Analysis of the Molecular Components and Different Steps of Autophagy-Related Pathways in Saccharomyces Cerevisiae.

Abstract

Autophagy is the primary intracellular pathway for degradation and recycling of long-lived proteins and organelles. It is critical for cell survival under various stress conditions, and essential for differentiation and development in higher eukaryotes; it clears unwanted intracellular proteins, damaged organelles and pathogens in defense against cancer, aging, neurodegeneration and pathogen infection. Much progress has been made on the identification and characterization of AuTophaGy-related (ATG) genes in different organisms. However, our understanding of the molecular mechanisms of autophagy remains limited, and difficult for therapeutic applications. In this study, to better understand the molecular mechanisms, a multiple knockout (MKO) yeast strain with 24 ATG genes deleted was generated and used for in vivo reconstitution of autophagy. The minimum requirements for different steps of autophagy-related pathways were determined, and new insights different from in vitro analyses were obtained—for instance, one piece of our data suggest that additional factors may regulate the deconjugation of Atg8–PE by the cystein protease Atg4 in vivo. The MKO strain was further converted into a yeast two-hybrid (Y2H) host strain. Both strains were used to study protein-protein interactions in the absence of other Atg proteins. Atg9 was found to self-interact in the absence of other Atg proteins, and this self-interaction was critical for its transport and function at the phagophore assembly site during autophagy. Also, Atg29 and Atg31 interacted with each other independent of other Atg proteins. In addition, the physiological significance of the PtdIns(3)P-binding FRRG motifs of two homologous proteins, Atg18 and Atg21, was analyzed. Our data suggest that both compensate for one another in the recruitment of Atg proteins, such as Atg8 and Atg16. Using the MKO strain, we also found that Atg18 and Atg21 protect Atg8–PE from Atg4-mediated cleavage. Thus, the MKO strain has proven to be, and will continue to be very useful in our understanding of autophagy.