Atg45 is an autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy in yeast.

The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.

Receptor-mediated cargo hitchhiking on bulk autophagy.

While the molecular mechanism of autophagy is well studied, the cargoes delivered by autophagy remain incompletely characterized. To examine the selectivity of autophagy cargo, we conducted proteomics on isolated yeast autophagic bodies, which are intermediate structures in the autophagy process. We identify a protein, Hab1, that is highly preferentially delivered to vacuoles. The N-terminal 42 amino acid region of Hab1 contains an amphipathic helix and an Atg8-family interacting motif, both of which are necessary and sufficient for the preferential delivery of Hab1 by autophagy. We find that fusion of this region with a cytosolic protein results in preferential delivery of this protein to the vacuole. Furthermore, attachment of this region to an organelle allows for autophagic delivery in a manner independent of canonical autophagy receptor or scaffold proteins. We propose a novel mode of selective autophagy in which a receptor, in this case Hab1, binds directly to forming isolation membranes during bulk autophagy.

A nuclear membrane-derived structure associated with Atg8 is involved in the sequestration of selective cargo, the Cvt complex, during autophagosome formation in yeast.

Macroautophagy (hereafter autophagy) is a conserved intracellular degradation mechanism required for cell survival. A double-membrane structure, the phagophore, is generated to sequester cytosolic cargos destined for degradation in the vacuole. The mechanism involved in the biogenesis of the phagophore is still an open question. We focused on 4 autophagy-related (Atg) proteins (Atg2, Atg9, Atg14, and Atg18), which are involved in the formation of the phagophore in order to gain a more complete understanding of the membrane dynamics that occur during formation of the autophagosome. The corresponding mutants, while defective in autophagy, nonetheless generate the membrane-bound form of Atg8, allowing us to use this protein as a marker for the nascent autophagosome precursor membrane. Using electron microscopy (EM), we discovered in these atg mutants a novel single-membrane structure (~120 to 150 nm in size). Electron tomography revealed that this structure originates from a part of the nuclear membrane, and we have named it the alphasome. Our data suggest that the alphasome is associated with Atg8, and sequesters selective cargo, the Cvt complex, during autophagy. Abbreviations: 3D: three-dimensional; AB: autophagic body; AP: autophagosome; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; EM: electron microscopy; IEM: immunoelectron microscopy; L: lipid droplet; N: nucleus; NM: nuclear membrane; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor aminopeptidase I; rER: rough endoplasmic reticulum; TEM: transmission electron microscopy; V: vacuole; VLP: virus-like particle.

A substrate localization model for the selective regulation of TORC1 downstream pathways.

Target of rapamycin complex 1 (TORC1) is a protein kinase complex conserved in eukaryotes that coordinates diverse cellular processes critical for cell growth to environmental conditions. Previous studies have shown that TORC1 is localized mainly in the lysosome/vacuoles, and its localization is important for signaling to downstream pathways. We recently demonstrated that signaling to Sch9, an S6K-related substrate of TORC1 in budding yeast, was selectively suppressed upon oxidative stress, which was mediated by the delocalization of phosphatidylinositol 3, 5-bisphosphate (PI[3,5]P2) from vacuolar membranes following stress. We propose that TORC1 downstream pathways can be regulated separately via the modulation of organelle localization of a specific target protein.

Vacuole-mediated selective regulation of TORC1-Sch9 signaling following oxidative stress.

Target of rapamycin complex 1 (TORC1) is a central cellular signaling coordinator that allows eukaryotic cells to adapt to the environment. In the budding yeast, Saccharomyces cerevisiae, TORC1 senses nitrogen and various stressors and modulates proteosynthesis, nitrogen uptake and metabolism, stress responses, and autophagy. There is some indication that TORC1 may regulate these downstream pathways individually. However, the potential mechanisms for such differential regulation are unknown. Here we show that the serine/threonine protein kinase Sch9 branch of TORC1 signaling depends specifically on the integrity of the vacuolar membrane, and this dependency originates in changes in Sch9 localization reflected by phosphatidylinositol 3,5-bisphosphate. Moreover, oxidative stress induces the delocalization of Sch9 from vacuoles, contributing to the persistent inhibition of the Sch9 branch after stress. Thus, our results establish that regulation of the vacuolar localization of Sch9 serves as a selective switch for the Sch9 branch in divergent TORC1 signaling. We propose that the Sch9 branch integrates the intrinsic activity of TORC1 kinase and vacuolar status, which is monitored by the phospholipids of the vacuolar membrane, into the regulation of macromolecular synthesis.

Dynamic relocation of the TORC1-Gtr1/2-Ego1/2/3 complex is regulated by Gtr1 and Gtr2.

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.