Maho Hamasaki

Autophagosomes form at ER–mitochondria contact sites

Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate—the endoplasmic reticulum (ER), mitochondria and plasma membrane. Here we show that autophagosomes form at the ER–mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER–mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane fraction under starvation conditions. Disruption of the ER–mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER–mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER–mitochondria contact site is important in autophagosome formation.

Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import

ABSTRACT Two isoforms of the peroxisomal targeting signal type 1 (PTS1) receptor, termed Pex5pS and (37-amino-acid-longer) Pex5pL, are expressed in mammals. Pex5pL transports PTS1 proteins and Pex7p-PTS2 cargo complexes to the initial Pex5p-docking site, Pex14p, on peroxisome membranes, while Pex5pS translocates only PTS1 cargoes. Here we report functional Pex5p domains responsible for interaction with peroxins Pex7p, Pex13p, and Pex14p. An N-terminal half, such as Pex5pL(1-243), comprising amino acid residues 1 to 243, bound to Pex7p, Pex13p, and Pex14p and was sufficient for restoring the impaired PTS2 import of pex5 cell mutants, while the C-terminal tetratricopeptide repeat motifs were required for PTS1 binding. N-terminal Pex5p possessed multiple Pex14p-binding sites. Alanine-scanning analysis of the highly conserved seven (six in Pex5pS) pentapeptide WXXXF/Y motifs residing at the N-terminal region indicated that these motifs were essential for the interaction of Pex5p with Pex14p and Pex13p. Moreover, mutation of several WXXXF/Y motifs did not affect the PTS import-restoring activity of Pex5p, implying that the binding of Pex14p to all of the WXXXF/Y sites was not a prerequisite for the translocation of Pex5p-cargo complexes. Pex5p bound to Pex13p at the N-terminal part, not to the C-terminal SH3 region, via WXXXF/Y motifs 2 to 4. PTS1 and PTS2 import required the interaction of Pex5p with Pex14p but not with Pex13p, while Pex5p binding to Pex13p was essential for import of catalase with PTS1-like signal KANL. Pex5p recruited PTS1 proteins to Pex14p but not to Pex13p. Pex14p and Pex13p formed a complex with PTS1-loaded Pex5p but dissociated in the presence of cargo-unloaded Pex5p, implying that PTS cargoes are released from Pex5p at a step downstream of Pex14p and upstream of Pex13p. Thus, Pex14p and Pex13p very likely form mutually and temporally distinct subcomplexes involved in peroxisomal matrix protein import.

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L‐Leucyl‐L‐leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy‐dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.

Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant‐negative inactive mutant of Myotubularin‐related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy‐related PtdIns3P‐binding proteins, GFP‐DFCP1 and GFP‐WIPI‐1α (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock‐down of MTMR3 increased autophagosome formation, and overexpression of wild‐type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3‐kinase and PI 3‐phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.

Starvation Triggers the Delivery of the Endoplasmic Reticulum to the Vacuole via Autophagy in Yeast

Autophagy is a survival mechanism necessary for eukaryotic cells to overcome nutritionally challenged environments. When autophagy is triggered, cells degrade nonselectively engulfed cytosolic proteins and free ribosomes that are evenly distributed throughout the cytoplasm. The resulting pool of free amino acids is used to sustain processes crucial for survival. Here we characterize an autophagic degradation of the endoplasmic reticulum (ER) under starvation conditions in addition to cytosolic protein degradation. Golgi membrane protein was not engulfed by the autophagosome under the same conditions, indicating that the uptake of ER by autophagosome was the specific event. Although the ER exists in a network structure that is mutually connected and resides predominantly around the nucleus and beneath the plasma membrane, most of autophagosome engulfed ER. The extent of the ER uptake by autophagy was nearly identical to that of the soluble cytosolic proteins. This phenomenon was explained by the appearance of fragmented ER membrane structures in almost all autophagosomes. Furthermore, ER dynamism is required for this process: ER uptake by autophagosomes occurs in an actin‐dependent manner.

Where do they come from? Insights into autophagosome formation

Autophagosomes (APs) are unique organelles that enwrap cytoplasmic components when necessary. APs then fuse with lysosomes and enclosed materials are degraded. Although approximately 30 autophagy‐related genes (ATG) required for AP formation have been identified, fundamental questions on the membrane source or dynamics during the formation remain unresolved. Here, we present a comprehensive overview of the putative membrane sources identified to date.

Critical Roles for Lipomannan and Lipoarabinomannan in Cell Wall Integrity of Mycobacteria and Pathogenesis of Tuberculosis

ABSTRACT Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis. IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target. Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.

Rubicon inhibits autophagy and accelerates hepatocyte apoptosis and lipid accumulation in nonalcoholic fatty liver disease in mice

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It encompasses a spectrum ranging from simple steatosis to fatty liver with hepatocellular injury, termed nonalcoholic steatohepatitis. Recent studies have demonstrated hepatic autophagy being impaired in NAFLD. In the present study, we investigated the impact of Rubicon, a Beclin1‐interacting negative regulator for autophagosome‐lysosome fusion, in the pathogenesis of NAFLD. In HepG2 cells, BNL‐CL2 cells, and murine primary hepatocytes, Rubicon was posttranscriptionally up‐regulated by supplementation with saturated fatty acid palmitate. Up‐regulation of Rubicon was associated with suppression of the late stage of autophagy, as evidenced by accumulation of both LC3‐II and p62 expression levels as well as decreased autophagy flux. Its blockade by small interfering RNA attenuated autophagy impairment and reduced palmitate‐induced endoplasmic reticulum stress, apoptosis, and lipid accumulation. Rubicon was also up‐regulated in association with autophagy impairment in livers of mice fed a high‐fat diet (HFD). Hepatocyte‐specific Rubicon knockout mice generated by crossing Rubicon floxed mice with albumin‐Cre transgenic mice did not produce any phenotypes on a normal diet. In contrast, on an HFD, they displayed significant improvement of both liver steatosis and injury as well as attenuation of both endoplasmic reticulum stress and autophagy impairment in the liver. In humans, liver tissues obtained from patients with NAFLD expressed significantly higher levels of Rubicon than those without steatosis. Conclusion: Rubicon is overexpressed and plays a pathogenic role in NAFLD by accelerating hepatocellular lipoapoptosis and lipid accumulation, as well as inhibiting autophagy. Rubicon may be a novel therapeutic target for regulating NAFLD development and progression. (Hepatology 2016;64:1994‐2014).

Autophagosome–lysosome fusion in neurons requires INPP5E, a protein associated with Joubert syndrome

Autophagy is a multistep membrane traffic pathway. In contrast to autophagosome formation, the mechanisms underlying autophagosome–lysosome fusion remain largely unknown. Here, we describe a novel autophagy regulator, inositol polyphosphate‐5‐phosphatase E (INPP5E), involved in autophagosome–lysosome fusion process. In neuronal cells, INPP5E knockdown strongly inhibited autophagy by impairing the fusion step. A fraction of INPP5E is localized to lysosomes, and its membrane anchoring and enzymatic activity are necessary for autophagy. INPP5E decreases lysosomal phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P2), one of the substrates of the phosphatase, that counteracts cortactin‐mediated actin filament stabilization on lysosomes. Lysosomes require actin filaments on their surface for fusing with autophagosomes. INPP5E is one of the genes responsible for Joubert syndrome, a rare brain abnormality, and mutations found in patients with this disease caused defects in autophagy. Taken together, our data reveal a novel role of phosphoinositide on lysosomes and an association between autophagy and neuronal disease.

Atg9A trafficking through the recycling endosomes is required for autophagosome formation

ABSTRACT Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N- and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation. Highlighted Article: Atg9A trafficking through the recycling endosome is required for autophagy, and is mediated by TRAPPIII and intrinstic Atg9A-sorting motifs that bind to AP2.