Takayuki Sekito

Hierarchy of Atg proteins in pre‐autophagosomal structure organization

Autophagy is a bulk degradation process that is conserved in eukaryotic cells and functions in the turnover of cytoplasmic materials and organelles. When eukaryotic cells face nutrient starvation, the autophagosome, a double‐membraned organelle, is generated from the pre‐autophagosomal structure (PAS). In the yeast Saccharomyces cerevisiae, 16 ATG (autophagy‐related) genes are essential for autophagosome formation. Most of the Atg proteins are involved in the PAS, leading to autophagosome production. However, the mechanism of PAS organization remains to be elucidated. Here, we performed a systematic and quantitative analysis by fluorescence microscopy to develop a hierarchy map of Atg proteins involved in PAS organization. This analysis suggests that Atg17p is the most basic protein in PAS organization: when it is specifically targeted to the plasma membrane, other Atg proteins are recruited to that location, suggesting that Atg17p acts as a scaffold protein to organize Atg proteins to the PAS.

Atg9 vesicles are an important membrane source during early steps of autophagosome formation

Atg9-containing vesicles assemble to the preautophagosomal structure and eventually are incorporated into the autophagosomal outer membrane.

The Atg18-Atg2 Complex Is Recruited to Autophagic Membranes via Phosphatidylinositol 3-Phosphate and Exerts an Essential Function

Atg18 is essential for both autophagy and the regulation of vacuolar morphology. The latter process is mediated by phosphatidylinositol 3,5-bisphosphate binding, which is dispensable for autophagy. Atg18 also binds to phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro. Here, we investigate the relationship between PtdIns(3)P-binding of Atg18 and autophagy. Using an Atg18 variant, Atg18(FTTG), which is unable to bind phosphoinositides, we found that PtdIns(3)P binding of Atg18 is essential for full activity in both selective and nonselective autophagy. Atg18(FTTG) formed a complex with Atg2 in a normal manner, and Atg18-Atg2 complex formation occurred in cells in the absence of PtdIns(3)P, indicating that Atg18-Atg2 complex formation is independent of PtdIns(3)P-binding of Atg18. Atg18 localized to endosomes, the vacuolar membrane, and autophagic membranes, whereas Atg18(FTTG) did not localize to these structures. The localization of Atg2 to autophagic membranes was also lost in Atg18(FTTG) cells. These data indicate that PtdIns(3)P-binding of Atg18 is involved in directing the Atg18-Atg2 complex to autophagic membranes. Connection of a 2×FYVE domain, a specific PtdIns(3)P-binding domain, to the C terminus of Atg18(FTTG) restored the localization of Atg18-Atg2 to autophagic membranes and full autophagic activity, indicating that PtdIns(3)P-binding by Atg18 is dispensable for the function of the Atg18-Atg2 complex but is required for its localization. This also suggests that PtdIns(3)P does not act allosterically on Atg18. Taken together, Atg18 forms a complex with Atg2 irrespective of PtdIns(3)P binding, associates tightly to autophagic membranes by interacting with PtdIns(3)P, and plays an essential role.

Atg17 recruits Atg9 to organize the pre‐autophagosomal structure

Autophagy is a degradation system of cytoplasmic proteins and organelles via formation of double‐membrane vesicles called autophagosomes. In the yeast Saccharomyces cerevisiae, autophagosomes are formed via the pre‐autophagosomal structure (PAS) in a manner dependent on Atg proteins. Under nutrient‐rich condition, Atg9 is recruited to the PAS by binding to Atg11 for the Cvt pathway. However, because Atg9 is recruited to the PAS in atg11Δ cells in starved condition and autophagy is induced, autophagy‐specific mechanism for the Atg9 recruitment to the PAS has been assumed. Here, we demonstrate that, in autophagy‐inducing condition, Atg9 is recruited to the PAS in a manner dependent on Atg17. Atg9 physically interacts with Atg17 in the presence of rapamycin. This interaction requires Atg1, a protein kinase essential for autophagy. Consistently, the Atg17‐dependent PAS localization of Atg9 requires Atg1. However, its kinase activity is dispensable for this process. It rather regulates the equilibrium of assembly and disassembly of Atg9 at the PAS.

RTG-dependent mitochondria to nucleus signaling is negatively regulated by the seven WD-repeat protein Lst8p.

In cells with reduced mitochondrial function, RTG1, 2 and 3 are required for expression of genes involved in glutamate synthesis. Glutamate negatively regulates RTG‐dependent gene expression upstream of Rtg2p, which, in turn, acts upstream of the bHLH/Zip transcription factors, Rtg1p and Rtg3p. Here we report that some mutations [lst8‐(2–5)] in LST8, an essential gene encoding a seven WD40‐repeat protein required for targeting of amino acid permeases (AAPs) to the plasma membrane, bypass the requirement for Rtg2p and abolish glutamate repression of RTG‐dependent gene expression. The lst8‐1 mutation, however, which reduces plasma membrane expression of AAP, cannot bypass the Rtg2p requirement, but still suppresses glutamate repression of RTG target gene expression. We show that Lst8p negatively regulates RTG gene function, acting at two sites, one upstream of Rtg2p, affecting glutamate repression of RTG‐dependent gene expression through Ssy1p, an AAP‐like sensor of external amino acids, and the other between Rtg2p and Rtg1p–Rtg3p. These data, together with genome‐wide transcription profiling, reveal pathways regulated by glutamate, and provide insight into the regulation of cellular responses to mitochondrial dysfunction.

Novel families of vacuolar amino acid transporters

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed.

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe

We have identified the Schizosaccharomyces pombe SPBC3E7.06c gene (fnx2 +) from a homology search with the fnx1 + gene involving in G0 arrest upon nitrogen starvation. Green fluorescent protein‐fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane. Uptake of histidine or isoleucine by S. pombe cells was inhibited by concanamycin A, a specific inhibitor of the vacuolar H+‐ATPase. Amino acid uptake was also defective in the vacuolar ATPase mutant, suggesting that vacuolar compartmentalization is critical for amino acid uptake by whole cells. In both Δfnx1 and Δfnx2 mutant cells, uptake of lysine, isoleucine or asparagine was impaired. These results suggest that fnx1 + and fnx2 + are involved in vacuolar amino acid uptake in S. pombe.

Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids. Graphical Abstract In the budding yeast Saccharomyces cerevisiae, basic amino acids that are highly accumulated in vacuoles are extruded by vacuolar amino acid transporter Avt4.

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1∆ mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids. Avt1p is a proton/amino acid antiporter involved in vacuolar uptake of histidine and various neutral amino acids

A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition. Graphical Abstract Disruption or overexpression of AVT7 encoding a member of AVT transporter family affects net uptake of glutamine and proline by vacuolar membrane vesicles.