Fig. 6. Post-translational processing of LC3. (A) Amino acid sequence alignment of the C-terminal segments of LC3 and its homologues. Residues identical to LC3 are shaded in black. Rn, Rattus norvegicus; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Lb, Laccaria bicolor. The sequence of HsLC3 was deduced from an EST clone (au96a10.y1). (B) Constructions of proteins used in (C). Myc epitope tags at the N-terminus, HA epitope tags at the C-terminus and a hypothetical cleavage site Gly120 residue are indicated. To construct LC3G120A, a single point mutation was introduced into LC3, which leads to an amino acid substitution from glycine to alanine at position 120. To construct LC3ΔC22, the 22 C-terminal residues were deleted by PCR. LC3ΔC22,G120A was also produced by site-directed mutagenesis of LC3ΔC22. The ΔC22 mutants were tagged only with the Myc epitopes at the N-teminus. (C) HeLa cells were transiently transfected with Myc-LC3-HA (lanes 1–3 and 13–15), Myc-LC3G120A-HA (lanes 4–6 and 16–18), Myc-LC3ΔC22 (lanes 7–9) or Myc-LC3ΔC22, G120A (lanes 10–12). The cells were labelled with [35S]methionine/cysteine for 4 min and chased for 0, 6 and 90 min at 37°C. The cell lysates were immunoprecipitated with anti-Myc epitope antibody (lanes 1–12) or anti-HA epitope antibody (lanes 13–18) and the immunoprecipitates were analysed by SDS–PAGE and a bioimage analyser.