Fig. 2. Rab7 localised in the membranes of autophagic vacuoles. (A) Subcellular fractionation of rat liver. Rab7 was detected by western blotting in both autophagic vacuolar membranes and lysosomal/late endosomal membranes. The characterisation of the fractions has previously been published (Kabeya et al., 2000). LC3II only is shown, as no LC3I was detected in the membrane fractions. (B-F) Localisation of GFP-Rab7 by immunoelectron microscopy. HeLa cells were transfected with pEGFP-Rab7 and grown for 1 day. The cells were starved of amino acids for 2 hours and prepared for immunogold labelling with anti-GFP (10 nm gold) and anti-Lamp1 (5 nm gold) antibodies. (B) Western blotting was used to show that all GFP was still connected to Rab7 in these conditions. Lane 1, cells transfected with GFP, and lane 2, cells transfected with GFP-Rab7. Early autophagic vacuoles (AVi) showed labelling for GFP-Rab7 in the outer and inner membranes (C). (D-F) Late autophagic vacuoles (AVd) with their limiting membranes labelled for GFP-Rab7. Some GFP-Rab7 was also present in the contents, in agreement with the association of GFP-Rab7 with both the outer and inner limiting membranes of the early vacuoles (C). Some AVd were also weakly labelled for Lamp1 (E and F, arrowheads), or Lamp1 was present in vesicles close to them (F, arrow). The AVd in D contains a partially degraded mitochondrion (m). PM, plasma membrane.