HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates.
In yeast, Atg4/Apg4 is a unique cysteine protease responsible for the cleavage of the carboxyl terminus of Atg8/Apg8/Aut7, a reaction essential for its lipidation during the formation of autophagosomes. However, it is still unclear whether four human Atg4 homologues cleave the carboxyl termini of the three human Atg8 homologues, microtubule-associated protein light chain 3 (LC3), GABARAP, and GATE-16. Using a cell-free system, we found that HsAtg4B, one of the human Atg4 homologues, cleaves the carboxyl termini of these three Atg8 homologues. In contrast, the mutant HsAtg4B(C74A), in which a predicted active site Cys(74) was changed to Ala, lacked proteolytic activity, indicating that Cys(74) is essential for the cleavage activity of cysteine protease. Using phospholipase D, we showed that the modified forms of endogenous LC3 and GABARAP are lipidated and therefore were designated LC3-PL and GABARAP-PL. When purified glutathione S-transferase-tagged HsAtg4B was incubated in vitro with a membrane fraction enriched with endogenous LC3-PL and GABARAP-PL, the mobility of LC3-PL and GABARAP-PL was changed to those of the unmodified proteins. These mobility shifts were not seen when Cys(74) of HsAtg4B was changed to Ala. Overexpression of wild-type HsAtg4B decreased the amount of LC3-PL and GABARAP-PL and increased the amount of unmodified endogenous LC3 and GABARAP in HeLa cells. Expression of CFP-tagged HsAtg4B (CFP-HsAtg4B) and YFP-tagged LC3 in HeLa cells under starvation conditions resulted in a significant decrease in the punctate pattern of distribution of YFP-tagged LC3 and an increase in its cytoplasmic distribution. RNA interference of HsAtg4B increased the amount of LC3-PL in HEK293 cells. Taken together, these results suggest that HsAtg4B negatively regulates the localization of LC3 to a membrane compartment by delipidation.
Tanida
Isei
I
Department of Biochemistry, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Sou
Yu-shin
YS
Ezaki
Junji
J
Minematsu-Ikeguchi
Naoko
N
Ueno
Takashi
T
Kominami
Eiki
E
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003470:Culture Media; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010587:Phagocytosis; D010739:Phospholipase D / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011963:Receptors, GABA-A / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014162:Transfection
C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D003470:Culture Media; D008055:Lipids; D008164:Luminescent Proteins; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D011963:Receptors, GABA-A; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D010739:Phospholipase D; C474025:ATG4A protein, human; D003546:Cysteine Endopeptidases; D003545:Cysteine; D000409:Alanine