Fig. 3: SPA1 interacts with eIF2α in the cytosol in a light-dependent manner through its kinase domain. | Nature Communications

Fig. 3: SPA1 interacts with eIF2α in the cytosol in a light-dependent manner through its kinase domain.

From: The phosphorylation of carboxyl-terminal eIF2α by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis

Fig. 3

a Mapping of the interaction domains for SPA1 using the yeast-two-hybrid assays. Left panel shows the full-length and various domains of SPA1. Right, interactions between SPA1 and eIF2α proteins in GAL4 yeast two-hybrid assays. The full-length of eIF2α homologs and different forms of SPA1 were fused with the GAL4 binding domain (BD) and activation domain (AD), respectively. The AD domain without SPA1 conjugation (AD only) is used as negative control. b Co-immunoprecipitation (Co-IP) assay shows that two homologs of eIF2α interact with SPA1 in response to light. Four-day-old etiolated seedlings were incubated either under dark (D) or illuminated under white light for 4 h (L4h) before sampling. The total proteins were extracted and incubated with GFP-Trap. The precipitated proteins were examined by immunoblotting using antibodies against cMyc and GFP. The numbers below the blot of TAP-SPA1 indicate the relative band with three biological repeats intensities of co-precipitated TAP-SPA1 normalized to precipitated eIF2α-GFP. The GFP only was used as negative control. c Co-IP assay shows that the cytosolic TAP-SPA1 interacts with native form of eIF2α after light treatment. Four-day-old etiolated seedlings were incubated either under dark (D) or illuminated under white light for 4 h (L4h) before sampling. The total proteins were extracted and separated into nuclear (Nu) or cytosolic (Cyt) proteins. The cytosolic proteins were incubated with anti-cMyc. The precipitated proteins were examined by immunoblotting using antibodies against cMyc and eIF2α. The nuclear proteins in L4h TAP-SPA1 and cytosolic proteins in L4h Col-0 were used as negative control. Anti-tubulin and anti-histone-H3 antibodies were used to detect cytosolic and nuclear marker protein, respectively. Three biological repeats of data showed the same results. d BiFC assay shows the interaction of SPA1 with eIF2α homologs under light condition with three biological repeats. The dark-treated samples were incubated under dark for 2 h before observation. The YFP, Chl, 4’, 6’-diaminophenylindole (DAPI; nucleus staining), red fluorescence protein (RFP; chlorophyll fluorescence), merge (merged image) and bright field were shown for each type of transformation combination. Bar = 10 μm.

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