Autophagy is a lysosome-mediated degradation system that involves formation of a double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes. In response to various stress conditions, autophagy nonselectively degrades a portion of the cytosol to provide energy and nutrients, serving as a cell survival mechanism. Autophagy also acts as a quality-control system in selectively removing damaged organelles, protein aggregates, and invading pathogens. Dysregulated autophagy is linked to a variety of human diseases, including neurodegeneration and tumor progression.
Genetic screens in yeast have identified a group of autophagy (ATG) genes essential for autophagosome formation. These Atg proteins include the Atg1 protein kinase complex and the Vps34 class III phosphatidylinositol 3-kinase (PI(3)K) complex involved in initiating formation of a cup-shaped isolation membrane. The two ubiquitin-like conjugation systems (Atg8–phosphatidylethanolamine conjugate and Atg12–Atg5 conjugate) are required for autophagosome expansion. The autophagy pathway in higher eukaryotes involves more complex membrane dynamics. Multiple sources, including the phosphatidylinositol 3-phosphate (PI(3)P)-enriched subdomains of the endoplasmic reticulum (ER), known as omegasomes, have been shown to contribute to autophagosomal membranes. Yeast autophagosomes directly fuse with the single large acidic vacuole, but nascent autophagosomes in higher eukaryotes undergo maturation processes before fusion with lysosomes. It is conceivable that, in addition to highly conserved Atg proteins, the more elaborate autophagic machinery in higher eukaryotes has acquired components that are absent from yeast. So far, very little is known about metazoan-specific autophagy genes. Systematic genetic screens for autophagy genes have not previously been carried out in multicellular organisms. My lab established Caenorhabditis elegans (C. elegans) as a multicellular genetic model system to delineate the machinery, regulation, and physiological functions of autophagy.
Establishing C. elegans as a Genetic Model to Study Autophagy
We demonstrated that autophagic activity is required for degradation of a variety of protein aggregates during C. elegans embryogenesis. For example, germ P granules, specialized protein aggregates derived from oocytes, are found exclusively in germ-line precursor cells in embryos. We showed that the P granule components PGL-1 and PGL-3 are degraded by autophagy in somatic cells. In autophagy mutants, PGL-1 and PGL-3 are not removed and accumulate into numerous aggregates, called PGL granules, in somatic cells (Figure). In another example, the C. elegans p62 homolog, SQST-1, is selectively degraded by autophagy. In wild-type animals, SQST-1 is weakly expressed and diffusely localized in the cytoplasm. In autophagy mutants, SQST-1 is strongly expressed and forms a large number of aggregates, which are distinct from PGL granules.
Identifying Novel Autophagy Genes
We performed genetic screens for mutants with defective degradation of PGL granules or SQST-1 aggregates. From 30,000 genomes screened, we obtained ~120 mutations that caused defective degradation of PGL-granule components in somatic cells and SQST-1 during embryogenesis. Characterization of these mutants revealed that we had identified mutant alleles of yeast Atg homologs. We also found that the roles of several yeast Atg proteins, including Atg14 and Atg13, are mediated by highly divergent functional homologs.
We also isolated multiple autophagy genes specific to multicellular organisms, including epg-3, -4, -5, and -6. epg-3 encodes the homolog of mammalian vacuole membrane protein 1 (VMP1), which is highly expressed in acute pancreatitis. epg-4 encodes the homolog of EI24/PIG8, a target of the tumor suppressor protein p53. The human homolog of epg-5, mEPG5, is frequently mutated in breast tumors. epg-6 encodes a WD40 repeat protein with PtdIns(3)P binding activity. epg-3, -4, and -6 are essential for the progression of omegasomes to autophagosomes. We further demonstrated that mammalian homologs of EPG-3, -4, -5, and -6 are also essential for starvation-induced autophagy, indicating that C. elegans is a suitable model for identifying metazoan-specific autophagy genes.
Another layer of complexity of the autophagic machinery in higher eukaryotes is conferred by the presence of multiple homologs of the same yeast Atg proteins. For example, at least eight Atg8 homologs are present in mammals; their roles in autophagy, however, are unknown. We are addressing the functional redundancy and functional divergence of the two Atg8 homologs, the two Atg4 homologs, and the two Atg16 homologs in the C. elegans autophagy pathway.
Taking advantage of the powerful C. elegans genetics, the availability of genetic null alleles of autophagy genes, and the distinct phenotypes in different autophagy mutants, we are establishing the hierarchical order of atg and epg genes in degradation of protein aggregates, a process referred to as aggrephagy. This analysis will reveal how ATG and EPG proteins coordinate to degrade protein aggregates.
Identifying Genes Required for Degradation of Specific Types of Protein Aggregates
The mechanism by which protein aggregates are selectively degraded by autophagy under physiological conditions is poorly understood. We isolated ~30 mutations that caused a defect only in degradation of PGL granules or SQST-1 aggregates. We showed that the coiled-coil domain protein SEPA-1 is required for degradation and formation of PGL granules. SEPA-1 itself forms aggregates. SEPA-1 directly associates with PGL-3, indicating that SEPA-1 functions as a receptor in recruiting PGL-1 and PGL-3 into the PGL granule. EPG-2 acts as an adaptor in bridging the PGL granule to the autophagic machinery. Loss of function of epg-2 disrupts the association of PGL granules with LGG-1/Atg8 puncta. The cargo/receptor/adaptor complex (PGL-1 and PGL-3/SEPA-1/EPG-2) is degraded by autophagy. We are also studying genes specifically required for degradation of SQST-1 aggregates. Understanding how PGL granules and SQST-1 aggregates are selectively removed will provide insights into the mechanisms by which autophagosomes are formed around the substrate.
Studying How Autophagy Activity Is Regulated During Animal Development
How autophagic activity is regulated under physiological conditions and how it is integrated with various signaling pathways during animal development remains largely unknown. We isolated a mutation in rpl-43, encoding large ribosomal subunit 43, which causes accumulation of SQST-1 aggregates in the larval intestine. Under starvation conditions, the SQST-1 aggregates in rpl-43 mutants are degraded by autophagy. We performed a genome-wide RNAi screen to identify genes whose inactivation suppresses accumulation of SQST-1 aggregates in rpl-43 mutants. The identified genes are being analyzed to investigate how they integrate into the autophagic machinery.
We are also identifying negative regulators of autophagy by isolating suppressors for null and hypomorphic alleles of autophagy genes.
Investigating the Physiological Functions of Autophagy
The wealth of knowledge about C. elegans developmental biology and the invariant cell lineage make C. elegans an excellent model to study the physiological functions of autophagy. We are studying the role of autophagy in cell death, in removal of cell corpses, and in many other biological processes.
Characterizing the Role of Autophagy Proteins in Autophagy-Independent Processes
Autophagosome formation shares characteristics with other membrane-mediated trafficking pathways, such as membrane curvature, fusion, and reformation of vacuolar structures. We are investigating whether autophagy genes play a role in autophagy-independent membrane trafficking processes.
The molecular understanding of autophagy has originated almost exclusively from yeast genetic studies. Our studies have established C. elegans as one of the premier genetic models to identify essential autophagy genes specific to higher eukaryotes and to study the regulation and physiological functions of autophagy. These studies will provide insights into the molecular mechanism of autophagosomal induction, expansion, and maturation and the mechanism underlying various diseases, including neurodegeneration, tumorigenesis, and developmental disorders.
Grants from the Ministry of Science and Technology, China, provided partial support for these projects.
As of January 17, 2012