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 HsAtg4BC74A, in which a predicted active site Cys74 was changed to Ala, lacked proteolytic activity, indicating that Cys74 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 Cys74 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.

[1]  T. Yoshimori.  Autophagy: a regulated bulk degradation process inside cells. , 2004, Biochemical and biophysical research communications.

[2]  V. Lelyveld,et al.  A Single Protease, Apg4B, Is Specific for the Autophagy-related Ubiquitin-like Proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L* , 2003, Journal of Biological Chemistry.

[3]  T. Nemoto,et al.  The Mouse APG10 Homologue, an E2-like Enzyme for Apg12p Conjugation, Facilitates MAP-LC3 Modification* , 2003, Journal of Biological Chemistry.

[4]  Daniel J Klionsky,et al.  A unified nomenclature for yeast autophagy-related genes. , 2003, Developmental cell.

[5]  Y. Tomino,et al.  MAP‐LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  M. Kadowaki,et al.  Amino acids as regulators of proteolysis. , 2003, The Journal of nutrition.

[7]  Z. Elazar,et al.  The COOH Terminus of GATE-16, an Intra-Golgi Transport Modulator, Is Cleaved by the Human Cysteine Protease HsApg4A* , 2003, The Journal of Biological Chemistry.

[8]  Eric Ogier-Denis,et al.  Autophagy: a barrier or an adaptive response to cancer. , 2003, Biochimica et biophysica acta.

[9]  Xose S. Puente,et al.  Human Autophagins, a Family of Cysteine Proteinases Potentially Implicated in Cell Degradation by Autophagy* , 2003, The Journal of Biological Chemistry.

[10]  M. Komatsu,et al.  GATE-16 and GABARAP are authentic modifiers mediated by Apg7 and Apg3. , 2003, Biochemical and biophysical research communications.

[11]  D. Klionsky,et al.  Autophagy in yeast: a review of the molecular machinery. , 2002, Cell structure and function.

[12]  R. Lockshin,et al.  Caspase-independent cell deaths. , 2002, Current opinion in cell biology.

[13]  M. Komatsu,et al.  Human Apg3p/Aut1p Homologue Is an Authentic E2 Enzyme for Multiple Substrates, GATE-16, GABARAP, and MAP-LC3, and Facilitates the Conjugation of hApg12p to hApg5p* , 2002, The Journal of Biological Chemistry.

[14]  D. Klionsky,et al.  Autophagy in the Eukaryotic Cell , 2002, Eukaryotic Cell.

[15]  A. Tolkovsky,et al.  Mitochondrial disappearance from cells: a clue to the role of autophagy in programmed cell death and disease? , 2002, Biochimie.

[16]  M. Thumm,et al.  The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants , 2001, Molecular Genetics and Genomics.

[17]  C. Thummel.  Steroid-triggered death by autophagy. , 2001, BioEssays : news and reviews in molecular, cellular and developmental biology.

[18]  A. Gingras,et al.  The target of rapamycin (TOR) proteins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Y. Ohsumi,et al.  Ubiquitin and proteasomes: Molecular dissection of autophagy: two ubiquitin-like systems , 2001, Nature Reviews Molecular Cell Biology.

[20]  Takeshi Tokuhisa,et al.  Dissection of Autophagosome Formation Using Apg5-Deficient Mouse Embryonic Stem Cells , 2001, The Journal of cell biology.

[21]  T. Ueno,et al.  The Human Homolog of Saccharomyces cerevisiae Apg7p Is a Protein-activating Enzyme for Multiple Substrates Including Human Apg12p, GATE-16, GABARAP, and MAP-LC3* , 2001, The Journal of Biological Chemistry.

[22]  Takeshi Noda,et al.  A ubiquitin-like system mediates protein lipidation , 2000, Nature.

[23]  Takeshi Noda,et al.  LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing , 2000, The EMBO journal.

[24]  Takeshi Noda,et al.  The Reversible Modification Regulates the Membrane-Binding State of Apg8/Aut7 Essential for Autophagy and the Cytoplasm to Vacuole Targeting Pathway , 2000, The Journal of cell biology.

[25]  A. Porat,et al.  GATE‐16, a membrane transport modulator, interacts with NSF and the Golgi v‐SNARE GOS‐28 , 2000, The EMBO journal.

[26]  Takeshi Noda,et al.  Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast , 1999, The Journal of cell biology.

[27]  A. Matsuura,et al.  Apg10p, a novel protein‐conjugating enzyme essential for autophagy in yeast , 1999, The EMBO journal.

[28]  N. Mizushima,et al.  Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy. , 1999, Molecular biology of the cell.

[29]  D. Klionsky,et al.  Apg7p/Cvt2p is required for the cytoplasm-to-vacuole targeting, macroautophagy, and peroxisome degradation pathways. , 1999, Molecular biology of the cell.

[30]  Weiping Yuan,et al.  Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein. , 1999, Molecular biology of the cell.

[31]  N. Brandon,et al.  GABAA-receptor-associated protein links GABAA receptors and the cytoskeleton , 1999, Nature.

[32]  J. Hammarback,et al.  Gene localization and developmental expression of light chain 3: A common subunit of microtubule‐associated protein 1A (MAP1A) and MAP1B , 1996, Journal of neuroscience research.

[33]  J. Hammarback,et al.  Molecular characterization of light chain 3. A microtubule binding subunit of MAP1A and MAP1B. , 1994, The Journal of biological chemistry.

[34]  D. Sulzer,et al.  Autophagy in neurons: a review. , 2002, Histology and histopathology.

[35]  R. Lüllmann-Rauch,et al.  Disease model: LAMP-2 enlightens Danon disease. , 2001, Trends in molecular medicine.