|dc.description.abstracteng||Macroautophagy is a conserved catabolic pathway for the removal and recycling of cytosolic components, damaged or surplus organelles, protein aggregates or intracellular pathogens to maintain cellular homeostasis. It is characterized by the formation of a double-membrane-layered vesicle, called autophagosome, that engulfs intracellular material. In yeast, autophagosome formation is initiated at the pre-autophagosomal structure (PAS). Here, a double-membrane structure, the phagophore, elongates and closes to form the autophagosome. Finally, the outer membrane of the autophagosome fuses with the vacuole releasing the inner membrane together with the cargo in the vacuolar lumen for degradation.
Atg8 is a key component for autophagosome biogenesis and selective cargo recruitment to the phagophore. In this study, a series of GFP-Atg8 variants, including mutants that are unable to bind Atg8-interacting motifs (AIMs), were used as baits for co-immunoprecipitation (CoIPs) and following mass spectrometry analysis to find novel Atg8 interaction partners. This allowed rapid validation of the numerous proteins identified in mass spectrometry.
This approach identified Atg26 as an AIM-dependent interaction partner of Atg8. Atg26 is a sterol glucosyltransferase with a PH and GRAM domain. So far, the function of Atg26 in S. cerevisiae was unknown. In this study, using bioinformatics, a C-terminal Atg8-interacting motif (AIM) was predicted in Atg26. Interestingly, this motif was necessary for recruitment of Atg26 to the phagophore and its autophagic degradation, which are common features of known AIM-containing proteins.
To uncover the function of Atg26 in S. cerevisiae, CoIPs with GFP-Atg26 as bait were done. Here, the major coat protein Gag of the S. cerevisiae virus L-A was identified as an Atg26 interaction partner. L-A is a dsRNA virus of the Totiviridea family. It has a single 4.6 kb dsRNA genome with two overlapping ORFs, where ORF1 encodes the major coat protein Gag and ORF2 is a RNA-dependent RNA polymerase (Pol) that is expressed as a 180 kDa Gag-Pol fusion protein. L-A virus-like particles (VLP) are made up of 120 Gag subunits (from which about 2 are Gag-Pol fusion proteins), containing genomic dsRNA inside.
In this study, using truncated versions of Atg26 as baits for CoIPs and pull down assays the Gag binding domain of Atg26 was narrowed down to the PH domain and an undefined following region of Atg26. Atg26 recruitment to the phagophore by its C-terminal AIM and its interaction with L-A Gag suggested an involvement of Atg26 in selective autophagic degradation of the L-A virus. Here, GFP-tagged L-A Gag, showing comparable features as endogenous L-A Gag in binding studies and microscopic analyses, was established as a tool to measure autophagic removal of L-A Gag. During starvation, GFP-tagged Gag was degraded by autophagy, while deletion of ATG26 caused a 50% reduction of the autophagic rate. Thus, this study showed for the first time that L-A Gag is degraded by autophagy and attributes a direct role in this process to Atg26. Indeed, the selective adapter Atg11 and the PROPPINs Atg21 and Hsv2, which are typical regulators of selective autophagy, are also involved in processing of GFP-Gag. These observations indicate that L-A Gag is degraded by selective autophagy.
Taken together, this study suggests that Atg26 might recruit L-A Gag or complete L-A VLPs to the phagophore for degradation by selective autophagy.||de