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The E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE) to drive autophagosome formation in Saccharomyces cerevisiae. In this study, we show that Atg3 localizes to the pre-autophagosomal structure (PAS) and the isolation membrane (IM), providing crucial evidence that Atg8-PE conjugates are produced on these structures. We also find that mutations in the Atg8-family interacting motif (AIM) of Atg3 significantly impairs the PAS/IM localization of Atg3, resulting in inefficient IM expansion. It is suggested that the AIM-mediated PAS/IM localization of Atg3 facilitates membrane expansion in these structures probably by ensuring active production of Atg8-PE on the membranes., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In Saccharomyces cerevisiae, under nitrogen-starvation conditions, the α-mannosidase Ams1 is recognized by the autophagic receptor Atg34 and transported into the vacuole, where it functions as an active enzyme. In this study, we identified Hrr25 as the kinase that phosphorylates Atg34 under these conditions. Hrr25-mediated phosphorylation does not affect the interaction of Atg34 with Ams1, but instead promotes Atg34 binding to the adaptor protein Atg11, which recruits the autophagy machinery to the Ams1-Atg34 complex, resulting in activation of the vacuolar transport of Ams1. Our findings reveal the regulatory mechanism of a biosynthetic pathway mediated by the autophagy machinery., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The Atg2-Atg18 complex is essential for autophagosome formation in Saccharomyces cerevisiae. In this paper, we show that partial induction of autophagy can proceed in cells expressing engineered variants of Atg2 capable of localizing to the pre-autophagosomal structure (PAS) in the absence of Atg18. Specifically, through the construction of fusion proteins, we show that the fusion to Atg2 of either the phosphatidylinositol 3-phosphate-binding FYVE domain or the core autophagy protein Atg8 allowed limited Atg18-independent recovery of autophagosome formation. These results indicate that effective targeting of Atg2 to the PAS can compensate for loss of Atg18 function in autophagy., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Autophagy is a system for degradation of bulk cellular components in lytic compartments, vacuoles, or lysosomes when eukaryotic cells face with nutrient starvation. In this review, we focus on the pre-autophagosomal structure (PAS), a functional entity responsible for autophagosome formation in Saccharomyces cerevisiae, and discuss its relevance to autophagy in mammalian cells., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Autophagy is a bulk degradation system conserved among most eukaryotes. Recently, autophagy has been shown to mediate selective degradation of various targets such as aggregated proteins and damaged or superfluous organelles. Structural studies have uncovered the conserved specific interactions between autophagic receptors and Atg8-family proteins through WXXL-like sequences, which we term the Atg8-family interacting motif (AIM). AIM functions in various autophagic receptors such as Atg19 in the cytoplasm-to-vacuole targeting pathway, p62 and neighbor of BRCA1 gene 1 (NBR1) in autophagic degradation of protein aggregates, and Atg32 and Nix in mitophagy, and may link the target-receptor complex to autophagic membranes and/or their forming machineries., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Autophagy is a bulk degradation process conserved among eukaryotes. In macro-autophagy, autophagosomes sequester cytoplasmic components and deliver their contents to lysosomes/vacuoles. Autophagosome formation requires the conjugation of Atg8, a ubiquitin-like protein, to phosphatidylethanolamine (PE). Here we report that the amino (N)-terminal region of Atg3, an E2-like enzyme for Atg8, plays a crucial role in Atg8-PE conjugation. The conjugating activities of Atg3 mutants lacking the 7 N-terminal amino acid residues or containing a Leu-to-Asp mutation at position 6 were severely impaired both in vivo and in vitro. In addition, the amino-terminal region is critical for interaction with the substrate, PE.

Autophagy is a degradation process accompanied by dynamic membrane organization. In the yeast, Saccharomyces cerevisiae, about 30 ATG (autophagy-related) genes have been identified as important genes for autophagy. Among them, 17 are indispensable for formation of the autophagosome, an organelle enclosed by a double lipid bilayer during starvation-induced autophagy. Recently, a central structure for autophagosome generation, termed the pre-autophagosomal structure, was identified. Despite intensive study, many questions regarding the mechanisms underlying autophagosome formation remain unanswered. In this review, we will give an overview of recent studies on the mechanisms of autophagosome formation and discuss these unresolved questions.

Autophagosome formation is a central event in macroautophagy. The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system. In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like). Apg12 is finally conjugated to Apg5 via an isopeptide bond. The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed. The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified. Here, we report the identification and characterization of the mouse Apg10 ortholog. A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10. We designated this protein mouse Apg10 (mApg10). We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5. The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system. This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation.

Vacuolar membrane H(+)-translocating pyrophosphatase (H(+)-PPase) was purified from pumpkin seedlings. Its enzymatic properties including molecular size of constituting polypeptide (75 kDa) were very similar to those of mung bean H(+)-PPase [(1989) J. Biol. Chem. 264, 20068-20073]. The native, functional molecular size of the pumpkin H(+)-PPase was estimated to be 135-139 kDa from gel permeation HPLC of the purified enzyme in the presence of detergent and from radiation inactivation of the enzyme in vacuolar membranes. It is concluded that native, functional pumpkin H(+)-PPase, and also probably H(+)-PPases from other plants, is a dimer of 75 kDa subunits.

The functional molecular masses of the vacuolar membrane H+-ATPase in Saccharomyces cerevisiae under two kinetic conditions for ATP hydrolysis were measured by radiation inactivation. When vacuolar membrane vesicles were exposed to gamma-rays from 60Co, the activities catalyzing a single-cycle and multi-cycles of ATP hydrolysis both decreased as single-exponential functions of the radiation dosage. By applying the target theory, the functional molecular masses for single- and multi-cycle hydrolyses of ATP were determined to be approx. 0.9-1.1 X 10(5) and 4.1-5.3 X 10(5) Da, respectively. N,N'-Dicyclohexylcarbodiimide (DCCD) did not inhibit the former reaction but strongly inhibited the latter. It is suggested that the ATPase with a minimal composite of subunits a and b, in which subunit c is not necessarily involved operationally, can catalyze single-cycle hydrolysis of ATP, whereas for multi-cycle hydrolysis of ATP, the ATPase requires a properly organized oligomeric structure with subunits a-c, which may direct a positive cooperative mechanism of ATP hydrolysis and coupled H+ translocation in a DCCD-sensitive manner.