HS1BP3 inhibits autophagy by regulation of PLD1

Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3’-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization.     

Receptor‐mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy

Autophagosome formation requires multiple autophagy‐related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome‐wide CRISPR screens identifying genes required for NBR1 flux in ATG7^KO cells. We find that ATG7‐independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1''s ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin‐independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor‐mediated clustering of upstream autophagy factors drives continued autophagosome formation.     

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.     

A novel ER-localized transmembrane protein,EMC6, interacts with RAB5A and regulates cell autophagy

Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.     

Swapping of interaction partners with ATG5 for autophagosome maturation

Autophagy is a tightly regulated lysosome-mediated catabolic process in eukaryotes that maintains cellular homeostasis. A distinguishable feature of autophagy is the formation of double-membrane structures, autophagosome, which envelopes the intracellular cargoes and finally degrades them by fusion with lysosomes. So far, many structures of Atg proteins working on the autophagosome formation have been reported, however those involved in autophagosome maturation, a fusion with lysosome, are relatively unknown. One of the molecules in autophagosome maturation, TECPR1, has been identified and recently, structural studies on both ATG5-TECPR1 and ATG5-ATG16L1 complexes revealed that TECPR1 and ATG16L1 share the same binding site on ATG5. These results, in combination with supporting biochemical and cellular biological data, provide an insight into a model for swapping ATG5 partners for autophagosome maturation. [BMB Reports 2015; 48(3): 129-130]     

Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site

Autophagosome formation is governed by sequential functions of autophagy-related (ATG) proteins. Although their genetic hierarchy in terms of localization to the autophagosome formation site has been determined, their temporal relationships remain largely unknown. In this study, we comprehensively analyzed the recruitment of mammalian ATG proteins to the autophagosome formation site by live-cell imaging, and determined their temporal relationships. Although ULK1 and ATG5 are separated in the genetic hierarchy, they synchronously accumulate at pre-existing VMP1-positive punctate structures, followed by recruitment of ATG14, ZFYVE1, and WIPI1. Only a small number of ATG9 vesicles appear to be associated with these structures. Finally, LC3 and SQSTM1/p62 accumulate synchronously, while the other ATG proteins dissociate from the autophagic structures. These results suggest that autophagosome formation takes place on the VMP1-containing domain of the endoplasmic reticulum or a closely related structure, where ULK1 and ATG5 complexes are synchronously recruited.     

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.     

FIP200 regulates targeting of Atg16L1 to the isolation membrane

Autophagosome formation is a dynamic process that is strictly controlled by autophagy‐related (Atg) proteins. However, how these Atg proteins are recruited to the autophagosome formation site or autophagic membranes remains poorly understood. Here, we found that FIP200, which is involved in proximal events, directly interacts with Atg16L1, one of the downstream Atg factors, in an Atg14‐ and phosphatidylinositol 3‐kinase‐independent manner. Atg16L1 deletion mutants, which lack the FIP200‐interacting domain, are defective in proper membrane targeting. Thus, FIP200 regulates not only early events but also late events of autophagosome formation through direct interaction with Atg16L1.     

Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

MAP1B‐LC1 prevents autophagosome formation by linking syntaxin 17 to microtubules

In fed cells, syntaxin 17 (Stx17) is associated with microtubules at the endoplasmic reticulum–mitochondria interface and promotes mitochondrial fission by determining the localization and function of the mitochondrial fission factor Drp1. Upon starvation, Stx17 dissociates from microtubules and Drp1, and binds to Atg14L, a subunit of the phosphatidylinositol 3‐kinase complex, to facilitate phosphatidylinositol 3‐phosphate production and thereby autophagosome formation, but the mechanism underlying this phenomenon remains unknown. Here we identify MAP1B‐LC1 (microtubule‐associated protein 1B‐light chain 1) as a critical regulator of Stx17 function. Depletion of MAP1B‐LC1 causes Stx17‐dependent autophagosome accumulation even under nutrient‐rich conditions, whereas its overexpression blocks starvation‐induced autophagosome formation. MAP1B‐LC1 links microtubules and Stx17 in fed cells, and starvation causes the dephosphorylation of MAP1B‐LC1 at Thr217, allowing Stx17 to dissociate from MAP1B‐LC1 and bind to Atg14L. Our results reveal the mechanism by which Stx17 changes its binding partners in response to nutrient status.     

Effects of potassium on expression of renal sodium transporters in salt-sensitive hypertensive rats induced by uninephrectomy

Dietary potassium is an important modulator of systemic blood pressure (BP). The purpose of this study was to determine whether dietary potassium is associated with an altered abundance of major renal sodium transporters that may contribute to the modulation of systemic BP. A unilateral nephrectomy (uNx) was performed in male Sprague-Dawley rats, and the rats were fed a normal-salt diet (0.3% NaCl) for 4 wk. Thereafter, the rats were fed a high-salt (HS) diet (3% NaCl) for the entire experimental period. The potassium-repleted (HS+KCl) group was given a mixed solution of 1% KCl as a substitute for drinking water. We examined the changes in the abundance of major renal sodium transporters and the expression of mRNA of With-No-Lysine (WNK) kinases sequentially at 1 and 3 wk. The systolic BP of the HS+KCl group was decreased compared with the HS group (140.3 ± 2.97 vs. 150.9 ± 4.04 mmHg at 1 wk; 180.3 ± 1.76 vs. 207.7 ± 6.21 mmHg at 3 wk). The protein abundances of type 3 Na(+)/H(+) exchanger (NHE3) and Na(+)-Cl(-) cotransporter (NCC) in the HS+KCl group were significantly decreased (53 and 45% of the HS group at 1 wk, respectively; 19 and 8% of HS group at 3 wk). WNK4 mRNA expression was significantly increased in the HS+KCl group (1.4-fold of control at 1 wk and 1.9-fold of control at 3 wk). The downregulation of NHE3 and NCC may contribute to the BP-attenuating effect of dietary potassium associated with increased urinary sodium excretion.     

Role of 3-O-sulfated heparan sulfate in virus-induced polykaryocyte formation

One way herpes simplex virus type-1 (HSV-1) spreads in vivo is by polykaryocytes formation. Here we demonstrate that polykaryocyte production during HSV-1 spread in cultured human corneal fibroblasts (CF) required heparan sulfate (HS) and more specifically 3-O sulfated HS (3-OS HS). The polykaryocyte formation heavily depended on the expression of HS on target (CF) cells but not on glycoprotein expressing effector cells. Furthermore, we provide the first visual evidence of 3-OS HS and HSV-1 gD colocalization during the membrane fusion process. Taken together our results provide novel insight into the significance of HS in polykaryocyte formation.     

Recycling endosomes contribute to autophagosome formation

Autophagosome formation is a complex cellular process, which requires major membrane rearrangements leading to the creation of a relatively large double-membrane vesicle that directs its contents to the lysosome for degradation. Although various membrane compartments have been identified as sources for autophagosomal membranes, the molecular mechanism underlying these membrane trafficking steps remains elusive. To address this question we performed a systematic analysis testing all known Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins for their ability to inhibit autophagosome formation by disrupting a specific membrane trafficking step. TBC proteins are thought to act as inhibitors of Rab GTPases, which regulate membrane trafficking events. Up to 11 TBC proteins inhibit autophagy when overexpressed and one of these, TBC1D14, acts at an early stage during autophagosome formation and is involved in regulating recycling endosomal traffic. We found that the early acting autophagy proteins ATG9 and ULK1 localize to transferrin receptor (TFR)-positive recycling endosomes (RE), which are tubulated by excess TBC1D14 leading to an inhibition of autophagosome formation. Finally, transferrin (TF)-containing recycling endosomal membranes can be incorporated into newly forming autophagosomes, although it is likely that most of the autophagosome membrane is subsequently acquired from other sources.     

Recycling endosomes contribute to autophagosome formation

Autophagosome formation is a complex cellular process, which requires major membrane rearrangements leading to the creation of a relatively large double-membrane vesicle that directs its contents to the lysosome for degradation. Although various membrane compartments have been identified as sources for autophagosomal membranes, the molecular mechanism underlying these membrane trafficking steps remains elusive. To address this question we performed a systematic analysis testing all known Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins for their ability to inhibit autophagosome formation by disrupting a specific membrane trafficking step. TBC proteins are thought to act as inhibitors of Rab GTPases, which regulate membrane trafficking events. Up to 11 TBC proteins inhibit autophagy when overexpressed and one of these, TBC1D14, acts at an early stage during autophagosome formation and is involved in regulating recycling endosomal traffic. We found that the early acting autophagy proteins ATG9 and ULK1 localize to transferrin receptor (TFR)-positive recycling endosomes (RE), which are tubulated by excess TBC1D14 leading to an inhibition of autophagosome formation. Finally, transferrin (TF)-containing recycling endosomal membranes can be incorporated into newly forming autophagosomes, although it is likely that most of the autophagosome membrane is subsequently acquired from other sources.     

How autophagy eats large mitochondria: Autophagosome formation coupled with mitochondrial fragmentation

Mitochondrial autophagy (mitophagy) is thought to be a multi-step pathway wherein mitochondria are first divided into small fragments, which are subsequently recognized by the phagophore. DNM1L (dynamin 1 like) plays a pivotal role in mitochondrial division; however, its role in mitophagy remains controversial. In our recent study, we examined the contribution of DNM1L to mitophagy and showed that mitophagy and mitochondrial division occur even in DNM1L-defective cells. Furthermore, time-lapse imaging of mitophagy showed that DNM1L-independent mitochondrial division occurs concomitantly with autophagosome formation. Upstream factors of autophagosome formation, i.e., RB1CC1/FIP200, ATG14, and WIPIs, are required for mitochondrial division, whereas ATG5 and ATG3 are dispensable. These results indicate that a portion of the tubular mitochondria is first recognized and then divided into small fragments by a phagophore-mediated event, independently of DNM1L. This autophagic process suggests that autophagy has the potential to degrade substrates larger than autophagosomes.     

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.     

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.