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J. Biol. Chem., Vol. 279, Issue 46, 47704-47710, November 12, 2004
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From the Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan
Received for publication, June 23, 2004 , and in revised form, August 19, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In mammalian cells, the Atg8-like modification system for formation of autophagosomes seems to be conserved. The carboxyl terminus of rat LC3 is cleaved, exposing Gly120, a residue that is essential for the cleavage and modification of LC3-I to LC3-II (3). Modification of rat LC3-I to LC3-II is facilitated under starvation conditions. During ultracentrifugation, rat LC3-II is fractionated into the 100,000 x g pellet, and LC3-II localizes to autophagosomes.
Human (h) LC3/MAP1LC3B, one of three orthologs of rat MAP1LC3 (microtubule-associated protein 1 light chain 3), shows significant homology to rat LC3 (3, 9), especially at residues 1–120, which are 98.3% identical (see Fig. 1A). hAtg7 and hAtg3 can form E1- and E2-substrate intermediates, respectively, with hLC3/MAP1LC3B (9–11), and transient expression of hAtg7 and hAtg3 along with MAP1LC3B results in an increase in MAP1LC3B-II (11). We recently showed that endogenous MAP1LC3B-II isolated from HeLa cells is an LC3-phospholipid (PL) conjugate (MAP1LC3B-PL); that purified hAtg4B cleaves the carboxyl terminus of MAP1LC3B in vitro; and that, during ultracentrifugation, MAP1LC3B-PL is fractionated into the pellet (11, 12). Since the exposure of a carboxyl-terminal Gly of a ubiquitin-like modifier (e.g. ubiquitin, SUMO-1, Nedd8, Atg12, Atg8, 
-aminobutyric acid type A receptor-associated protein (GABARAP), and Ufm1) is essential for further ubiquitylation-like reactions, we hypothesized that the carboxyl-terminal Gly120 of MAP1LC3B is exposed in vivo and that MAP1LC3B is an authentic modifier, as is rat LC3.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmid Construction and Site-directed Mutagenesis—The mammalian expression vectors pCMV-hAtg7 (expressing wild-type hAtg7), pCMV-hAtg7CS (expressing mutant hAtg7C572S), and pGFP-hAtg3CS (expressing green fluorescent protein (GFP)-tagged hAtg3C264S mutant protein) have been described (9, 10). For mammalian cell expression of MAP1LC3B-3xFLAG protein, in which three repeats of the FLAG epitope were fused to the carboxyl terminus of wild-type MAP1LC3B, the open reading frame of MAP1LC3B was excised from pGEM-hLC3 (10) and introduced into p3xFLAG-CMV-14, and the resulting plasmid was designated pCMV-hLC3–3xFLAG. For expression of MAP1LC3BG120A-3xFLAG, in which Gly120 was changed to Ala, and MAP1LC3BK122A-3xFLAG, in which Lys122 was changed to Ala (see Fig. 1B), site-directed mutations were introduced into pCMV-hLC3–3xFLAG, and the resulting plasmids were designated pCMV-hLC3G120A-3xFLAG and pCMV-hLC3K122A-3xFLAG, respectively.
For expression of Myc-MAP1LC3B-His protein, in which wild-type MAP1LC3B was fused to the Myc epitope at its amino terminus and to His6 at its carboxyl terminus, oligonucleotides MAP1LC3B-ATGF (5'-AGATCTATGCCGTCGGAGAAGACC-3') and MAP1LC3B-His6R(5'-GTCGACTTAATGGTGATGGTGATGATGCACTGACAATTTCATCCCGAACGTCTC-3') and high fidelity thermostable DNA polymerase were used to amplify a DNA fragment from pGEM-hLC3 by PCR; the fragment was introduced into the pGEM-T vector; and the resulting plasmid was designated pGEM-hLC3-His6. The open reading frame of MAP1LC3B-His6 was excised from pGEM-hLC3-His6 and introduced into pCMV-Tag3B, and the resulting plasmid was designated pMyc-hLC3WT-His6. For expression of mutants Myc-MAP1LC3BG120A-His, Myc-MAP1LC3BK122R-His, and Myc-MAP1LC3BK122A-His (see Fig. 1D), the respective site-directed mutations were introduced into pMyc-hLC3WT-His6, and the resulting plasmids were designated pMyc-hLC3G120A-His6, pMyc-hLC3K122R-His6, and pMyc-hLC3K122A-His6, respectively.
Plasmid pTRX-MAP1LC3B-Myc, used for expression in E. coli of thioredoxin (TRX)-MAP1LC3B-Myc protein (see Fig. 2A), in which wild-type MAP1LC3B was fused to TRX-His6 at its amino terminus and to the Myc epitope at its carboxyl terminus, has been described (12). For expression of mutants TRX-MAP1LC3BG120A-Myc (see Fig. 2A) and TRX-MAP1LC3B
G-Myc, in which Gly120 was deleted (Fig. 2A), site-directed mutations were introduced into pTRX-MAP1LC3B-Myc, and the resulting plasmids were designated pTRX-MAP1LC3BG120A-Myc and pTRX-MAP1LC3BG-Myc, respectively.
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promoter (see Fig. 3), the open reading frame of MAP1LC3B was introduced into plasmid pEF4/Myc-His, and the resulting plasmid was designated pEF-hLC3. For expression of mutants MAP1LC3B-TF, MAP1LC3B-TFG, MAP1LC3BG120A, and MAP1LC3BK122A (see Fig. 3A), site-directed mutations were introduced into pEF-hLC3, and the resulting plasmids were designated pEF-hLC3TF, pEF-hLC3TFG, pEF-hLC3G120A, and pEF-hLC3K122A, respectively.
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Cell Culture and Transfection—HEK293 and HeLa cells were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. 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—Anti-MAP1LC3B (hLC3) antibody was prepared as described (12); this antibody shows little cross-reactivity with human GABARAP and GATE-16. Anti-Myc antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA); anti-FLAG antibody M2 was from Sigma; anti-His antibody was from Molecular & Biological Laboratories Co. Ltd. (Nagoya, Japan); and anti-TRX antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).
Other Techniques—Our in vitro assay for cleavage of the carboxyl terminus of TRX-MAP1LC3B-Myc has been described (12).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To minimize any artificial effect of an amino-terminal tag on the LC3 modification, we first investigated carboxyl-terminal cleavage of MAP1LC3B using a series of mutant MAP1LC3B-3xFLAG proteins, in which MAP1LC3B was fused to three repeats of the FLAG epitope at its carboxyl terminus (Fig. 1B). MAP1LC3B-3xFLAG was expressed in HEK293 cells; total cell lysate proteins were separated on 12.5% SDS-polyacrylamide gel; and MAP1LC3B in the lysate was assayed by immunoblotting with anti-MAP1LC3B antibody. Two bands corresponding to MAP1LC3B and MAP1LC3B-PL were recognized by anti-MAP1LC3B antibody (Fig. 1C, left panel, WT), but not by anti-FLAG antibody (right panel, WT). Expression of mutant MAP1LC3BG120A-3xFLAG, in which Gly120 of MAP1LC3B was changed to Ala, resulted in a single band (of slower mobility compared with wild-type MAP1LC3B) that was recognized by immunoblotting with both anti-MAP1LC3B and anti-FLAG antibodies (Fig. 1C, G120A).
We next investigated the effect of MAP1LC3B Lys122 on the cleavage reaction. Expression of MAP1LC3BK122A-3xFLAG, in which Lys122 of MAP1LC3B was changed to Ala, resulted in two bands corresponding to MAP1LC3B and MAP1LC3B-PL, both of which were recognized by immunoblotting with anti-MAP1LC3B antibody, but not with anti-FLAG antibody (Fig. 1C, K122A versus WT), indicating that Lys122 has no effect on the carboxyl-terminal cleavage of MAP1LC3B. We obtained similar results for MAP1LC3BG120A and MAP1LC3BK122A fused to the GFP protein at the carboxyl terminus and expressed in HeLa and HEK293 cells (data not shown). These results were consistent with our hypothesis derived from biochemical evidence (9, 10, 12, 15).
To clarify in a straightforward manner whether the carboxyl terminus of Myc-MAP1LC3B-His is cleaved in vivo, we constructed a series of mutant Myc-MAP1LC3B-His proteins (Fig. 1D). When Myc-MAP1LC3B-His protein was expressed in HEK293 cells, two bands corresponding to the cytosolic and lipidated forms of Myc-MAP1LC3B were recognized by immunoblotting with anti-Myc antibody, but not with anti-His antibody (Fig. 1E, WT). Similar results were obtained when Myc-MAP1LC3BK122A-His and Myc-MAP1LC3BK122R-His were expressed in HEK293 cells (Fig. 1E, K122R and K122A). Expression of mutant Myc-MAP1LC3BG120A-His resulted in a single band (of faster mobility compared with wild-type Myc-MAP1LC3B-His on SDS-polyacrylamide gel) that was recognized by both anti-Myc and anti-His antibodies (Fig. 1E, G120A). Mutation of Gly120 to Ala showed that His6 at the carboxyl terminus affects the mobility of non-cleaved mutant Myc-MAP1LC3BG120A-His on SDS-polyacrylamide gel. These results indicate that cleavage of the carboxyl terminus of MAP1LC3B in HEK293 cells requires Gly120, but not Lys122.
Gly120 Is Essential for the Carboxyl-terminal Cleavage of MAP1LC3B by hAtg4B in Vitro—We further investigated the significance of Gly120 for carboxyl-terminal cleavage using an in vitro assay for cleavage of MAP1LC3B by hAtg4B (Fig. 2). We prepared three recombinant proteins: TRX-MAP1LC3B-Myc (in which MAP1LC3B was fused to TRX-His6 at its amino terminus and to the Myc epitope at its carboxyl terminus), TRX-MAP1LC3BG120A-Myc, and TRX-MAP1LC3BG-Myc (in which Gly120 was deleted) (Fig. 2A). When wild-type TRX-MAP1LC3B-Myc was incubated with FLAG-hAtg4B at 37 °C for 30 and 60 min, a band corresponding to the protein, which was recognized by immunoblotting with anti-TRX antibody, had a faster mobility on SDS-polyacrylamide gel than before incubation (Fig. 2B, lanes 1–3), and this faster band was not recognized by anti-Myc antibody. When TRX-MAP1LC3BG120A-Myc and TRX-MAP1LC3BG-Myc were used as substrates for FLAG-hAtg4B, they were recognized by both anti-TRX and anti-Myc antibodies, even after incubation for 60 min (Fig. 2B, lanes 4–9), and both showed little shift in mobility. In each reaction mixture, FLAG-hAtg4B was recognized after incubation for 60 min (Fig. 2B, lanes 10–15). These results indicate that Gly120 of MAP1LC3B is essential for its carboxyl-terminal cleavage by FLAG-hAtg4B in vitro.
Gly120, but Not Lys122, of MAP1LC3B Is Essential for Formation of E1- and E2-Substrate Intermediates—We previously showed that when the mammalian Atg7C567S active-site mutant is expressed together with GFP-MAP1LC3B, the two proteins form a stable E1-substrate intermediate (15). In addition, mutant hAtg7C572S (in which the active-site Cys572 for the E1-like reaction was changed to Ser) forms stable intermediates with the other Atg8 orthologs, human GATE-16 and GABARAP, with Gly116 in the latter two proteins being essential for the E1 reaction (11). It was not clear, however, whether Gly120 (or Lys122) of MAP1LC3B is essential for the formation of the hAtg7C572S-MAP1LC3B (E1-substrate) intermediate. To avoid any effect of an amino-terminal tag on MAP1LC3B in its E1 and E2 reactions with hAtg7 and hAtg3, we constructed a series of mutant MAP1LC3B proteins without any tag (Fig. 3A) and expressed these proteins together with hAtg7C572S in HEK293 cells (Fig. 3B, lanes 6–10). Total cell lysate proteins were separated on 7% SDS-polyacrylamide gel, and MAP1LC3B and hAtg7 were recognized by immunoblotting with their respective antibodies. When wild-type MAP1LC3B and hAtg7C572S were expressed, both the hAtg7C572S-MAP1LC3B (E1-substrate) intermediate and the free proteins were recognized (Fig. 3B, lane 6 versus lane 1). E1-substrate intermediates were also observed when hAtg7C572S was expressed together with MAP1LC3B-TFG, in which the five carboxyl-terminal amino acids (MKLSV) were deleted to expose Gly120, or MAP1LC3BK122A (Fig. 3B, lanes 8 and 10). When MAP1LC3B, MAP1LC3B-TFG, or MAP1LC3BK122A alone was expressed, however, little intermediate was observed (Fig. 3B, lanes 1, 3, and 5). Similarly, there was little intermediate when hAtg7C572S was expressed together with MAP1LC3B-TF, in which the six carboxyl-terminal amino acids (GMKLSV) were deleted (Fig. 3B, lane 7 versus lane 8), or with MAP1LC3BG120A (lane 9). Similar results were obtained when a series of mutant FLAG-MAP1LC3B and GFP-MAP1LC3B proteins were expressed instead of MAP1LC3B (data not shown). These findings indicate that MAP1LC3B can react with hAtg7 after carboxyl-terminal cleavage of the latter and that exposure of Gly120 is essential for the E1-like reaction mediated by hAtg7, whereas Lys122 contributes little to it.
We previously showed that MAP1LC3B forms an E2-substrate intermediate with hAtg3C264S, in which the active-site Cys264 was changed to Ser, in the presence of wild-type hAtg7 (10). It was unclear, however, whether Gly120 (or Lys122) of MAP1LC3B is essential for the formation of the hAtg3C264S-MAP1LC3B (E2-substrate) intermediate. To determine whether Gly120 is essential for formation of this intermediate, we expressed a series of mutant MAP1LC3B proteins together with wild-type hAtg7 and mutant GFP-hAtg3C264S (Fig. 3B, lanes 11–15). After separation of total cell lysate proteins on 7% SDS-polyacrylamide gel, MAP1LC3B, hAtg7, and GFP-hAtg3 were recognized by immunoblotting using specific antibodies. When MAP1LC3B-TFG or MAP1LC3BK122A was expressed together with hAtg7 and GFP-hAtg3C264S, GFP-hAtg3C264S-MAP1LC3B (E2-substrate) intermediates were observed by immunoblotting, similar to the results observed with wild-type MAP1LC3B (Fig. 3B, upper and lower panels, lanes 11, 13, and 15). In contrast, when MAP1LC3B-TF or MAP1LC3BG120A was expressed together with hAtg7 and GFP-hAtg3C264S, little E2-substrate intermediate was detected by immunoblotting (Fig. 3B, lanes 12 and 14). These results indicate that MAP1LC3B can react with hAtg3 and that the exposure of Gly120 is essential for this E2 reaction, whereas Lys122 contributes little to it.
In contrast to a report that the carboxyl terminus of Myc-MAP1LC3B-His is not cleaved (13), we found that the carboxyl terminus of wild-type Myc-MAP1LC3B-His was cleaved in vivo (Fig. 1E). If the Gly120 of Myc-MAP1LC3B-His is exposed after carboxyl-terminal cleavage, Myc-MAP1LC3B-His will form E1- and E2-substrate intermediates with hAtg7C572S and hAtg3C264S, respectively. We therefore investigated whether Myc-MAP1LC3B-His can react with hAtg7C572S and hAtg3C264S. As shown for untagged MAP1LC3B (Fig. 3B), Myc-MAP1LC3B-His formed these intermediates, whereas Lys122 was not essential for this reaction (Fig. 3C).
Overexpression of MAP1LC3B Increases the MAP1LC3B-PL Conjugate, and the Conjugate Fractionates into the Pellet—We have shown that, in HeLa cells, endogenous MAP1LC3B-PL is a lipidated form of endogenous MAP1LC3B (12), and the lipidated protein fractionated into the 100,000 x g pellet (Fig. 4A) (11, 12). If recombinant MAP1LC3B is lipidated by a ubiquitylation-like reaction after its carboxyl-terminal cleavage, overexpression of MAP1LC3B would result in increases in the lipidated and cytosolic forms of MAP1LC3B. We therefore investigated whether the mobility of recombinant MAP1LC3B-PL is similar to that of endogenous MAP1LC3B-PL on SDS-polyacrylamide gel. When we expressed untagged MAP1LC3B under the control of the elongation factor 1 promoter in HeLa cells, recombinant and endogenous proteins showed similar mobility (Fig. 4B, lanes 1 and 3 versus lanes 2 and 4). After subcellular fractionation by centrifugation at 100,000 x g for 1 h, a band corresponding to MAP1LC3B-PL was observed in the pellet (Fig. 4B, lanes 6 and 7), suggesting that recombinant MAP1LC3B-PL is a lipidated form, similar to the endogenous protein, and that this band does not represent a degradation product of MAP1LC3B, findings in agreement with our previous observations (10–12, 16, 17). Similar results were observed when MAP1LC3B-TFG and MAP1LC3BK122A were expressed in HeLa cells (data not shown).
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We have also shown that GFP-MAP1LC3B, in which MAP1LC3B was fused to GFP at its amino terminus, forms E1- and E2-substrate intermediates with mammalian Atg7 and Atg3, respectively (10, 16). However, it is unclear whether GFP-MAP1LC3B would be recognized as two bands (cytosolic GFP-MAP1LC3B and membrane-bound GFP-MAP1LC3B-PL) on SDS-polyacrylamide gel or whether GFP-MAP1LC3B-PL is fractionated into the pellet. When GFP-MAP1LC3B was expressed in HeLa cells and probed with anti-GFP antibody, two bands were observed (Fig. 4D). These bands were of closer mobility than the two bands observed with FLAG-MAP1LC3B because the GFP tag is larger than the FLAG tag. Subcellular fractionation indicated that GFP-MAP1LC3B-PL was in the pellet, along with the lipidated forms of the endogenous and other recombinant proteins (Fig. 4D, Sup and Ppt). These results indicate that, like the endogenous protein, recombinant MAP1LC3B is lipidated in vivo.
RNA Interference of hLC3/MAP1LC3B Inhibits Expression of Endogenous LC3 in HEK293 Cells—Endogenous MAP1LC3B and MAP1LC3B-PL were recognized by immunoblotting with anti-MAP1LC3B antibody. However, this antibody may show significant cross-reaction with MAP1LC3A or MAP1LC3C. To determine whether this is the case, we used a vector expressing MAP1LC3B-specific short hairpin RNA under the control of a human H1 promoter (pSi-hLC3) to interfere with MAP1LC3B mRNA. In the presence of this hairpin, bands recognized by anti-MAP1LC3B antibody and corresponding to endogenous MAP1LC3B and MAP1LC3B-PL will be simultaneously decreased. Furthermore, the employed hairpin sequence shows little identity to the DNA sequences of MAP1LC3A and MAP1LC3C, and a BLAST search found no other identical sequences. When we transfected HEK293 cells with pSi-hLC3, cultured the cells for 48 h, and immunoblotted the total cell lysates with anti-MAP1LC3B antibody, we found that the quantities of both endogenous MAP1LC3B and MAP1LC3B-PL were significantly decreased compared with control cells (Fig. 5). Since RNA interference specifically inhibits mRNA containing 100% identical sequence, these results indicate that endogenous MAP1LC3B-PL and MAP1LC3B are derived from authentic hLC3/MAP1LC3B mRNA and not from MAP1LC3A or MAP1LC3C.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Following cleavage, both the cytosolic and lipidated forms of Myc-MAP1LC3B-His were recognized as two bands by immunoblotting with anti-Myc antibody, but not with anti-His antibody. Both Myc-MAP1LC3B-His and Myc-MAP1LC3BK122A-His formed E1- and E2-substrate intermediates with hAtg7C572S and hAtg3C264S, respectively, as previously shown for untagged MAP1LC3B, FLAG-MAP1LC3B, and GFP-MAP1LC3B (Fig. 3 and Ref. 10). RNA interference of MAP1LC3B mRNA showed that anti-MAP1LC3B antibody recognized endogenous MAP1LC3B, but not MAP1LC3A and MAP1LC3C. In addition, this antibody recognized endogenous MAP1LC3B as two bands corresponding to the cytosolic and membrane-bound forms and recognized the endogenous lipidated form of MAP1LC3B in 19 other human cell lines.2 These results demonstrate that the carboxyl terminus of Myc-MAP1LC3B-His is cleaved to expose Gly120 for further lipidation in a manner similar to that observed with endogenous MAP1LC3B. Considering the biochemical properties of MAP1LC3B, hAtg4B (a cysteine protease that cleaves MAP1LC3B at its carboxyl terminus, exposing Gly120), hAtg7 (an E1-like enzyme for MAP1LC3B), and hAtg3 (an E2-like enzyme for MAP1LC3B) shown here and previously (9–12, 15–18), it is reasonable that the carboxyl-terminal cleavage of recombinant MAP1LC3B, even Myc-MAP1LC3B-His, occurs prior to ubiquitylation-like modification in a manner similar to that shown for endogenous MAP1LC3B.
Mutants MAP1LC3B-TFG and MAP1LC3BK122A and wild-type MAP1LC3B can form E1- and E2-substrate intermediates with hAtg7C572S and hAtg3C264S, respectively, whereas mutant MAP1LC3B-TF cannot, suggesting that MAP1LC3B is cleaved at Met121 to expose Gly120. We obtained similar results using a series of Myc-MAP1LC3B-His proteins. Since Gly120 is exposed after carboxyl-terminal cleavage, it is not likely that Lys122 contributes to the E1 and E2 reactions because, in vivo, little of the remaining cytosolic MAP1LC3B would contain carboxyl-terminal residues 121–125 (MKLSV). When wild-type MAP1LC3B was overexpressed in HeLa cells, the mobility of recombinant MAP1LC3B and MAP1LC3B-PL was similar to that of the endogenous proteins, and recombinant MAP1LC3B-PL proteins were fractionated into the pellet. These findings indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 and that this residue, and not Lys122, is essential for further ubiquitylation-like modifications mediated by hAtg7 and hAtg3. Finally, MAP1LC3B is modified to MAP1LC3B-PL, i.e. hLC3/MAP1LC3B is an authentic modifier, as is rat LC3. Therefore, even if Lys122 of MAP1LC3B is modified prior to carboxyl-terminal cleavage, it could not contribute to the modifier function of MAP1LC3B or to its activity as a promising autophagosomal marker.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Biochemistry, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: 81-3-5802-1031; Fax: 81-3-5802-5889; E-mail: kominami{at}med.juntendo.ac.jp.
1 The abbreviations used are: LC3, light chain 3; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; h, human; PL, phospholipid; GABARAP, -aminobutyric acid type A receptor-associated protein; GFP, green fluorescent protein; TRX, thioredoxin.
2 I. Tanida, T. Ueno, and E. Kominami, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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