Molecular cloning and characterization of rat LC3A and LC3B—Two novel markers of autophagosome

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Abstract

Rat microtubule-associated protein light chain 3 (LC3) is a homologue of yeast Atg8, an essential component of autophagy. Following synthesis, the C-terminus of rat LC3 is cleaved by a cysteine protease-Atg4, to produce LC3-I, which is located in cytosolic fraction. LC3-I can be converted to LC3-II through the processing by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme). LC3-II is modified by phosphatidylethanolamine on C-terminus and binds tightly to autophagosomal membrane. Here we reported the cloning of two novel variants of rat LC3, named LC3A and LC3B, respectively, and LC3B is an alternative splicing variant of LC3. LC3A, LC3B, and LC3 showed different expression patterns in rat tissues, suggesting a functional divergence among these proteins. When LC3A and LC3B were overexpressed, both exhibited two forms (18 and 16 kDa, representing types of I and II, separately), which might be due to post-translational modification including the characteristic C-terminal cleavage at these two proteins as similar to that found in rat LC3 and yeast Atg8. Subcellular localization demonstrated that both LC3A and LC3B are colocalized with LC3 and associated with the autophagic membranes. Mutation analysis further revealed that the conserved Gly120 residues of LC3A and LC3B are essential for their characteristic C-terminal cleavage and localization to autophagic membranes. Present data suggested that LC3A and LC3B could also be used as two novel autophagosomal markers.

Section snippets

Materials and methods

Cloning of LC3A and LC3B cDNAs. The amino acid sequences of human LC3A (GenBank Accession No.: AF276658), LC3B (GenBank Accession No.: AF087871) and LC3C (GenBank Accession No.: AF276659) were used to search the rat expressed sequence tag (EST) database at GenBank (http://www.ncbi.nlm.nih.gov). Homologous ESTs were obtained and assembled into three EST contigs and checked manually. Two pairs of primers (named LC3A/B/-A and B; see Table 1) were designed based on the contig sequences and used for

Identification and isolation of LC3A and LC3B

Mann and Hammarback [5] purified the bovine brain LC3 by molecular sieve chromatography from salt-extracted MAPs, and the sequence of the first 20 amino acids of gel-purified bovine LC3 was determined by Edman degradation. They obtained two amino acid sequences, and they used the major sequence PSDRPFKQRRSFADDVKEVQ to search the GenBank database and cloned the rat LC3 gene, but the sequence similarity is significantly low [5]. We previously identified three human LC3 family members (LC3A, LC3B,

Conclusion

In present work, we cloned LC3A and LC3B, two novel variants of rat LC3. The expression patterns of LC3, LC3A, and LC3B were different in 12 normal rat adult tissues. LC3A and LC3B undergo a characteristic C-terminal cleavage and are associated with autophagic membranes. The conserved Gly120 residues of LC3A and LC3B are essential for their post-translational modification and localization to autophagic membranes. Present data also suggested that LC3A and LC3B could be used as two novel

Acknowledgments

This work was supported by the National 973 Program and 863 High Technology Program of China, as well as the National Natural Science Foundation of China.

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