Fig. 1: TRB1 and TRB2 proteins interact with VAP27 at ER-mitochondrial interface. | Nature Communications

Fig. 1: TRB1 and TRB2 proteins interact with VAP27 at ER-mitochondrial interface.

From: TraB family proteins are components of ER-mitochondrial contact sites and regulate ER-mitochondrial interactions and mitophagy

Fig. 1

a Schematic illustration of TRB1 protein in Arabidopsis; it contains two ATG8 interacting motifs (AIM), one TRAB homology domain and a C-terminal transmembrane domain. b GFP-TRB1 and VAP27-1-RFP co-localize at ER-derived donut-shaped membrane structures in N. benthamiana leaf epidermal cells. c GFP-TRB2 co-localizes with VAP27-1-RFP at the ER network and ring-shaped membrane structures. d GFP-TRB1 (green), VAP27-1-YFP (Red), and Mito-CFP (blue) co-expressed in N. benthamiana leaf epidermal cells, demonstrating that the ER derived donut-shaped structures superimpose with the mitochondrial outer membrane. e GFP-TRB2 (green), VAP27-1-YFP (Red), and Mito-mTAGBFP2 (blue) co-expressed in N. benthamiana leaf epidermal cells, the ER derived ring-shaped structures superimpose with the mitochondrial outer membrane. f Control cells expressing VAP27-1-RFP alone. g, h Triple expression of GFP-TRB1, VAP27-1-RFP, and mTAGBFP-HDEL in N. benthamiana. Recruitment of mTAGBFP2-HDEL-labelled ER to TRB1-VAP27-labelled hybrid structures was identified (g). However, this phenotype was not found in the absence of TRB1 (h), supporting the idea that TRB1-VAP27 interaction promotes ER-mitochondrial association. i FRET-FLIM further proved the interactions between TRB1/VAP27-1 and TRB2/VAP27-1. The fluorescence life-time of GFP-TRB1 (control) and GFP-TRB2 changes significantly (life-time reduces from 2.50 ± 0.01 to 2.31 ± 0.02 ns; and from 2.50 ± 0.01 to 2.21 ± 0.02 ns, respectively) in the presence of RFP-VAP27-1. j The interaction between VAP27-1 and TRB1 is confirmed by a GFP-Trap assay. VAP27-1-RFP was only pulled-down in the presence of GFP-TRB1 (right) but not with free GFP (left). P, pellet; S, supernatant; T, total. k Split-YFP based BiFC study showed cYFP-VAP27-1 and nYFP-TRB1 producing signals when co-expressed in N. benthamiana. l The YFP signal generated from VAP27-TRB1 BiFC colocalised with Mito-CFP and RFP-HDEL, demonstrating the interaction to be ER and mitochondrial-localised. Microscopy studies were repeated at least 3 times for every experiment. n = 10 independent biological samples for every FRET-FLIM analysis, error bars are SEM, *** P < 0.001 in two-tailed Student’s t tests (scale bar = 10 μm).

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