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
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m401461200
ISSN1083-351X
AutoresIsei Tanida, Yu‐shin Sou, Junji Ezaki, Naoko Minematsu‐Ikeguchi, Takashi Ueno, Eiki Kominami,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoIn 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. 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. Autophagy is the bulk degradation of proteins and organelles, essential for cellular maintenance and cell viability. In yeast, the Atg/Apg/Aut/Cvt (cytoplasm-to-vacuole targeting) proteins involved in autophagy have been identified and characterized (1Ohsumi Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 211-216Crossref PubMed Scopus (1035) Google Scholar, 2Huang W.P. Klionsky D.J. Cell Struct. Funct. 2002; 27: 409-420Crossref PubMed Scopus (159) Google Scholar). Recently, the nomenclature for yeast autophagy-related genes was unified to ATG from APG, AUT, or CVT (3Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar). The Atg4/Apg4/Aut2 protein has been shown to be a unique cysteine protease essential for autophagy and Cvt (cytoplasm-to-vacuole) transport in yeast (4Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1489) Google Scholar, 5Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (731) Google Scholar). Atg4 cleaves Atg8/Apg8/Aut7 near its carboxyl terminus to expose a carboxyl-terminal Gly. This proteolytic reaction is indispensable for the ensuing conjugation of Atg8 with phosphatidylethanolamine (PE), 1The abbreviations used are: PE, phosphatidylethanolamine; CFP, cyan fluorescent protein (a variant of green fluorescent protein); DMEM, Dulbecco's modified Eagle's medium; dsRNA, double stranded RNA; GABARAP, GABAA receptor-associated protein; GABARAP-Myc, thioredoxin-His6-GABARAP-Myc fusion protein; GATE-16, Golgi-associated ATPase enhancer of 16 kDa; GABARAP-Myc, thioredoxin-His6-GABARAP-Myc fusion protein; GABARAP-PL, GABARAP-phospholipid conjugate (GABARAP-II); GST, glutathione S-transferase; LC3, microtubule-associated protein light chain 3; LC3-Myc, thioredoxin-His6-LC3-Myc fusion protein; LC3-PL, LC3-phospholipid conjugate (LC3-II); PL, phospholipid; RNAi, RNA interference; TM, LC3-PL- and GABARAP-PL-enriched pellet; TRX, thioredoxin; YFP, yellow fluorescent protein (a variant of green fluorescent protein); YFP-LC3, YFP-tagged LC3; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; CMV, cytomegalovirus; FCS, fetal calf serum; ds-HsAtg4B, dsRNA of HsATG4B for RNA interference.1The abbreviations used are: PE, phosphatidylethanolamine; CFP, cyan fluorescent protein (a variant of green fluorescent protein); DMEM, Dulbecco's modified Eagle's medium; dsRNA, double stranded RNA; GABARAP, GABAA receptor-associated protein; GABARAP-Myc, thioredoxin-His6-GABARAP-Myc fusion protein; GATE-16, Golgi-associated ATPase enhancer of 16 kDa; GABARAP-Myc, thioredoxin-His6-GABARAP-Myc fusion protein; GABARAP-PL, GABARAP-phospholipid conjugate (GABARAP-II); GST, glutathione S-transferase; LC3, microtubule-associated protein light chain 3; LC3-Myc, thioredoxin-His6-LC3-Myc fusion protein; LC3-PL, LC3-phospholipid conjugate (LC3-II); PL, phospholipid; RNAi, RNA interference; TM, LC3-PL- and GABARAP-PL-enriched pellet; TRX, thioredoxin; YFP, yellow fluorescent protein (a variant of green fluorescent protein); YFP-LC3, YFP-tagged LC3; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; CMV, cytomegalovirus; FCS, fetal calf serum; ds-HsAtg4B, dsRNA of HsATG4B for RNA interference. which is mediated by Atg7/Apg7/Cvt2, an E1-like enzyme, and Atg3/Apg3/Aut1, an E2-like enzyme (4Ichimura Y. 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Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (731) Google Scholar, 10Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (712) Google Scholar). Interestingly, expression of a mutant form of Atg8 that has a carboxyl-terminal Gly (Atg8-G) in an atg4 mutant results in significantly more accumulation of the lipidated form of Atg8 than is found in a wild-type strain (5Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (731) Google Scholar). Taken together, these findings suggest that Atg4 may be important for the delipidation of Atg8-PE as well as for the cleavage of the carboxyl-terminal region of Atg8. 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EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5395) Google Scholar). At least four human Atg4 homologues, HsAtg4A/HsApg4A/autophagin-2, HsAtg4B/HsApg4B/autophagin-1, HsAutl1/autophagin-3, and autophagin-4, have been reported (30Scherz-Shouval R. Sagiv Y. Shorer H. Elazar Z. J. Biol. Chem. 2003; 278: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 32Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Since the carboxyl-terminal regions of LC3, GABARAP, and GATE-16 are cleaved soon after translation, it is possible that one or more of these Atg4 homologues is the cysteine protease that cleaves these proteins. HsAtg4A has recently been shown to cleave the carboxyl terminus of GATE-16 (30Scherz-Shouval R. Sagiv Y. Shorer H. Elazar Z. J. Biol. Chem. 2003; 278: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), and autophagin-3/HsAutl1 has been shown to have N-ethylmaleimide-sensitive proteolytic activities for a synthetic substrate, 7-(methoxycoumarin-4-yl)-acetyl-Thr-Phe-Gly-Met-N-3-(2,4-dinitrophenyl)-l-α,β-diaminopropionyl-NH2 (32Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The overexpression of HsAtg4B or HsAutl1 has been found to suppress the Atg– and Cvt– phenotypes of the atg4 mutant (32Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Moreover, one of the two Drosophila Atg4 homologues has been shown to play an essential role in the Notch-signaling pathway (33Thumm M. Kadowaki T. Mol. Genet. Genomics. 2001; 266: 657-663Crossref PubMed Scopus (40) Google Scholar), suggesting that the four human Atg4 homologues may have divergent functions. Although HsAtg4A and HsAutl1 have been independently characterized as cysteine proteases, HsAtg4B has not yet been biochemically characterized, nor have its effects on LC3, GABARAP, and GATE-16 been determined, whereas HsAtg4B has specific interactions with the carboxyl termini of LC3, GABARAP, GATE-16, and Atg8L (29Hemelaar J. Lelyveld V.S. Kessler B.M. Ploegh H.L. J. Biol. Chem. 2003; 278: 51841-51850Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). It is not known if LC3-II and GABARAP-II are lipidated forms of their respective proteins in mammals; nor has it been shown whether Atg4 and human Atg4 homologues directly delipidate Atg8-PE, LC3-II, and GABARAP-II in yeast and mammals. We have therefore investigated whether HsAtg4B can cleave the carboxyl termini of LC3, GABARAP, and GATE-16 in a cell-free system. We also investigated whether LC3-II and GABARAP-II are phospholipase D-sensitive forms (i.e. lipidated forms of LC3 and GABARAP, respectively) and whether HsAtg4B has delipidating activity in vitro. We also determined whether overexpression of HsAtg4B affects endogenous LC3 modification and the localization of YFP-tagged LC3 (YFP-LC3) in HeLa cells and whether RNA interference of HsAtg4B affects LC3 modification. Strains, Media, and Materials—Escherichia coli strain JM109, the host for plasmids and protein expression, was grown in LB medium in the presence of antibiotics as required. The plasmid pGEM-T was purchased from Promega (Madison, WI); pCMV-Tag2B was from Stratagene (La Jolla, CA); pEGFP-C1, pECFP-C1, and pEYFP-C1 were from BD Biosciences Clontech (Palo Alto, CA); and pGEX-4T-1 was from Amersham Biosciences. Plasmid Construction and Site-directed Mutagenesis—Based on the DNA sequence of HsATG4A and HsAUTL1 (GenBank™ accession numbers AB066214 and AJ320169, respectively), the open reading frame of each was amplified by high fidelity PCR with human brain Marathon-ready cDNA as template. The cDNA was synthesized from RNA extracted from a normal, whole brain of a 50-year-old Caucasian male. The primers were designed to introduce a BglII site 5′ to the start codon and a SalI site 3′ to the termination codon. Each resulting DNA fragment was cloned in pGEM-T, and the resulting plasmids were designated as pGEM-HsATG4A and pGEM-HsAUTL1, respectively. Similarly, based on the DNA sequence of HsATG4B/HsAPG4B (GenBank™ accession number AB066215), its open reading frame was amplified by high fidelity PCR with human brain Marathon-ready cDNA as template, with the primers designed to introduce a BamHI site 5′ to the start codon and a SalI site 3′ to the termination codon. The resulting DNA fragment was cloned in pGEM-T, and the resultant plasmid was designated as pGEM-HsAtg4B. To express CFP-HsAtg4A, CFP-HsAutl1, and CFP-HsAtg4B under the control of the CMV promoter in mammalian cells, the cloned DNA fragments of pGEM-HsATG4A, pGEM-HsAUTL1, and pGEM-HsATG4B were introduced into the mammalian expression vector, pECFP-C1 (BD Biosciences Clontech) and designated as pCFP-HsATG4A, pCFP-HsAUTL1, and pCFP-HsATG4B, respectively. To express FLAG-tagged proteins, the cloned DNA fragments of pGEM-HsATG4A, pGEM-HsAUTL1, and pGEM-HsATG4B were introduced into the vector, pCMV-Tag2B (Stratagene), and designated as pTag2B-HsATG4A, pTag2B-HsAUTL1, and pTag2B-HsATG4B, respectively. To express GST-tagged proteins in E. coli, the cloned DNA fragments of pGEM-HsATG4A, pGEM-HsAUTL1, and pGEM-HsATG4B were introduced into the vector, pGEX-4T-1 (Amersham Biosciences) and designated as pGEX-HsATG4A, pGEX-HsAUTL1, and pGEX-HsATG4B, respectively. The human Atg8 homologues LC3, GABARAP, and GATE-16 were fused with thioredoxin-His6 at the amino terminus and with the Myc epitope at the carboxyl terminus by introducing six His repeats into the amino-terminal region of each Atg8 homologue by high fidelity PCR and subsequently introducing the Myc epitope directly 3′ to each sequence. Each resultant DNA fragment was cloned in the vector pThioHisA (Invitrogen), and each construct was used to transform E. coli (Fig. 1B). To express YFP-LC3, a BglII-SalI fragment of pGEM-LC3 was introduced into the BglII-SalI site of the vector pEYFP-C1 (BD Biosciences Clontech), and the resulting construct was designated as pYFP-LC3. The Cys residue at amino acid 74 of HsAtg4B was replaced by Ala using the Gene-Editor in vitro site-directed mutagenesis system (Promega) and the oligonucleotide, 5′-CCCACCTCGGACACAGGCTGGGGCGGCCATGCTGCGGTGTGGACAGATGATCTTTGCCC-3′ (HsA-TG4BCA oligonucleotide), according to the manufacturer's directions. Cell Culture and Transfection—HEK293 and HeLa cells obtained from the ATCC (Manassas, VA), were grown in DMEM containing 10% fetal calf serum (FCS). Cells were transfected with the indicated constructs using LipofectAMINE 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, the cells were harvested for further analyses. Antibodies—Rabbits were immunized with GST-HsAtg4A, GST-HsAtg4B, and GST-HsAutl1 to obtain antisera against HsAtg4A, HsAtg4B, and HsAutl1, respectively. Each antibody was affinity-purified by chromatography on each thioredoxin (TRX)-linked antigen-Sepharose and designated as anti-HsAtg4A, anti-HsAtg4B, and anti-HsAutl1 antibodies, respectively. Anti-LC3 and anti-GABARAP antibodies were prepared as described previously. Anti-LC3 antibody shows little cross-reactivity with GABARAP and GATE-16. Anti-GABARAP antibody shows little cross-reactivity with LC3 and GATE-16. Anti-Myc antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA), and anti-TRX antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Purification of Recombinant Proteins—The purification of GST fusion proteins from E. coli was performed according to the manufacturer's protocol (Amersham Biosciences). The purification of LC3-Myc, GABARAP-Myc, and GATE-16-Myc, each with a His6 tag, was performed using TALON purification kits according to the manufacturer's protocol (BD Biosciences Clontech). Assay for Cleavage of the Carboxyl Terminus of LC3-Myc, GABARAP-Myc, and GATE-16-Myc by Human Atg4 Homologues—FLAG-tagged HsAtg4A/autophagin-2 (FLAG-HsAtg4A), FLAG-tagged HsAtg4B/autophagin-1 (FLAG-HsAtg4B), and FLAG-tagged HsAutl1/autophagin-3 (FLAG-HsAutl1) were expressed in HEK293 cells using pTag2B-HsAtg4A, pTag2B-HsAtg4B, and pTag2B-HsAutl1, respectively. Cells were lysed in phosphate-buffered saline-dithiothreitol buffer (20 mm sodium phosphate, pH 7.2, 150 mm NaCl, 1 mm dithiothreitol) containing a Complete™ proteinase inhibitor mixture tablet (Roche Applied Science), and supernatant was prepared from each lysate as described previously (34Nemoto T. Tanida I. Tanida-Miyake E. Minematsu-Ikeguchi N. Yokota M. Ohsumi M. Ueno T. Kominami E. J. Biol. Chem. 2003; 278: 39517-39526Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Supernatant (0.4 μg) of each lysate was added to 3.6 μg purified LC3-Myc, GABARAP-Myc, or GATE-16-Myc in phosphate-buffered saline-dithiothreitol buffer containing a Complete™ proteinase inhibitor mixture tablet, and the mixture was incubated at 37 °C for the indicated times (0, 30, and 60 min). To stop the reaction, Laemmli's sample buffer was added to the mixture, and the mixture was boiled for 5 min. Proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anti-Myc and anti-TRX antibodies. Preparation of a Pelletable Fraction Enriched with Modified Forms of LC3 (LC3-II) and GABARAP (GABARAP-II) as Substrates of the Delipidation Assay—Endogenous LC3 and GABARAP in HeLa cells were present as modified forms, even under nutrient-rich conditions. 2I. Tanida, T. Ueno, and E. Kominami, unpublished results. HeLa cells were cultured in DMEM containing 10% FCS in the presence of E64d (10 μg/ml) and pepstatin A (10 μg/ml) to enrich for LC3-II and GABARAP-II. The cells were lysed in phosphate-buffered saline by sonication, and total pellets were prepared by ultracentrifugation at 100,000 × g for 1 h. The LC3-II- and GABARAP-II-enriched pellet fraction (TM fraction) was employed as substrates for treatment with phospholipase D and GST-HsAtg4B. Assay for Delipidation of LC3-II and GABARAP-II by Phospholipase D—Phospholipase D (from Streptomyces chromofuscus) was purchased from Sigma. LC3-II was solubilized from 45 μg of the TM fraction in TX solution (2% Triton X-100, 20 mm sodium phosphate, pH 7.5, 150 mm NaCl), 10 units of phospholipase D were added, and the mixture was incubated at 37 °C for the indicated times. To stop the reaction, Laemmli's sample buffer was added to the mixture, and the mixture was boiled for 5 min. Total proteins were separated by 12.5% SDS-PAGE, and LC3 was assayed by immunoblotting with an anti-LC3 antibody. Delipidation of LC3-II (membrane-bound form) was recognized as a change in mobility. To assay delipidation of GABARAP-II, the latter was solubilized from 90 μg of the TM fraction in NP solution (0.5% Nonidet P-40, 20 mm sodium phosphate, pH 7.5, 150 mm NaCl), and the sample was treated with phospholipase D as described above. GABARAP was assayed by immunoblotting with an anti-GABARAP antibody. Delipidation of GABARAP-II was recognized as a change in mobility. Assay for Delipidation of LC3-II and GABARAP-II by Purified GST-HsAtg4B—To assay delipidation of LC3-II and GABARAP-II by GST-HsAtg4B, 0.45 μg of GST-HsAtg4B was added in place of phospholipase Dto45 μg of the TM fraction, and the assay was performed as described above. Fluorescent Microscopy—HeLa cells expressing CFP-HsAtg4B and YFP-LC3 were fixed according to the manufacturer's protocol (BD Biosciences Clontech), and cyan and yellow fluorescences in HeLa cells were observed with a Zeiss Axioplan2 fluorescence microscope with filters XF114-2 and XF104-2. RNA Interference Analysis of HsAtg4B—Double-stranded RNAs of HsAtg4B were produced by a BLOCK-it™ Complete Dicer RNAi kit with a DNA template containing the region from start codon to 500 base of HsAtg4B cDNA as a template according to the manufacturer's protocol (Invitrogen). For RNA interference of HsAtg4B, 2 μg of dsRNA of HsAtg4B/60-mm culture dish was transfected into HEK293 cells using LipofectAMINE 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. After 24 h, the cells were harvested for further analyses. For plasmid-based RNA interference of HsATG4B, two oligonucleotides, siT4 (5′-GATCCGAAGCTTGCTGTCTTCGATTTCAAGAGAATCGAAGACAGCAAGCTTCTTTTTTGGAAA-3′) and siB4 (5′-AGCTTTTCCAAAAAAGAAGCTTGCTGTCTTCGATTCTCTTGAAATCGAAGACAGCAAGCTTCG-3′), were synthesized. After annealing these oligonucleotides, the resultant fragment was introduced into a BamHI-HindIII site of pSilencer H1–3.0 (Ambion, Austin, TX), and the resulting construct was designated as pSi-HsATG4B. For RNA interference of HsA-tg4B, 6 μg of pSi-HsATG4B per 60-mm culture dish was transfected into HEK293 cells using LipofectAMINE 2000 transfection reagent (Invitrogen). After 24 h, the cells were harvested for further analyses. Other Techniques—Quantification of the images was performed using Aqua Lite (Hamamatsu Photonics, Tokyo, Japan) and NIH Image (Dr. W. Raeband, National Institutes of Health) programs on Endeavor (EPSON direct, Tokyo, Japan) and Macintosh G4 (Apple Computer, New York) computers, respectively. The Cys74 Residue in HsAtg4B Is an Authentic Active Site Cysteine Essential for the Ability of This Protein to Cleave the Carboxyl Termini of the Three Atg8 Homologues—Although the carboxyl termini of LC3, GABARAP, and GATE-16 are cleaved to expose a Gly soon after translation (21Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5395) Google Scholar, 30Scherz-Shouval R. Sagiv Y. Shorer H. Elazar Z. J. Biol. Chem. 2003; 278: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 31Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (84) Google Scholar), it is not known whether HsAtg4B is responsible for this activity. However, the region containing a predicted active site Cys within HsAtg4B shows a significant homology with that within yeast Atg4 (Fig. 1A). Therefore, we focused on HsAtg4B and determined its proteolytic activity on the carboxyl termini of LC3, GABARAP, and GATE-16. FLAG-HsAtg4B was expressed in HEK293 cells, the cells were lysed, and the lysate was fractionated by ultracentrifugation, with the resulting supernatant used as enzyme mixture. HsAtg4B in the supernatant was recognized by immunoblotting with an anti-FLAG-antibody (Fig. 1B, pTag2B-HsATG4B wt, +). The substrates, LC3-Myc, GABARAP-Myc, and GATE-16-Myc with TRX-His6 at the amino terminus and with the Myc epitope at the carboxyl terminus were expressed in E. coli and affinity-purified by a nickel-chelating resin. We incubated FLAG-HsAtg4B with each Myc-linked Atg8 homologue and determined the carboxyl-terminal cleavage activity by immunoblotting using an anti-Myc antibody (Fig. 1C, WB: anti-Myc, HsAtg4B versus vector control). FLAG-HsAtg4B cleaved the carboxyl terminus of Myc conjugate LC3, GABARAP, and GATE-16 in a time-dependent manner. Using an anti-TRX antibody, we were able to confirm that the amino-terminal TRX tag within each substrate remains unchanged (Fig. 1C, anti-TRX), indicating that the carboxyl-terminal Myc tag itself was cleaved by HsAtg4B. Essentially the same results were obtained when purified GST-HsAtg4B, instead of the supernatant expressing FLAG-HsAtg4B, was incubated with Myc-linked Atg8 homologues (data not shown). The cleavage is sensitive to N-ethylmaleimide, but not to phenylmethylsulfonyl fluoride or pepstatin A (data not shown). These results show that the HsAtg4B protease cleaves the carboxyl termini of all
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