Autophagosome biogenesis requires two ubiquitin-like conjugation systems. One couples ubiquitin-like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin-like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P-containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 b-propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled-coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6-motif in the N-terminal helical domain of Atg8, but not its AIM-binding site. Accordingly, the Atg8 AIM-binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P-dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.

Mitochondria are turned over by an autophagic process termed mitophagy. This process is considered to remove damaged, superfluous and aged organelles. However, little is known about how defective organelles are recognized, what types of damage induce turnover, and whether an identical set of factors contributes to degradation under different conditions. Here we systematically compared the mitophagy rate and requirement for mitophagy-specific proteins during post-log-phase and rapamycin-induced mitophagy. To specifically assess mitophagy of damaged mitochondria, we analyzed cells accumulating proteins prone to degradation due to lack of the mitochondrial AAA-protease Yme1. While autophagy 32 (Atg32) was required under all tested conditions, the function of Atg33 could be partially bypassed in post-log-phase and rapamycin-induced mitophagy. Unexpectedly, we found that Uth1 was dispensable for mitophagy. A re-evaluation of its mitochondrial localization revealed that Uth1 is a protein of the inner mitochondrial membrane that is targeted by a cleavable N-terminal pre-sequence. In agreement with our functional analyses, this finding excludes a role of Uth1 as a mitochondrial surface receptor.

Introduction ERAS pathway has been proposed as the standard of care in elective abdominal surgery. Guidelines on ERAS in emergency surgery have been recently published; however, few evidences are still available in the literature. The aim of this study was to evaluate the feasibility of an enhanced recovery protocol in a large cohort of patients undergoing emergency surgery and to identify possible factors impacting postoperative protocol compliance. Methods This is a prospective multicenter observational study including patients who underwent major emergency general surgery for either intra-abdominal infection or intestinal obstruction. The primary endpoint of the study is the adherence to ERAS postoperative protocol. Secondary endpoints are 30-day mortality and morbidity rates, and length of hospital stay. Results A total of 589 patients were enrolled in the study, 256 (43.5%) of them underwent intestinal resection with anastomosis. Major complications occurred in 92 (15.6%) patients and 30-day mortality was 6.3%. Median adherence occurred on postoperative day (POD) 1 for naso-gastric tube removal, on POD 2 for mobilization and urinary catheter removal, and on POD 3 for oral intake and i.v. fluid suspension. Laparoscopy was significantly associated with adherence to postoperative protocol, whereas operative fluid infusion > 12 mL/Kg/h, preoperative hyperglycemia, presence of a drain, duration of surgery and major complications showed a negative association. Conclusions The present study supports that an enhanced recovery protocol in emergency surgery is feasible and safe. Laparoscopy was associated with an earlier recovery, whereas preoperative hyperglycemia, fluid overload, and abdominal drain were associated with a delayed recovery.

The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification.

How Atg15 is activated exclusively in the vacuolar lumen is still elusive. The overexpression of active, membrane-bound Atg15 variants reduced cell growth in different deletion strains. This was still the case in some mutants of the v-ATPase or different transport pathway. It is thus unlikely that the vacuolar pH or a high substrate specificity of Atg15 is responsible for its activation. Based on sequence homology, Atg15 was identified as a α/ß-hydrolase with a potential catalytic lid, which could be controlled by a yet unknown additional component such as a colipase. Figure 2.1: Life cycle of Saccharomyces cerevisiae (1) In the haploid phase, the mating types Mat a and Mat α bud by mitotic divisions. (2) Mating of two haploid cells results in a diploid cell. (3) Generation of 4-8 haploid spores after sporulation and start of a new cycle. The phosphatidylinositol 3-kinase complexThe membranes of autophagosomes contain a specific lipid molecule, phosphatidylinositol 3-phosphate (PI3P or PtdIns3P) (Obara et al., 2008a). Up to date, Vps34 is the only identified phosphatidylinositol 3-kinase specifically generating PI3P in yeast. Vps34 is organized in two complexes: PtdIns 3-kinase complex I and II (Schu et al., 1993). Both complexes share the components Vps34, essential during very early steps of phagophore formation (Legakis et al., 2007;Yamamoto et al., 2012). Using overexpressed Atg9, the peripheral pool was described as vesicles and tubular structures adjacent to mitochondria (Mari et al., 2010). However, a more recent study postulated that the Atg9 peripheral pool is organized in cytosolic and highly motile Atg9-vesicles when expressed under its endogenous promoter. It was further postulated that each Atg9 vesicle contains approximately 27 molecules of Atg9 (Yamamoto et al., 2012). Up to now, the real nature of the peripheral pool of Atg9 is still under debate (Mari et al., 2010;Ohashi and Munro, 2010;Yamamoto et al., 2012). in the formation of the Atg12-Atg5 complex. (Mizushima et al., 1998;Noda et al., 2011;Tanida et al., 1999). In contrast to the ubiquitin conjugation system, this reaction does not need a ligase acting as E3-like enzyme. Here, Atg10 is able to directly mediate the formation of covalently bound Atg12-Atg5 (Yamaguchi et al., 2012). It has to be mentioned that Atg5 also consists of two ubiquitin-like domains Autophagy was first described as a mechanism for unspecific degradation of cytosolic components especially necessary during starvation. Diverse genetic screens extended this point of view toward the existence of different selective forms of autophagy. These receptor-mediated selective autophagic pathways have to be discriminated from unspecific bulk macroautophagy (Suzuki, 2012).Pexophagy describes the selective autophagic pathway to remove peroxisomes. Similar to Atg19 in the Cvt pathway, Atg36 acts as a cargo receptor for peroxisomes in S. cerevisiae (Motley et al., 2012). Atg36 contains binding sites for Atg11 and Atg8 and interacts with Pex3. Pex3 is a protein of the peroxisomal membran...