Investigating the Effect of the Atg8 Conjugation System on Autophagosome Size and Number.

Autophagy is the cellular degradation process in which cellular contents are encapsulated by double-membrane vesicles, autophagosomes, and delivered to the vacuole to be degraded and recycled. This process is important for cell health and homeostasis. It involves the upregulation of many autophagy-related proteins, including Atg3 and Atg10. We are investigating the role of these proteins in the model system S. Cerevisiae is to measure the size and number of autophagosomes formed when insufficient amounts of that protein are available. Atg8 levels are known to affect only autophagosome size. However, we have found that levels of Atg7, the most upstream protein in the Atg8-PE conjugation pathway, affect both autophagosome size and number. Atg3 and Atg10 are intermediate proteins involved downstream of Atg7 and upstream of Atg8. Atg3 directly conjugates Atg8 to PE, while Atg10 conjugates Atg12 to Atg5, and the resulting Atg12-5 complex stimulates the activity of Atg3. It is possible that the Atg12-5 complex has additional roles independent of Atg8 that explain the effect of Atg7 levels on autophagosome number. If so, we would expect that Atg3 activity would correlate only with autophagosome size, while Atg10 might control size and number. We are generating Atg3 and Atg10 active site mutants and testing the functionality of these mutants by performing western blots and enzymatic assays. Mutants that give a partial loss of autophagic activity will be selected for later measurement of autophagosome size and number.