antibodies. (F) SH3P2-GFP, CFP-NBR1 were transiently co-expressed together with wild-type or mutants CNX-mCherry-ATG8f in Arabidopsis protoplast. SH3P2-GFP was recruited by wild-type and the Y26E R29E variant of CNX-mCherry-ATG8f, but not by the Y50A L51A variant. In contrast, both Y26E R29E and Y50A L51A variants compromised the recruitment of CFP-NBR1.