ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes

Diao, Jiajie; Liu, Rong; Rong, Yueguang; Zhao, Minglei; Zhang, Jing; Lai, Ying; Zhou, Qiuhong; Wilz, Livia; Li, Jianxu; Vivona, Sandro; Pfuetzner, Richard A.; Brunger, Axel T.; Zhong, Qing

doi:10.1038/nature14147

Cited by 489 publications

(490 citation statements)

References 39 publications

Supporting

Mentioning

456

Contrasting

Unclassified

Order By: Relevance

“…Surprisingly, the phosphorylation sites in VAMP8 that we identified face the 3 other α‐helices of the SNARE complex (Fig 2A) (Fasshauer et al , 1998; Sutton et al , 1998; Diao et al , 2015) as part of the 16 conserved SNARE layers (Fasshauer et al , 1998) that are buried inside the SNARE complex (T47 at layer +3, S54 at layer +5, and T53 close to layer +5) (Figs 2B–D and EV2C). An additional site was listed as a putative phosphorylation site in the PhosphoSitePlus (Hornbeck et al , 2015) database (S61 at layer +7).…”

Section: Resultsmentioning

confidence: 86%

“…Incomplete zippering of the SNARE complex toward the C‐terminal end may result in docked vesicles that fail to fuse with the plasma membrane (Hernandez et al , 2012). To test the fusogenic activity of VAMP8, we used in vitro ensemble content‐ and lipid‐mixing assays with SNARE proteins reconstituted in proteoliposomes (Materials and Methods) (Diao et al , 2015). We mixed liposomes containing purified VAMP8 together with liposomes containing both purified SNAP23 and STX3 (Figs 3A and EV5A) (Sakiyama et al , 2009; Brochetta et al , 2014).…”

Section: Resultsmentioning

confidence: 99%

“…DNA plasmids used for SNARE reconstitution were human full‐length VAMP8, STX3, and SNAP23. The previously described VAMP8 (Diao et al , 2015) plasmid was mutated using QuickChange site‐directed mutagenesis kit (Stratagene) to create VAMP8Glu (T48E, T54E, S55E, these are the human amino acids that correspond to rat amino acids T47E, T53E, S54E). Full‐length human STX3 fused with a N‐terminal, TEV protease‐cleavable, hexa‐histidine tag in expression vector pJ414 was synthesized (DNA2.0).…”

Section: Methodsmentioning

confidence: 99%

“…A similar analysis was done for SNARE‐Qa, SNARE‐Qb, and SNARE‐Qc (NCBI cd15840, cd15841, cd15842). Accessible surface area for each amino acid was calculated from the crystal structure of PDB: 4WY4 (Diao et al , 2015) using STRIDE (http://webclu.bio.wzw.tum.de/stride) (Heinig & Frishman, 2004). PyMol was used to visualize the crystal structure.…”

Section: Methodsmentioning

confidence: 99%

“…Human full‐length VAMP8, STX3, and SNAP23 were expressed and purified according to previously reported protocols (Studier, 2005; Diao et al , 2015) with modifications. Briefly, SNAP23 and VAMP8 were expressed as N‐terminal GST fusions in BL21 (DE3) cells.…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

Self Cite

View full text Add to dashboard Cite

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α‐helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

show abstract

Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

The lysosomal membrane protein LAMP‐2 is dispensable for PINK1/Parkin‐mediated mitophagy

et al. 2019

View full text Add to dashboard Cite

Selective autophagy for the elimination of aberrant mitochondria, termed mitophagy, can be regulated by the kinase PINK1 and the ubiquitin ligase Parkin. The lysosome‐associated membrane protein 2 (LAMP‐2) plays diverse functions in non‐selective autophagy, chaperone‐mediated autophagy and selective autophagy for the degradation of RNA/DNA. In the present study, we investigated whether LAMP‐2 plays important roles during PINK1/Parkin‐mediated mitophagy. The results obtained clearly show that knockdown of LAMP‐2 does not cause defects in mitophagy in HeLa cells stably expressing Parkin, indicating that LAMP‐2 is dispensable for PINK1/Parkin‐mediated mitophagy. The present study is the first to determine the potential role of LAMP‐2 in PINK1/Parkin‐mediated mitophagy, thereby providing more insight into the sophisticated process of mitophagy.

show abstract

Rational Design of Biocompatible Ir(III) Photosensitizer to Overcome Drug‐Resistant Cancer via Oxidative Autophagy Inhibition

Park,

Nam,

Kim

et al. 2024

Advanced Science

View full text Add to dashboard Cite

Autophagy is a crucial quality control mechanism that degrades damaged cellular components through lysosomal fusion with autophagosomes. However, elevated autophagy levels can promote drug resistance in cancer cells, enhancing their survival. Downregulation of autophagy through oxidative stress is a clinically promising strategy to counteract drug resistance, yet precise control of oxidative stress in autophagic proteins remains challenging. Here, a molecular design strategy of biocompatible neutral Ir(III) photosensitizers is demonstrated, B2 and B4, for precise reactive oxygen species (ROS) control at lysosomes to inhibit autophagy. The underlying molecular mechanisms for the biocompatibility and lysosome selectivity of Ir(III) complexes are explored by comparing B2 with the cationic or the non‐lysosome‐targeting analogs. Also, the biological mechanisms for autophagy inhibition via lysosomal oxidation are explored. Proteome analyses reveal significant oxidation of proteins essential for autophagy, including lysosomal and fusion‐mediator proteins. These findings are verified in vitro, using mass spectrometry, live cell imaging, and a model SNARE complex. The anti‐tumor efficacy of the precise lysosomal oxidation strategy is further validated in vivo with B4, engineered for red light absorbance. This study is expected to inspire the therapeutic use of spatiotemporal ROS control for sophisticated modulation of autophagy.

show abstract

ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes

Abstract: Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by

Cited by 489 publications

References 39 publications

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

The lysosomal membrane protein LAMP‐2 is dispensable for PINK1/Parkin‐mediated mitophagy

Rational Design of Biocompatible Ir(III) Photosensitizer to Overcome Drug‐Resistant Cancer via Oxidative Autophagy Inhibition

Contact Info

Product

Resources

About