Shintaro Kira

TRAPPIII is responsible for vesicular transport from early endosomes to Golgi, facilitating Atg9 cycling in autophagy

Summary Autophagy is a bulk protein-degradation process that is regulated by many factors. In this study, we quantitatively assessed the contribution of each essential yeast gene to autophagy. Of the contributing factors that we identified, we focused on the TRAPPIII complex, which was recently shown to act as a guanine-nucleotide exchange factor for the Rab small GTPase Ypt1. Autophagy is defective in the TRAPPIII mutant under nutrient-rich conditions (Cvt pathway), but starvation-induced autophagy is only partially affected. Here, we show that TRAPPIII functions at the Golgi complex to receive general retrograde vesicle traffic from early endosomes. Cargo proteins in this TRAPPIII-dependent pathway include Atg9, a transmembrane protein that is essential for autophagy, and Snc1, a SNARE unrelated to autophagy. When cells were starved, further disruption of vesicle movement from late endosomes to the Golgi caused defects in Atg9 trafficking and autophagy. Thus, TRAPPIII-dependent sorting pathways provide Atg9 reservoirs for pre-autophagosomal structure and phagophore assembly sites under nutrient-rich conditions, whereas the late endosome-to-Golgi pathway is added to these reservoirs when nutrients are limited. This clarification of the role of TRAPPIII elucidates how general membrane traffic contributes to autophagy.

Reciprocal conversion of Gtr1 and Gtr2 nucleotide-binding states by Npr2-Npr3 inactivates TORC1 and induces autophagy

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To investigate the mechanisms that regulate autophagy, we performed a genome-wide screen using a yeast deletion-mutant collection, and found that Npr2 and Npr3 mutants were defective in autophagy. Their mammalian homologs, NPRL2 and NPRL3, were also involved in regulation of autophagy. Npr2-Npr3 function upstream of Gtr1-Gtr2, homologs of the mammalian RRAG GTPase complex, which is crucial for TORC1 regulation. Both npr2∆ mutants and a GTP-bound Gtr1 mutant suppressed autophagy and increased Tor1 vacuole localization. Furthermore, Gtr2 binds to the TORC1 subunit Kog1. A GDP-bound Gtr1 mutant induced autophagy even under nutrient-rich conditions, and this effect was dependent on the direct binding of Gtr2 to Kog1. These results revealed that 2 molecular mechanisms, Npr2-Npr3-dependent GTP hydrolysis of Gtr1 and direct binding of Gtr2 to Kog1, are involved in TORC1 inactivation and autophagic induction.

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

Ego2 is characterized as a new subunit of Ego protein complex (the yeast Ragulaor counterpart) that is a scaffold of Gtr (the yeast Rag counterpart) and TORC1. Gtr1 and Gtr2 regulate the dynamic translocation of the Ego/Gtr/TORC1 supercomplex between the vacuolar limiting membrane and perivacuolar foci. This localization shift is closely associated with the TORC1 activity level.

Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.

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

TORC1 modulates proteosynthesis, nitrogen metabolism, stress responses, and autophagy. Here it is shown that the Sch9 branch of TORC1 signaling depends specifically on vacuolar membranes and that this specificity allows the cells to regulate selectively the outputs of divergent downstream pathways in response to oxidative stress.

Ole1, fatty acid desaturase, is required for Atg9 delivery and isolation membrane expansion during autophagy in Saccharomyces cerevisiae

ABSTRACT Macroautophagy, a major degradation pathway of cytoplasmic components, is carried out through formation of a double-membrane structure, the autophagosome. Although the involvement of specific lipid species in the formation process remains largely obscure, we recently showed that mono-unsaturated fatty acids (MUFA) generated by stearoyl-CoA desaturase 1 (SCD1) are required for autophagosome formation in mammalian cells. To obtain further insight into the role of MUFA in autophagy, in this study we analyzed the autophagic phenotypes of the yeast mutant of OLE1, an orthologue of SCD1. Δole1 cells were defective in nitrogen starvation-induced autophagy, and the Cvt pathway, when oleic acid was not supplied. Defects in elongation of the isolation membrane led to a defect in autophagosome formation. In the absence of Ole1, the transmembrane protein Atg9 was not able to reach the pre-autophagosomal structure (PAS), the site of autophagosome formation. Thus, autophagosome formation requires Ole1 during the delivery of Atg9 to the PAS/autophagosome from its cellular reservoir. Summary: Isolation membrane expansion in yeast autophagy requires mono-unsaturated fatty acids generated by Ole1, fatty acid desaturase during delivery of Atg9 to the PAS/autophagosome from its cellular reservoir.

Chapter Seventeen – Quantitative Assay of Macroautophagy Using Pho8△60 Assay and GFP-Cleavage Assay in Yeast

It is intrinsically difficult to directly measure a specific protein reduction that is mediated by nonselective autophagy, because the degradation proceeds at a relatively slow pace, and the residual nondegraded part becomes the background. Several methods for measuring nonselective autophagy have been reported in the past. One classical simple method is called bulk degradation assay, which measures the release of degraded amino acids after radioisotope labeling of the total cellular proteins. In 1995, we developed a quantitative Pho8△60 assay in the yeast, Saccharomyces cerevisiae, for studying autophagy, which is widely used for its advantages that are described in the following sections. Another method used in recent times is the GFP-based processing assay in yeast. We will describe these two methods in this chapter.