Fig. 5
AtEH1/Pan1 interacts with VAP27-1 and is involved in the formation of autophagosomes at the ER-PM contact sites. a–d Immunogold TEM labelling on Arabidopsis root cells. Autophagosome-like structures that are labelled by anti-AtEH/Pan1, anti-NAP1, anti-ATG8 or anti-ATG4 gold particles are found at the site where the ER and PM are connected (Scale bars: 500 nm). e Consistent with this observation, ATG8e-labelled autophagosomes can be found associated with the ER-PM contact sites that are labelled by VAP27-1-YFP. f GFP-AtEH1/Pan1-labelled autophagosomes can be recruited to VAP27-1-YFP containing ER-PM contact sites (Scale bar: 10 μm; images are 3D projections, Z = 18 μm, 35 slices). g Using FRET-FLIM, GFP-AtEH1/Pan1 is shown to interact with RFP-VAP27-1. The fluorescence life-time of the GFP-AtEH1/Pan1 donor protein (control) is on average 2.70 ± 0.02 ns, whereas it significantly reduces to 2.58 ± 0.02 ns in the presence of RFP-VAP27-1 (test condition). h The interaction between VAP27-1 and AtEH1/Pan1 is further confirmed using a GFP-trap based co-precipitation assay. RFP-VAP27-1 is found in the pellet fraction in the presence of GFP-AtEH1/Pan1. As control, RFP-VAP27-1 is not able to be precipitated by the GFP-trap. N ≥ 12 for every FRET-FLIM analysis; error bars are S.D., ***P < 0.001 in Student’s t-test
