Figure 5.
Human ATG4B can cleave cross-kingdom ATG8s in vitro. Purified recombinant maltose binding protein (MBP) fused to different ATG4s (MBP-ATG4) and MBP-alone were incubated with various C-ATG8- or LC3A-ShR synthetic substrates as described in the Materials and Methods section. The reaction mixtures were separated on SDS-PAGE gel and blots were probed with anti-ShR. Arrows and arrowheads indicate full-length synthetic substrate (C-ATG8-ShR) and cleaved byproduct ShR, respectively. The yeast Atg4 (A), Arabidopsis ATG4a (B), and tomato ATG4 (C) could process both yeast and plant ATG8s but not human HsLC3A. HsATG4B could process all species ATG8 synthetic substrates (D). The tomato ATG4 mutant (SlATG4178-188Δ) in which the plant specific domain is deleted decreased the efficiency of processing of plant ATG8s and completely inhibited processing of yeast Atg8 (E). Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; At, Arabidopsis thaliana; Sl, Solanum lycopersicum.