Journal: Nature plants

Article Title: Control of retrograde signalling by protein import and cytosolic folding stress.

doi: 10.1038/s41477-019-0415-y

Figure Lengend Snippet: Fig. 1 | GUN1 physically interacts with the ClpC1 and cpHSC70-1 chaperones. a, Co-IPs performed with rosette leaves from GUN1-GFP transgenic lines in the gun1-101 (C-1) or wild-type (OE-13) background. An RBCS transit peptide-fused GFP in the wild-type background (C-cpGFP) line was used as a co-IP control. Tic40, which cannot be detected by mass spectrometry in co-IPs, was included as a negative control. Of the total input, 10% was loaded in a separated lane and analysed as input control. Four independent immunoprecipitation experiments were performed with anti-GFP antibodies, and the immunoprecipitates were subjected to western blotting with anti-GFP antibodies (detecting cpGFP and GUN1-GFP) or antibodies against proteins associated with the Tic (anti-ClpC, anti-Tic110 and anti-Tic40) and Toc (anti-Toc159) complexes. b, Co-IP performed with de-etiolating seedlings grown without (control) or with 0.5 mM Lin. Of the total input, 10% was loaded in a separated lane as input control. Two independent co-IP experiments were performed and showed similar results. The anti-ClpC and anti-cpHSC70 antibodies (in a and b) do not distinguish between different isoforms of ClpC (ClpC1 and ClpC2) and cpHSC70 (cpHSC70-1 and cpHSC70-2), respectively. c, BiFC assays verifying the specific interaction of GUN1 with ClpC1 and cpHSC70-1. GUN1-YN co-expressed with ClpD-YC served as the negative control. ClpC1-YN co-expressed with Tic110-YC served as the positive control. For additional controls expressing the individual proteins as GFP fusions, see Supplementary Fig. 6. Three independent experiments were performed and showed similar results. Scale bars, 10 µm. d, The cphsc70-1 mutant shows a GUN phenotype. Expression of LHCB1.2 was determined by qRT–PCR and the relative gene expression is compared to that in the wild type (WT) grown under identical conditions. Data are presented as means ± s.d. (n = 3 biologically independent samples, indicated as open circles). In contrast to cphsc70-1, the clpc1 mutant shows a GUN phenotype only upon Lin (0.5 mM) treatment, but not upon NF (5 µM) treatment. See Supplementary Fig. 9a for expression data for additional PhANG genes and non-PhANG control genes.

Article Snippet: All other antibodies were obtained from commercial suppliers: GFP: Clonetech (632381, clone name: JL-8, dilution: 1:5000).

Techniques: Transgenic Assay, Co-Immunoprecipitation Assay, Control, Mass Spectrometry, Negative Control, Immunoprecipitation, Western Blot, Positive Control, Expressing, Mutagenesis, Quantitative RT-PCR, Gene Expression