Two ubiquitin-like conjugation systems essential for autophagy
Introduction
There are two major pathways of intracellular protein degradation. First, the ubiquitin-proteasome system in the cytosol is involved in degradation of short-lived, damaged or misfolded proteins [1], [2]. Target proteins to be degraded are first tagged with ubiquitin and then digested by the proteasome with strict recognition by the ubiquitin ligase system.
Long-lived proteins are believed to be degraded within a specific compartment, the lysosome/vacuole. So far several delivery routes to this lytic compartment have been proposed. The process of degradation of the cell’s own intracellular constituents in lysosomes is generally called autophagy in contrast to heterophagy, degradation of extracellular materials [3]. Macroautophagy (hereafter autophagy) is a major pathway in autophagy, and initiates by enwrapping a portion of cytoplasm by a membrane sac called the isolation membrane, to form a double membrane structure, the autophagosome [4]. The autophagosome then fuses with the lysosome, becoming the autolysosome and its inner membrane and contents are digested for reuse.
Autophagy is characterized as being nonselective resulting in the bulk degradation of cellular proteins. More than 90% of cellular proteins are long lived, thus the turnover of long-lived proteins is important to understand the physiology of the cell. Since the lysosome was discovered [5], electron microscopy has revealed that autophagy occurs in a variety of cells from different tissues and cultured cells, and it is now generally accepted that autophagy is a ubiquitous activity in eukaryotic cells.
However, the molecular mechanism of autophagy has remained elusive. There was no specific monitoring marker or quantitative assay system to detect autophagy in mammalian cells. Therefore, the genes involved in autophagy remained unidentified for a long time. Here, we will focus on the recent progress in the molecular dissection of autophagy. Discoveries of two ubiquitin-like conjugation reactions have begun to unravel the mystery of autophagy.
Section snippets
Discovery of autophagy in the yeast, Saccharomyces cerevisiae
It has been postulated that the vacuole is a lytic organelle in yeast like the lysosome [6]. It was shown that bulk protein turnover is induced upon nitrogen starvation, which is dependent upon vacuolar enzyme activities [7], [8]. Obvious questions are what kinds of substrates are degraded in the vacuole and what is the mechanism of sequestering those substrates into the vacuole?
In early 1990, we found that the yeast cell induces autophagy under various nutrient starvation conditions. When
Genetic approaches to yeast autophagy
Genetic approaches were taken to address the molecular mechanism of autophagy. The morphological change of the vacuole under starvation, accumulation of autophagic bodies, and an immunoscreen of cells that retain a cytosolic enzyme (fatty acid synthase) after starvation, were used to obtain autophagy-defective mutants (apg and aut). Later, two hybrid screens using Apg proteins as bait identified two more APG genes [12], [13]. Klionsky’s group isolated mutants defective in the maturation of one
Characterization of Atg proteins
All the atg mutants grew normally in rich medium, but failed to induce bulk protein degradation under nutrient-depletion conditions. A homozygous diploid with an atg mutation could not perform sporulation [19]. Another feature of autophagy-defective mutants is the loss of viability during nitrogen starvation. These mutants start to die after 2 days of starvation and almost completely lose viability after 1 week [19]. Cloning and identification of autophagy genes revealed that almost all are
The yeast Atg12 conjugation system
Atg12, a hydrophilic small protein of 186 amino acids with no apparent homology to ubiquitin, covalently links to Atg5 [24]. The mode of conjugation of Atg12 to Atg5 is quite similar to that of ubiquitination. The carboxy-terminal residue of Atg12 is a single glycine, which is activated by Atg7 in an ATP-dependent manner [24]. Then, Atg12 forms a conjugate with Atg7 through a thioester bond [25], [26], [27]. Atg7 shows restricted homology with the ubiquitin-E1 enzyme within the regions around
Atg8 conjugation system
The second ubiquitin-like protein essential for autophagy is Atg8 (Aut7/Apg8), a 117-amino acid protein. Atg8 was shown to be a good marker for membrane dynamics during autophagy because it resides on the membrane sac enwrapping the cytoplasm, autophagosome, and also the autophagic body [21]. Cell fractionation studies showed that Atg8 is mostly membrane bound; about half is peripherally bound to membrane but half behaves like an intrinsic membrane protein [30].
Epitope tagging analyses of Atg8
Other factors required for autophagy
Recent analyses of Atg proteins revealed that there are two kinase complexes in addition to ubiquitin-like conjugation systems. One is the Atg1 protein kinase associated with Atg13, Atg17, Cvt9 [32], [33]. The N-terminus contains a protein kinase domain, and kinase activity was detected in vitro. A kinase-negative atg1 mutant is defective in autophagy, implying that the kinase activity is essential for the function [13], [32]. The kinase activity is upregulated during induction of autophagy, so
Preautophagosomal structure, the site of autophagsosome formation
All the former APG genes have a function before or during the formation step of the autophagosome. The membrane dynamics of autophagy are distinct from the classical vesicular membrane trafficking. There are many fundamental questions relating to the molecular mechanism of autophagosome formation.
For a long time, the origin of the autophagosome membrane was proposed to be the ER. Freeze-fracture of the autophagsome indicates that both membranes are quite different from the ER, and only the
The Atg conjugation systems in mammalian cells
The two Atg conjugation systems are highly conserved in higher eukaryotic cells, suggesting that eukaryotes acquired the mechanism of autophagy at the beginning of its evolution. In both mouse and human, there is only one orthologue for each component of the Atg12 system. Atg12 is conjugated to Atg5 [36], which is catalyzed by Atg7 [37] and Atg10 [38], [39]. In mammalian cells, the Atg12–Atg5 conjugate forms an ∼800-kDa protein complex. Characterization of this complex revealed an additional
Role of Atg conjugation in mammalian autophagy
The function of the mammalian Atg12 system was shown using mouse embryonic stem (ES) cells. ES cells induce autophagy well by nutrient starvation and the size of the autophagosome in ES cells is larger than in other cell lines. First, the localization of Atg12–Atg5 was examined in detail using GFP-fused Atg5 [50]. A small fraction of cytosolic Atg12–Atg5·Atg16L complex localizes to the isolation membrane throughout its elongation process (Fig. 2). Atg12–Atg5 initially associates with a small
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