Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes.
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 Gly(120). 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 Gly(120) 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 MAP1LC3B(G120A)-3xFLAG and Myc-MAP1LC3B(G120A)-His, containing Ala in place of Gly(120). An in vitro assay showed that Gly(120) 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 x 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 Gly(120) for further ubiquitylation-like reactions.
Tanida
Isei
I
Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
Ueno
Takashi
T
Kominami
Eiki
E
D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071193:Autophagy-Related Protein 7; D002458:Cell Fractionation; D002460:Cell Line; D005998:Glycine / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000378:metabolism
C114830:ATG7 protein, S cerevisiae; C477012:MAP1LC3A protein, human; C502070:MAP1LC3B protein, human; C502071:MAP1LC3C protein, human; D008869:Microtubule-Associated Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine; D000071193:Autophagy-Related Protein 7; D005998:Glycine