Introduction
Protein degradation, as well as synthesis, is essential to normal activity of the cell. To degrade intracellular proteins, all eukaryotic cells have two major mechanisms, namely the ubiquitin–proteasome system and autophagy. Autophagy is a pivotal physiological process for survival during starvation, differentiation and normal growth control, and may play a number of roles in various other cellular functions, via the turnover of cellular macromolecules and organelles. It is defined as the process of sequestrating cytoplasmic proteins or even entire organelles into the lytic compartment, i.e. the lysosome/vacuole (for reviews see
Seglen and Bohley, 1992;
Dunn, 1994;
Blommaart et al., 1997;
Klionsky and Ohsumi, 1999). The initial step of autophagy is the surrounding of cytoplasmic and organelle portions of the cell by a single isolation membrane. The fusion of the edges of the membrane sac with each other forms a closed double‐membrane structure, so‐called autophagosome or immature autophagic vacuole (AVi). Finally, the autophagosome fuses with a lysosome to become the autolysosome or the degradative autophagic vacuole (AVd). Within the AVd compartment, the sequestered content is degraded by lysosomal hydrolases. A convergence between autophagic and endosomal transport has been observed in the early stage of both pathways (
Tooze et al., 1990;
Liou et al., 1997). Although autophagy is a constitutive cellular event, it is enhanced in certain situations such as nutrient or serum starvation, hormonal stimulation and drug treatments.
While the morphology and regulation of autophagy have been investigated extensively for animals, including human, the molecular machinery underlying its process is poorly understood. To identify the proteins involved in autophagy, we isolated yeast mutants defective in autophagy (
Tsukada and Ohsumi, 1993). We identified 13
APG genes essential for autophagy and characterized their products (
Klionsky and Ohsumi, 1999). We also found by searching the expressed sequence tag (EST) database that most Apg proteins have related proteins in mammals, implying that the molecular basis of autophagy may well be conserved between yeast and human. As a matter of fact, we found a unique covalent modification of the human homologues of Apg12p (hApg12) with Apg5p (hApg5) (
Mizushima et al., 1998b), which is equivalent to the yeast Apg12p–Apg5p conjugation system essential for autophagy that we reported previously (
Mizushima et al., 1998a). Moreover,
Liang et al. (1999) showed recently that the human homologue of Apg6p, beclin 1, promoted autophagy in human breast carcinoma MCF7 cells. To elucidate the molecular mechanism of animal autophagy, we initiated the systematic characterization of the Apg homologues.
Here we analyse the intracellular localization and processing of the rat microtubule‐associated protein 1 light chain 3 (LC3). LC3 was identified originally as a protein that co‐purified with microtubule‐associated protein 1A and 1B from rat brain (
Mann and Hammarback, 1994). It shows 28% amino acid identity with Apg8/Aut7p, which is essential for yeast autophagy (
Liang et al., 1999). Our recent investigation on Apg8/Aut7p in yeast suggested that this protein plays a critical role in the formation of autophagosomes (
Kirisako et al., 1999). We found two forms of the LC3 molecules in cells. We suggest that one is in the cytoplasmic form and is processed into another form associated with the autophagosome membrane. To our knowledge, LC3 is the first mammalian protein localized in the autophagosome membrane to be identified.
Discussion
Autophagy is a dynamic process consisting of the formation and fusion of membrane compartments. Therefore, it is essential to identify protein components of autophagic membranes in order to unravel the mechanism of the phenomenon. We describe here that LC3‐II, a processed form of LC3, is localized in autophagosomes and autolysosomes by subcellular fractionation and immunogold electron microscopy. This is in agreement with the distribution of Apg8/Aut7p, a yeast homologue of LC3. We located Apg8/Aut7p by immunogold electron microscopy in autophagosomes, autophagic bodies and intermediate structures during autophagosome formation (
Kirisako et al., 1999).
Based on the results of this study, we propose that post‐translational modifications generate LC3‐I and LC3‐II. The initial step must be a cleavage at the C‐terminal region. Gly120 is important for the latter reaction. The amino acid segment Tyr121–Leu142, downstream of Gly120, should be cleaved off, although this has to be confirmed. Interestingly, it was reported previously that the genome of a cytopathogenic mutant of bovine viral diarrhoea virus contained an insertion sequence equivalent to LC3
ΔC22, which lacks Tyr121–Leu142 (
Meyers et al., 1998). The fact that a polyprotein translated from the virus was cleaved immediately after Gly120 of the inserted LC3
ΔC22 by some cellular protease is very compatible with our hypothesis. Recently, we found that Apg8/Aut7p was also cleaved immediately after Gly116, which corresponds to Gly120 in LC3, and this proteolytic processing was required for autophagosome formation (
Kirisako et al., 2000). In addition, we confirmed that a similar cleavage occurs in another Apg8/Aut7p homologue, Golgi‐associated ATPase enhancer of 16 kDa (GATE16: DDBJ/EMBL/GenBank accession No.
AF20262) (
Sagiv et al., 2000)/ganglioside expression factor‐2 (GEF2: DDBJ/EMBL/GenBank accession No.
AB003515). Thus, the processing at the C‐terminal region may be common in the Apg8/Aut7p family for their function. Furthermore, we could generate LC3‐I
in vitro from the recombinant LC3 in the presence of cell extracts (data not shown). The reaction was sensitive to
N‐ethylmaleimide, implying involvement of a cytosolic cysteine protease. We identified the yeast Apg4p as the cellular cysteine protease cleaving Apg8/Aut7p (
Kirisako et al., 2000). We have also identified human homologues of Apg4p and are now in the process of their characterization. It is not clear at present that the cleavage is sufficient to form LC3‐I. Although LC3‐II might be produced directly from proLC3, we favour the model that LC3‐I is converted to LC3‐II, based on the behaviour of two mutants, LC3
ΔC22 and LC3
ΔC22,G120A. In either case, the nature of the modification involved in the formation of LC3‐II remains cryptic, although Gly120 is evidently important. The modification should give LC3‐II a membrane protein‐like property. It is unclear at present whether LC3‐II is formed prior to its binding to the membranes or after targeting of LC3‐I to the membranes.
The cell culture in the serum‐ and amino acid‐free medium caused the increase in the amount of LC3‐II. Autophagy was induced under these conditions and we found a correlation between the rate of LC3‐II increase and the rate of autophagosome formation. Wortmannin and 3‐methyladenine, two inhibitors of autophagosome formation, suppressed the starvation‐induced increase of LC3‐II, whereas the drugs that accumulate autophagosomes, such as vinblastine and bafilomycin A1, had a strong LC3‐II‐increasing effect, which was synergistic with the starvation effect. Consequently, it is likely that the amount of LC3‐II reflects the number of autophagosomes. If so, LC3‐II may define the number of autophagosomes, i.e. it may regulate the formation of the compartments. In the later stage of autophagy, LC3‐II may degrade or recycle back to cytosolic LC3‐I, since autolysosomes were less labelled than autophagosomes, with gold particles representing LC3‐II in immunoelectron microscopy (the labelling density was 2.5 ± 2.0/μm and 36.4 ± 20.5/μm of membranes, respectively).
When we initiated the analysis of LC3, only one mammalian protein homologous to the yeast Apg8/Aut7p was known. However, two other homologues, GATE16 and γ‐aminobutyric acid A receptor‐associated protein (GABARAP;
Wang et al., 1999), were identified later in mammals. GATE16 was reported to be a modulator of intra‐Golgi membrane transport (
Sagiv et al., 2000) and GABARAP is known to bind to GABA
A receptors (
Wang et al., 1999). Therefore, LC3 seems to be the only one to play a role in autophagy. Since there is no homologue of Apg8/Aut7p in yeast, it may be an ancestor of an Apg8/Aut7p family including LC3, GABARAP and GATE16. The diversified members of the LC3 family in animals may have evolved to have a specialized function in different places inside the cell.
It was shown previously that purified recombinant LC3 binds to microtubules assembled from purified tubulin
in vitro (
Mann and Hammarback, 1994). This feature inspired us to consider a possible involvement of microtubules in the function of LC3 in autophagy. We also suspected that the increment of LC3‐II induced by vinblastine is attributable to the breakdown of microtubules. However, other microtubule‐depolymerizing reagents such as nocodazole and colchicine did not affect the amount of LC3‐II (data not shown), ruling out this possibility.
Lang et al. (1998) previously proposed that Apg8/Aut7p functions in the transport of autophagosomes by attaching to microtubules via another protein Apg4/Aut2p. Conversely, we provided evidence for a normal autophagy in yeast treated with nocodazole (
Kirisako et al., 1999). Although microtubules are not required for the Apg8/Aut7p function or autophagy in yeast, the situation in mammalian cells may be different: microtubules may assist efficient transport of autophagosomes in mammalian cells, which are much larger than yeast cells. To substantiate the association of LC3‐II with microtubules
in vivo, we counterstained cells expressing GFP–LC3 with antibody against tubulin. Most of the dots stained did not co‐localize with the microtubule meshes, although some seemed to have done so (our unpublished observation). The hypothesis that LC3‐II functions by linking autophagosomes to microtubule is still a possibility to be examined.
LC3‐II would be a good marker for autophagosomes, which so far have been defined by morphology but not by molecular composition. Detailed characterization of LC3 would provide us with clues to the many questions about the autophagy in mammals. We are now at a starting point for the molecular dissection of this mysterious organelle, the autophagosome.