Journal of Biological Chemistry
Volume 279, Issue 46, 12 November 2004, Pages 47704-47710
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Protein Synthesis, Post-Translation Modification, and Degradation
Human Light Chain 3/MAP1LC3B Is Cleaved at Its Carboxyl-terminal Met121 to Expose Gly120 for Lipidation and Targeting to Autophagosomal Membranes*

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Human light chain 3/MAP1LC3B, an autophagosomal ortholog of yeast Atg8, is conjugated to phospholipid (PL) via ubiquitylation-like reactions mediated by human Atg7 and Atg3. Since human Atg4B was found to cleave the carboxyl terminus of MAP1LC3B in vitro, we hypothesized that this exposes its carboxyl-terminal Gly120. It was recently reported, however, that when Myc-MAP1LC3B-His is expressed in HEK293 cells, its carboxyl terminus is not cleaved. (Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., and Kominami, E. (2004) J. Biol. Chem. 279, 36268–36276). To clarify this contradiction, we sought to determine whether the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 for further ubiquitylation-like reactions. When MAP1LC3B-3xFLAG and Myc-MAP1LC3B-His were expressed in HEK293 cells, their carboxyl termini were cleaved, whereas there was little cleavage of mutant proteins MAP1LC3BG120A-3xFLAG and Myc-MAP1LC3BG120A-His, containing Ala in place of Gly120. An in vitro assay showed that Gly120 is essential for carboxyl-terminal cleavage by human Atg4B as well as for formation of the intermediates Atg7-MAP1LC3B (ubiquitin-activating enzyme-substrate) and Atg3-MAP1LC3B (ubiquitin carrier protein-substrate). Recombinant MAP1LC3B-PL was fractionated into the 100,000 × g pellet in a manner similar to that shown for endogenous MAP1LC3B-PL. RNA interference of MAP1LC3B mRNA resulted in a decrease in both endogenous MAP1LC3B-PL and MAP1LC3B. These results indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 for further ubiquitylation-like reactions.

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This work was supported in part by Grants-in-aid for Scientific Research 15590254 (to I. T.), 16370063 (to T. U.), and 1246101 (to E. K.) and Grant-in-aid 12146205 for Scientific Research on Priority Areas (to E. K.) from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation (to E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.