The Function Of C. Elegans Atg-16Homolog In The Autophagy Pathway

Autophagy is a degradation system involving the enclosure of cytosolic components in a double-membrane autophagosome and their subsequent delivery to the vacuole/lysosome for degradation. A group of evolutionarily conserved Atg proteins act at distinct steps of autophagosome formation. The molecular understanding of autophagy pathway has originated almost exclusively from yeast genetic studies. However, little is known about essential autophagy components specific to higher eukaryotes. The presence of multiple homologs of the same yeast Atg genes endows an extra layer of complexity on the autophagic machinery in higher eukaryotes. Most Atg proteins have only one ortholog in C. elegans except for Atg4, Atg8and Atg16, which have two orthologs. The physiological function of individual homologs in the autophagy pathway remains poorly understood.In this study, we established C. elegans as a multicellular genetic model to delineate the autophagic machinery by demonstrating that a variety of protein aggregates are selectively removed by autophagy during embryogenesis. We carried out genetic screens in multicellular organisms to identify essential autophagy genes. Firstly, we isolated several autophagy genes conserved in yeast and higher eukaryotes. Secondly, we investigated the physiological function of these autophagy genes in C. elegans.In two different C. elegans genetic screens we identified atg-16.1and atg-16.2. Here we characterized the function of the two atg-16homologs, atg-16.1and atg-16.2, in the autophagy pathway in C. elegans. We showed that atg-16.2mutants exhibit a stronger autophagic defect than atg-16.1mutants. Atg-16.2; atg-16.1double mutants display a much more severe defect than either single mutant. ATG-16.1and ATG-16.2contain seven WD repeats at their C termini, which were also shared by mammalian ATG16L but were absent in yeast Atg16. ATG-16.2shared28.8%identity and48.3%similarity to human ATG16L1; ATG-16.1had29.7%identity and49.3%similarity to human ATG16L1. ATG-16.1and ATG-16.2interacted with themselves and each other and also directly associated with ATG-5. Atg-16.1mutant embryos exhibited a wild-type expression and distribution pattern of LGG-1/Atg8, while LGG-1puncta were markedly fewer in number and weaker in intensity in atg-16.2mutants. In atg-16.2; atg-16.1double mutants, the lipidated form of LGG-1accumulates, but LGG-1puncta are completely absent. ATG-16.2ectopically expressed on the plasma membrane provides novel sites of LGG-1puncta formation. We also demonstrated that the-COOH terminal WD repeats are dispensable for the role of. atg-16.2in aggrephagy (the degradation of protein aggregates by autophagy). Genetic epistasis analysis placed atg-16.2upstream of atg-2, epg-6and atg-18.Our study indicated that C. elegans ATG-16s are involved in specifying LGG-1puncta formation and the two ATG-16homologs have partially redundant yet distinct functions in the aggrephagy pathway.