Dissecting the molecular function of the ubiquitin-like Atg8 during autophagosome biogenesis in S. cerevisiae

Abstract

This study shows that Gyp1 affects the Cvt-pathway, demonstrated by analysis of pApe1 maturation, proper proceeding of post-log mitophagy and might also play a role during Atg8-dependent recognition of other targets. Two functions of Gyp1 are proposed. First, Gyp1 seems to regulate the efficient dissociation of the Ypt1-Atg1 complex during initial steps of phagophore assembly. In the absence of Gyp1, this complex is enriched and the recruitment of downstream proteins is impaired, as shown for Atg14 in this study. This indicates that dissociation of Ypt1 and Atg1 is a prerequisite for recruitment of Atg proteins and proceeding of selective autophagy. For this process, the GAP activity of Gyp1 is needed. Gyp5 and Gyp8 might compensate the loss of Gyp1 as additional deletion of the two other Ypt1 GAPs leads to a more severe impairment of the Cvt-pathway. Singledeletion of both proteins causes no defect. Thus, Gyp1 might function as the main GAP during the Cvt-pathway.The second proposed function of Gyp1 was detected in later steps of selective autophagy. Here, Gyp1 interacts in an AIM-dependent manner with Atg8. In the absence of Gyp1, the function of Atg8 is impaired, which was demonstrated in this study with the mitophagy receptor Atg32. Upon deletion of GYP1, Atg8 and Atg32 show a reduced binding, which indicates a role of Gyp1 during cargo recognition.The analysis of Gyp1 mutants revealed that this function is AIM-dependent but GAP activity independent.Thus, this study proposes a dual role for Gyp1 during selective autophagy. First, it leads to the efficient dissociation of the Ypt1-Atg1 complex and later Gyp1 interacts with Atg8, which seems to be important for proper binding of Atg8 to selective cargo receptors.Autophagy is defined as a specific transport mechanism of proteins to the degradative organelles, which are the vacuole in yeast and plants and the lysosome in higher eukaryotes. Autophagy was initially studied as a response to stress conditions especially starvation, but it plays an important role in general cellular homeostasis (Choi et al., 2013). Autophagy is involved in adaption to stress conditions, aging, immunity, host defense and programmed cell death (Deretic et al., 2013;Fîlfan et al., 2017). The contribution in those processes leads also to several diseases that are associated with autophagy. Cancer, neurodegeneration, pathogen infection, myopathies and diabetes are known to be linked to an impaired degradation of superfluous or damaged proteins and thusThe cytoplasm-to-vacuole targeting pathway (Cvt-pathway) transports resident hydrolases to the vacuole by using the autophagic machinery (Fig. 2.4). The precursor aminopeptidase I (pApe1 or prApe1 in figure 2.4), the best-studied cargo of the Cvt-pathway, is synthesized in the cytosol where it assembles into a dodecamer. pApe1 is synthesized with an N-terminal propeptide, which mediates the assembly of the dodecamers into large oligomers, the Ape1 complex. In contrast to unselective macroautophagy, the cargo is exclusively enclosed by the Cvt...