We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

/pdf/construction-of-escherichia-coli-k-12-in-frame-single-gene-4c68fx6pb1.pdf

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

/pdf/ampk-and-mtor-regulate-autophagy-through-direct-14wqsz16ke.pdf

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Autophagy: Renovation of Cells and Tissues

Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. Despite its simplicity, recent progress has demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles, which are sometimes complex. Autophagy consists of several sequential steps--sequestration, transport to lysosomes, degradation, and utilization of degradation products--and each step may exert different function. In this review, the process of autophagy is summarized, and the role of autophagy is discussed in a process-based manner.

http://genesdev.cshlp.org/content/21/22/2861.full.pdf

Autophagy: process and function

Author response: Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/ vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and

https://doi.org/10.1080/27694127.2023.2188523

Atg8 family proteins, LIR/AIM motifs and other interaction modes

ATG4 Proteases in Autophagy

ABSTRACT Recent studies have revealed that even the nucleus can be degraded by selective macroautophagy (hereafter macronucleophagy). In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a macronucleophagy receptor that mediates sequestration of nucleus-derived double-membrane vesicles (NDVs) into phagophores. The outer and inner membranes of these NDVs are derived from the outer and inner nuclear membranes (ONM and INM), respectively, and the lumen contains nucleoplasmic material. Little was known about the mechanisms underlying macronucleophagy, including how the two nuclear membranes are coordinately deformed to generate NDVs and what nuclear components are preferentially loaded into or rather eliminated from NDVs. We found that Atg39 links the ONM and INM through the ONM-embedded transmembrane domain and INM-associated amphipathic helices (APHs). These APHs are important for Atg39 anchoring to the NE and autophagosome formation-coupled Atg39 clustering in the NE. In addition, the overaccumulation of Atg39 in the NE caused NE protrusion toward the cytoplasm depending on the APHs. These results allowed us to propose the mechanism by which Atg39 conducts NDV formation in coordination with autophagosome formation during macronucleophagy.

Atg39 binding to the inner nuclear membrane triggers nuclear envelope deformation in piecemeal macronucleophagy

Eukaryotic cells have developed various organelles, and therein separated subsets of reactions, to perform diverse cellular functions in an organized manner. They have also been equipped with elaborate mechanisms to strictly control quality and quantity of organelles, and deficiencies in these mechanisms can lead to diseases in humans. Autophagy, an intracellular degradation pathway, proceeds through the following steps: (1) a double-membraned vesicle called the autophagosome is formed in the cytoplasm to sequester degradation targets, (2) the autophagosome fuses with the lysosomal or vacuolar membrane, and (3) the sequestered material is degraded by hydrolases in lysosomes/vacuoles.1 While autophagy randomly degrades cytoplasmic constituents under nutrient-limiting conditions, it can also selectively degrade certain molecules or structures via landmark proteins called autophagy receptors.2 These proteins localize to specific targets, and also bind to Atg8-family proteins on expanding autophagosomal membranes, leading to the sequestration of the targets by autophagosomes. Previous studies have shown that receptor-mediated selective autophagy largely contributes to degradation of some organelles, including mitochondria and peroxisomes, as well as protein aggregates and invading pathogens.2 However, how extensively selective autophagy is involved in organelle homeostasis and regulation remained to be investigated. In a recent study, we have revealed that when starved, yeast cells actively degrade their own endoplasmic reticulum (ER) and nucleus via selective autophagy, which involves 2 novel receptors.3

Hitoshi Nakatogawa

Papers

Author response: Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein

Atg8 family proteins, LIR/AIM motifs and other interaction modes

ATG4 Proteases in Autophagy

Atg39 binding to the inner nuclear membrane triggers nuclear envelope deformation in piecemeal macronucleophagy

Appetite for ER/nucleus destruction