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. 2019 Nov 7;182(4):1723–1742. doi: 10.1104/pp.19.00898

Figure 4.

Figure 4.

Analysis of RFA1 and RFA4 ubiquitination and the role of the Cys-361 residue of RFA4 in the ubiquitination and activity of the E3 ligase. A, Both RFA1 and RFA4 are themselves subjected to degradation via the 26S proteasome. Arabidopsis transgenic lines expressing HA-tagged RFA1 or RFA4 accumulate higher levels of these proteins after 50 μm MG132 treatment for 24 h. Values are averages ± sd obtained from three independent experiments (n = 5 plants in each experiment). *, P < 0.05 (Student’s t test) with respect to the corresponding mock-treated sample. B, Both RFA1 and RFA4 are ubiquitinated in vivo. The HA-RFA1 and HA-RFA4 proteins were immunoprecipitated from MG132-treated samples using anti-HA antibodies and subsequently analyzed by immunoblotting using anti-HA or anti-Ub (P4D1) antibody. C, The Cys-361Ala mutation markedly reduces the ubiquitination of RFA4 and stabilizes the protein compared with the wild type. Arabidopsis transgenic lines expressing HA-tagged RFA4ΔC or RFA4Cys-361AlaΔC accumulate higher levels of these proteins after 50 μm MG132 treatment for 24 h. Protein extracts were immunoprecipitated using anti-HA antibodies and subsequently analyzed by immunoblotting using anti-HA or anti-Ub (P4D1) antibodies. Values are averages ± sd obtained from three independent experiments (n = 5 plants in each experiment), and RFA4ΔC protein levels in mock-treated plants were taken as 100%. *, P < 0.05 (Student’s t test) compared with the corresponding mock-treated sample. D, Transient expression in N. benthamiana of HA-tagged RFA4ΔC or RFA4Cys-361AlaΔC reveals reduced ubiquitination of RFA4Cys-361AlaDC compared with the wild type. *, P < 0.05 (Student’s t test) compared with the wild type. E, In vivo ubiquitination of PYL4 by RFA4ΔC in N. benthamiana leaf cells. Agrobacteria encoding GFP-PYL4 were coinfiltrated in N. benthamiana cells with agrobacteria encoding either HA-RFA4ΔC or RFA4Cys-361AlaΔC. Protein extracts were immunoprecipitated using anti-GFP and subsequently immunoblotted with anti-GFP, anti-PYL4, anti-Ub, and anti-HA antibodies. *, P < 0.05 (Student’s t test) compared with the wild type. For D and E, values are averages ± sd obtained from three independent experiments (n = 5 leaves in each experiment).