Autophagy is the process by which portions of cytoplasm are enclosed by 
membranous organelles, phagophores, which deliver the sequestered cytoplasm to 
degradative autophagic vacuoles. Genes and proteins involved in phagophore 
manufacture have been extensively studied, but little is known about how mature 
phagophores proceed through the subsequent steps of expansion, closure and 
fusion. Here we have addressed these issues by combining our unique autophagic 
cargo sequestration assay (using the cytosolic enzyme lactate dehydrogenase as a 
cargo marker) with quantitative measurements of the lipidation-dependent 
anchorage and turnover of the phagophore-associated protein LC3. In isolated rat 
hepatocytes, amino acid starved to induce maximal autophagic activity, the two 
unrelated reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin 
(TG) both blocked cargo sequestration completely. However, whereas 3MA inhibited 
LC3 lipidation, TG did not, thus apparently acting at a post-lipidation step to 
prevent phagophore closure. Intriguingly, the resumption of cargo sequestration 
seen upon release from a reversible TG block was completely suppressed by 3MA, 
revealing that 3MA not only inhibits LC3 lipidation but also (like TG) blocks 
phagophore closure at a post-lipidation step. 3MA did not, however, prevent the 
resumption of lysosomal LC3 degradation, indicating that phagophores could fuse 
directly with degradative autophagic vacuoles without carrying cytosolic cargo. 
This fusion step was clearly blocked by TG. Furthermore, density gradient 
centrifugation revealed that a fraction of the LC3-marked phagophores retained 
by TG could be density-shifted by the acidotropic drug propylamine along with 
the lysosomal marker cathepsin B, suggesting physical association of some 
phagophores with lysosomes prior to cargo sequestration.