The human homolog of Saccharomyces cerevisiae Apg7p is a Protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3.

Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system. In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by vacuolar hydrolases. Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions. One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy. Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog. In this study, the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys(572) of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p.hApg12p intermediate. Overexpression of hApg7p enhances the formation of the hApg5p.hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system. Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p. Each of three human Apg8p counterparts, i.e. the Golgi-associated ATPase enhancer of 16 kDa, GABA(A) receptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7p(C572S) to form a stable intermediate via an ester bond. These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs.

Post-translational modifications regulate the functions and localization of target proteins, resulting in many significant intracellular events. One unique modification is the covalent attachment of modifier proteins, ubiquitin, ubiquitin-related proteins (SUMO-1/Smt3p and NEDD-8/RUB1), and Apg12p (for reviews, see Refs. [1][2][3][4][5][6]. The enzymatic processes of these modifications have been intensively studied in ubiquitylation. Ubiquitin forms conjugates with a target protein via a three step mechanism. First, ubiquitin is activated at its C-terminal Gly by the ubiquitin-activating enzyme (UBA1, E1 enzyme) to form a conjugate with the active site Cys in the E1 enzyme via a thiol ester bond. Next, ubiquitin is transferred from the E1 enzyme to one of several ubiquitin-conjugating enzymes (UBCs, E2 enzymes). In the last step, ubiquitin is attached to a Lys within the target protein via an isopeptide bond. This step is often catalyzed by a member of the ubiquitin-protein ligase family, an E3 enzyme. The reaction mechanism is basically common for each modifier protein.
Autophagy is a process of bulk degradation of cytoplasmic components by the lysosomal/vacuolar system (5,7,8). In the initial step of macroautophagy, a cup-shaped membrane sac surrounds cytosolic components to form an autophagosome (9). The outer membrane of the autophagosome fuses with a lysosome/vacuole (10). Taking advantage of yeast genetics, apg 1 and aut mutants were isolated as autophagy-defective mutants in the yeast, Saccharomyces cerevisiae (12,13). Surprisingly, most of the apg mutants overlap genetically with cvt mutants, which have a defect in the cytoplasm-to-vacuole targeting of aminopeptidase I, indicating that these genes function in a unique transport system under vegetative growth conditions in addition to starvation conditions (14 -16). A novel modifier protein, Apg12p, was discovered as an APG gene product (11). Apg12p shows little homology to ubiquitin, but it is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation. In this conjugation reaction, Apg7p and Apg10p function as E1-and E2-like enzymes for Apg12p, respectively (11,(17)(18)(19). After the formation of the Apg12p⅐ Apg5p conjugate, Apg16p attaches to Apg5p forming an Apg12p⅐Apg5p⅐Apg16p complex for autophagy (20). Unlike other modifier-conjugation systems, the unique character of the Apg12p-conjugation system is that it plays indispensable roles in the formation of membrane structures, including autophagosomes and Cvt-vesicles.
Apg7p, an authentic E1-like enzyme essential for Apg12p, plays an indispensable role in the initial step of the conjugation system, whereas the enzyme shows slight homology to other E1 enzymes (18). Apg7p interacts with Apg8p/Aut7p and Aut1p/ Apg3p in addition to Apg12p (21,22). 2 The dimerization of Apg7p via the C-terminal region is essential for these interactions, suggesting that Apg7p forms multimeric complexes with these proteins. 2 Apg8p/Aut7p is localized on autophagosomes and Cvt-vesicles (23). The AUT1/APG3 gene is a multicopy suppressor of the apg8/aut7 mutant (24). Apg8p/Aut7p also interacts with two ER-to-Golgi v-SNAREs (Bet1p and Sec22p) and vacuolar t-and v-SNAREs (Vam3p and Nyv1p, Ref. 25). Furthermore, more recent findings suggest that Apg8p/Aut7p, Aut1p/Apg3p, and Apg7p comprise a second protein-conjugation system indispensable for autophagy and Cvt pathways (22,26). The second modifier is Apg8p/Aut7p, and Apg7p and Aut1p/Apg3p are corresponding E1-and E2-like enzymes. These results suggest that Apg7p, because it is involved in two distinct conjugation systems, is a key enzyme for membrane formation and the targeting of autophagosomes and Cvt vesicles.
In mammalian cells, several homologs of yeast APG gene products have been reported. hApg12p conjugates with hApg5p (first identified as an apoptosis-specific protein), suggesting that the Apg12p conjugation system exists even in human cells (27,28). There are three candidates for mammalian Apg8p/ Aut7p homologs, GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), GABARAP (GABA receptor-associated protein), and MAP-LC3 (microtubule-associated protein light chain 3) (25, 29 -33). GATE-16 was first identified as a ganglioside expression factor, but was recently characterized as a soluble transport factor. GATE-16 interacts with NSF and the Golgi v-SNARE GOS-28 (33). The mRNA of GATE-16 is expressed ubiquitously but at significantly higher levels in brain tissue. The interaction of yeast Apg8p/Aut7p with two ER-to-Golgi v-SNAREs was proven as a functional GATE-16 homolog (25). GABARAP interacts with GABA A receptors, cytoskeleton, and gephyrin, suggesting functional importance in brain or neuronal cells (31,34). MAP-LC3 copolymerizes with tubulin and is a component of the MAP-1 complex, which is composed of light chains 1, 2, and 3 and heavy chains (29,30). Rat MAP-LC3 is localized on autophagosomal membranes, suggesting that rat MAP-LC3 is also a functional Apg8p/Aut7p homolog (35). These results suggest that mammalian Apg8p/Aut7p homologs have divergent functions in mammalian cells, especially in neuronal cells. For Apg7p, there are several clones in the EST database, and the sequence of hApg7p has been determined to be homologous to Pichia pastoris GSA7, which is essential for microautophagy (36). However, as yet, there is no biochemical evidence that hApg7p is an E1-like enzyme for hApg12p. There is a further question; which of MAP-LC3, GATE-16, and GABARAP is an authentic substrate for hApg7p? In this report, we show that hApg7p conjugates with hApg12p and all three Apg8p/Aut7p homologs as hApg7p substrate intermediates in mammalian cells.

Animals-Male
Wistar rats (250 -300 g) were maintained in an environmentally controlled room (lights on from 7:00 AM to 20:00 PM) for at least 2 weeks before experiments. All rats were fed a standard pellet laboratory diet and tap water ad labitum during this period.
Strains, Media, Materials, and Molecular Biological Techniques-Escherichia coli strain DH5␣ cells, the host for plasmids and protein expression, were grown in Luria Broth medium in the presence of antibiotics as required (37). Standard genetic and molecular biological techniques were performed as described (37,38). The yeast strain for two hybrid experiments was cultured in SD medium (0.67% yeast nitrogen base without amino acids, 2% glucose, and appropriate amino acids) as described previously (38). Protein was determined by BCA protein assay following the manufacturer's protocol (Pierce, Rockford, IL). The polymerase chain reaction was performed with a programmed temperature control system PC-701 (ASTEC, Fukuoka, Japan). The DNA sequence was determined with an ABI 373A DNA sequencer (PE Biosystems, Foster City, CA). DNA plasmid was transfected into mammalian cells with FuGene-6 transfection reagent according to the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany). Restriction enzymes were purchased from TOYOBO (Osaka, Japan) and New England BioLabs (Beverly, MA). Oligonucleotides were synthesized by the ESPEC oligo-service (Ibaraki, Japan). pGAD-C1 vector, pGBD-C1 vector, and PJ69 -4A strain were kind gifts from P. James (39). pcDNA3 was purchased from Invitrogen (Carlsbad, CA), pGEM-T was from PROMEGA (Madison, WI), pEGFP-C1 and pEGFP-N1 were from CLONTECH, and pBluescriptII (SKϩ) was from Stratagene (La Jolla, CA).
Plasmid Construction and Site-directed Mutagenesis-Based on the DNA sequence of the human APG7/GSA7 homolog (Genbank TM /EBI accession number AF094516), we cloned an open-reading frame of the human GSA7/APG7 cDNA by polymerase chain reaction with high fidelity (36,40), introduced the amplified DNA fragment into the SalI-NotI site of pBluescriptII (SKϩ), and designated the resultant plasmid for pSKhAPG7 plasmid. To construct an expression plasmid as hApg7p, a KpnI-NotI fragment (ϳ2.5 kilobase pairs) of the pSKhAPG7 plasmid was introduced into the pEGFP-N1 vector (CLONTECH) and designated pCMV-hAPG7.
To obtain a DNA fragment containing an open-reading frame of the human APG12 homolog, GATE-16, and GABARAP, polymerase chain reaction was performed with specific primers for their open-reading frames with high fidelity using a human brain cDNA library as a template, and the amplified fragment was introduced into pGEM-T vector (pGEM-hAPG12, pGEM-hGATE-16, and pGEM-hGABARAP). The isolated DNA fragments were introduced into pEGFP-C1 to express GFP fusion proteins (pGFP⅐hApg12p, pGFP⅐hGATE-16, and pGFP⅐hGABARAP).
Cys 572 within hApg7p was replaced by Ser, mutagenized by the Gene-Editor in vitro site-directed mutagenesis system (PROMEGA) with an oligonucleotide (hAPG7CS; 5Ј-CGGACCTTGGACCAGCAGAG-CACTGTGAGTCGTCCAGG-3Ј) according to the manufacturer's protocol. The expression plasmid for mutant Apg7p C572S was constructed as in the case of pCMV-hAPG7 and was designated pCMV-hAPG7C572S.
Antibodies-A polyclonal antibody against a synthetic polypeptide (VVAPGDSTRDRTL) corresponding to residues 550 -571 of hApg7p was raised in Japanese white rabbits (anti-hApg7p). The antibody was affinity-purified by chromatography on immobilized hApg7p-peptide-Sepharose. For the preparation of antibody against murine Apg12p homolog (mApg12p), rabbits were immunized with a maltose-binding protein⅐mApg12p fusion protein. The antibody to mApg12p was purified by affinity chromatography on maltose-binding protein⅐mApg12p-immobilized-Sepharose. The polyclonal anti-GFP antibody was purchased from CLONTECH.
Cloning of hMAP-LC3 cDNA-The advanced BLAST search program from the National Center for Biotechnology Information was used to search for homologs in the human and mouse EST database. Based on the DNA sequence of EST clones, we performed rapid amplification of the 5Ј-cDNA ends in Marathon-Ready cDNA (CLONTECH) by polymerase chain reaction with high fidelity (40). The amplified DNA fragment was introduced into pGEM-T vector, and the DNA sequence was determined. The DNA sequences of all five independent clones were identical, and the predicted amino acids of the clones show significant homology to rat and murine MAP-LC3. To express GFP⅐hMAP-LC3 in HEK293 cells, the cloned DNA fragment was introduced into pEGFP-C1 vector (pGFP⅐hMAP-LC3).
Expression of hApg Proteins in HEK293 Cells-HEK293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. For transfection, 2 ϫ 10 5 cells were seeded on 60-mm dishes. After incubation for 24 h at 37°C, the cells were transfected with a mixture of 2.5 g of plasmid DNA and 12 l of FuGene-6. For cotransfection, 1 g of each plasmid was used. The transfectant was harvested after incubation for an additional 48 h. 1 ϫ 10 6 cells were washed with 1 ml of phosphate-buffered saline, and resuspended in 200 l of phosphate-buffered saline containing Complete protease inhibitor mixture (Roche Diagnostics). The cell suspension was lysed by sonication for 10 s at 4°C. Proteins in the lysate were separated by reducing or nonreducing SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Immunoblot analysis was performed with anti-hApg7p and -GFP antibodies (CLONTECH), and the blots were developed by an enhanced chemiluminescence system (Amersham Pharmacia Biotech).
Glycerol Gradient Centrifugation-Livers were isolated from Wistar male rats, passed through a stainless steel mesh, and suspended in 5 volumes of 5 mM Tes-NaOH, pH 7.5, 0.3 M sucrose. The homogenate was centrifuged at 100,000 ϫ g for 1 h, and the supernatant was used as the cytosol fraction. Cytosol (0.4 ml) was loaded onto an 11.5-ml linear glycerol gradient (10 -40%) in 20 mM Tes-NaOH, pH 7.5, 0.15 M NaCl and centrifuged at 151,000 ϫ g for 15 h (Beckman SW-41 rotor). Fractions of 0.7 ml were collected from the bottom of the tubes. Rat Apg7p was immunoprecipitated from each fraction with anti-hApg7p and subjected to immunoblotting analysis, because hApg7p cross-reacts with rat Apg7p. Authentic thyroglobulin (670 kDa, 19 S), catalase (220 kDa, 11.2 S), aldolase (158 kDa, 7.4 S), and bovine serum albumin (67 kDa, 4.3 S) were used as internal S-value standards. Two-hybrid analysis was performed as described by James et al. (39).
To investigate whether hApg7p forms an enzyme-hApg12p intermediate, we employed site-directed mutagenesis of a predicted active site cysteine residue within hApg7p. Based on a homology search between yeast and human Apg7p, we predicted that the active site cysteine residue within hApg7p must be Cys 572 (Fig. 2A). If an active site cysteine residue within an E1-enzyme is changed to serine, an O-ester bond will be formed instead of a thiol ester bond. Therefore, we changed The cells were lysed, and hApg12p was immunoprecipitated with anti-mouse Apg12p antibody. The precipitate was analyzed by SDS-PAGE and subjected to autoradiography. Both pGFP⅐hAPG12 and one of pCMV-hAPG7 (pCMV-hAPG7) or vector control was transfected into HEK293 cells. When wild-type hApg7p was expressed in HEK293 cells, an hApg5p⅐GFP⅐hApg12p conjugate was recognized (hApg5p-GFPhApg12p conjugate) in addition to GFP⅐hApg12p (GFP-hApg12p).
Cys 572 within hApg7p to Ser by site-directed mutagenesis and expressed both the mutant hApg7p C572S and GFP-fused hApg12p (GFP⅐hApg12p) in HEK293 cells (Fig. 2B). Cell lysates expressing both proteins were prepared and analyzed by SDS-PAGE. hApg7p was recognized by immunoblot with anti-hApg7p antibody. Wild-type hApg7p and mutant hApg7p C572S were both expressed well in HEK293 cells (Fig. 2B, wild and C572S, hApg7p of ϳ80 kDa). When both hApg7p C572S and GFP⅐hApg12p were expressed in HEK293 cells, a higher molecular mass band consistent with a stable GFP⅐hApg12p⅐hApg7p C572S intermediate (ϳ140 kDa) appeared in addition to the band of ϳ80 kDa for free hApg7p (Fig. 2B, C572S). This higher molecular mass band was also recognized by immunoblotting with anti-GFP antibody in the presence or absence of reducing reagent (data not shown). These results indicate that hApg12p is an authentic substrate for hApg7p.
If hApg7p is an E1-like enzyme in the hApg12p-conjugation system, it is possible that the overexpression of hApg7p will influence the conjugation of hApg12p with hApg5p. To investigate this possibility, we expressed both hApg7p and GFP⅐hApg12p in HEK293 cells, metabolically labeled the cells with 35 S-labeled Met and Cys and prepared a cell lysate. hApg12p was immunoprecipitated with anti-mApg12p antibody, and the precipitates were analyzed by SDS-PAGE and autoradiography. When GFP⅐hApg12p alone was expressed, GFP⅐hAPG12p itself was immunoprecipitated with anti-mApg12p antibody (Fig. 2C, vector). In cells expressing both GFP⅐hApg12p and hApg7p, a high molecular weight peptide corresponding to the hApg5p⅐GFP⅐hApg12p conjugate was immunoprecipitated in addition to GFP⅐hApg12p with anti-mApg12p antibody (Fig. 2C, pCMV-hAPG7). The formation of the hApg5p⅐GFP⅐hApg12p conjugate was further confirmed by a second immunoprecipitation using anti-hApg5p antibody (data not shown). The overexpression of mutant hApg7p C572S did not enhance the conjugation (data not shown). These results indicate that hApg7p is an authentic protein-activating enzyme essential for the human Apg12pconjugation system.
hApg7p Forms a Homodimer-Komatsu et al. 2 have found that yeast Apg7p forms a homodimer via the C-terminal region. Considering the functional homology between yeast and human Apg7p, it is likely that hApg7p will also form a homodimer. To investigate this possibility, we conducted a cross-linking experiment. A HEK293 cell lysate expressing hApg7p was prepared and treated with a noncleavable cross-linker, disuccinimidyl suberate. After cross-linking, the lysate was analyzed by SDS-PAGE, and hApg7p was detected by immunoblotting with anti-hApg7p antibody. Before treatment with the cross-linking reagent, hApg7p was detected in the cell lysate as a band corresponding to ϳ80 kDa (Fig. 3A, pCMV-hAPG7, DSSϪ). After cross-linking, the amount of this 80-kDa band was decreased, and a broad band at ϳ160 kDa appeared (Fig. 3A, pCMV-hAPG7, DSSϩ).
We next analyzed endogenous Apg7p in rat liver cytosol by glycerol density gradient ultracentrifugation using the cross-reactivity of the anti-hApg7p antibody with rat Apg7p. The cytosolic fraction of a rat liver homogenate was prepared and subjected to a 10 -40% glycerol density gradient centrifugation. Rat Apg7p was immunoprecipitated with anti-hApg7p antibody. The resulting precipitates were analyzed by SDS-PAGE, and rat Apg7p was identified by immunoblotting with anti-hApg7p antibody. Rat Apg7p was collected in fractions 11-14 and sedimented mainly with a sedimentation coefficient of ϳ7.4 S (Fig. 3B,  fraction 13). A two-hybrid experiment also indicated that hApg7p interacts with itself (data not shown). Therefore we conclude that mammalian Apg7p forms a homodimer similar to yeast Apg7p.
All Three hApg8p Proteins (MAP-LC3, GATE-16, and GABARAP), Are Substrates of hApg7p-Recent findings have indicated that yeast Apg7p also functions as an activating enzyme for Apg8p and is essential for Apg8p targeting to autophagosomal membranes (22,26). 2 A BLAST search suggested that there are at least three Apg8p homologs, GATE-16 (human), GABARAP (human, mouse, and rat), and MAP-LC3 (rat) in mammalian cells. A BLAST search of the human EST database suggested that there are human MAP-LC3 homologs (Genbank TM /EBI accession numbers AI365977, AA476809, and AI382200). hMAP-LC3 was isolated from a human brain cDNA library by rapid amplification of the 5Ј-cDNA ends according to the information obtained from the BLAST search. The amino acid sequence of hMAP-LC3 shows 95.9% identity with its rat counterpart. The C-terminal regions of the three hApg8p proteins show significant homology to yeast Apg8p. Considering the significant homology between human and yeast Apg8p, these proteins may also be substrates for Apg7p.
To investigate whether these Apg8p homologs interact with hApg7p as substrates, we first performed a coimmunoprecipitation experiment. hMAP-LC3, hGATE-16, and hGABARAP were expressed as GFP fusion proteins together with hApg7p in COS7 cells. Cell lysates expressing both hApg7p and a GFP fusion protein were prepared. The GFP fusion proteins were immunoprecipitated using anti-GFP antibody. The precipitates were analyzed by SDS-PAGE, and hApg7p was recognized by immunoblotting using anti-hApg7p antibody. hApg7p coimmunoprecipitated with GFPhGATE-16, GFPhGABARAP, and GFPhMAP-LC3 but not with GFP alone (Fig. 4A). The results indicate that hGATE-16, hGABARAP, and hMAP-LC3 interact with hApg7p.
We next examined the formation of stable conjugates of mutant hApg7p C572S with hGATE-16, hGABARAP, and hMAP-LC3 via an Oester bond. hApg7p C572S was coexpressed with GFPhGATE-16, GFPh-GABARAP, or GFPhMAP-LC3 in COS7 cells, and the cell lysates were analyzed by SDS-PAGE under reducing conditions. The GFP⅐hApg8p homologs were recognized by immunoblotting with anti-GFP antibody. A high molecular weight band corresponding to a stable hApg7p C572S substrate intermediate was detected in cell lysates expressing both hApg7p C572S and a GFP⅐Apg8p homolog, but not in cells expressing both wild-type hApg7p and GFP⅐Apg8p homologs, indicating that a stable conjugate is formed between hApg7p C572S and the Apg8p homologs (Fig.  4B). These results indicate that all three hApg8p proteins, hGATE-16, hGABARAP, and hMAP-LC3, are authentic substrates for hApg7p.

DISCUSSION
In this study, we showed that the human Apg7p homolog is an authentic E1-like enzyme for the hApg12p conjugation system and that hGATE-16, hGABARAP, and hMAP-LC3 are substrates for hApg7p. GATE-16 as a soluble transport factor interacts with NSF and GOS-28, is localized in the Golgi, and is expressed in the largest amount in brain (33). GABARAP is GABA A receptor-associated protein that colocalizes with the GABA A receptor in cultured cortical neurons and interacts with gephyrin (31, 32, 34). MAP-LC3 is localized on autophagosomal membranes (35). Considering the divergent functions FIG. 3. Mammalian Apg7p forms a homodimer. A, appearance of an hApg7p band at ϳ160 kDa in addition to a band at an ϳ80 kDa with cross-linking. Cells expressing hApg7p were lysed, and the lysate was treated with 5 mM of disuccinimidyl suberate, as described under "Experimental Procedures." hApg7p was recognized by SDS-PAGE followed by immunoblotting with anti-hApg7p antibody. B, endogenous rat Apg7p homodimer in rat liver. The cytosolic fraction was prepared from rat liver and was separated by centrifugation through a 10 -40% glycerol gradient. Fractions were collected from the bottom of the gradients and assayed for the presence of rat Apg7p by immunoblotting using anti-hApg7p antibody. The positions of marker proteins in the gradient are indicated above the blot. kDa, molecular mass; S, sedimentation value. and intracellular localizations of the three Apg8p homologs, it is surprising that all three human Apg8p homologs are substrates for hApg7p. Because yeast Apg7p plays an indispensa-ble role in autophagy and the Cvt transport of aminopeptidase I, mammalian Apg7p must also be essential for autophagy and other forms of membrane transport, common phenomena involving the formation of cup-shaped and/or elongated membrane structures.
Because MAP-LC3 is localized on autophagosomal membranes in rat liver as in yeast Apg8p (35), at least two substrates, MAP-LC3 and hApg12p, play major roles in autophagy in mammalian cells. At present, there has been no report of a mammalian Cvt-like pathway. Considering the strong expression of GATE-16 and GABARAP in brain and neuronal cells, a Cvt-like pathway and/or other membrane transport pathways in which GATE-16 and GABARAP function as protein modifiers may also exist in these tissues. It is difficult to explain how hApg7p distinguishes the four substrates and regulates the multiple interactions among the substrates. There must be some regulatory factors associated with the hApg7p homodimer to form multimeric complexes. Further candidates related to hApg7p will be sought by a two-hybrid experiment using a human brain cDNA library and coimmunoprecipitation of rat Apg7p with anti-hApg7p antibody in several rat tissues.
At present, the target proteins of GATE-16, GABARAP, and MAP-LC3 remain unknown. There is no report that these proteins conjugate with other proteins. We have recognized no targeting protein with which MAP-LC3 forms a conjugate. Kabeya et al. (35) reported that MAP-LC3 is processed to several forms in cultured mammalian cells, so there may exist some unknown mechanism. Further biochemical analysis of hApg8p is necessary.
It has become more and more evident that the Apg machinery plays an important role in at least brain and cardiac and skeletal muscles. Clinical and biochemical analyses of a group of severe inheritable neurodegenerative disorders, neuronal ceroid-lipofuscinosis, have suggested that lysosomal degradation via autophagy occurs actively during neuronal development (for a review see Ref. 41). Furthermore, clinical, genetic and biochemical analyses of X-linked vacuolar cardiomyopathy, myopathy in humans and LAMP-2-deficient mice have indicated that autophagic processes play an indispensable role in normal mammalian bodies (42,43). In addition, autophagy is activated by apoptotic signaling in sympathetic neurons (44). In view of the ubiquitous distribution of hApg12p and human Apg8p homologs, GATE-16, GABARAP, and MAP-LC3, and the multiple reactivity of hApg7p with these different substrates, it is possible that mammalian Apg7p plays an essential role in various stages of development and apoptosis in addition to autophagy. We are now investigating the possible tissue-specific functions of hApg7p using APG7 gene-knockout mice. These studies will contribute to the understanding of the physiological functions of mammalian Apg7p.